INTERNATIONAL REVIEW OF CYTOLOGY,Vol. 54
An Enzyme Profile of the Nuclear Envelope I. B. ZBARSKY Biochemistry Laboratory, N . K . Koltzov Institute of Developmental Biology, Academy of Sciences of the USSR. Moscow, USSR
I. Introduction . . . . . . . . . . . . . . . . . . . . A. Historical Aspects . . . . . . . . . . . . . . . . B. Progress in Studies on the NE . . . . . . . . . , . . C. Probable Functions of the NE in Relation to Its Enzyme Profile 11. Enzyme Profile of NEs . . . . . . . . . . . . , . . A. Hydrolytic Enzymes and Transferases . . . . , . . B. Oxidoreductases . . . . . . . . . . . . . . . . . C. Enzyme Profile of the NE in Comparison with That of Other Cell StNCtUES . . . . . . . . . . . . . . . . . . . 111. The Problem of Oxidation and Oxidative Phosphorylation in the NE A. Nuclear Oxidation and Oxidative Phosphorylation . . . . . B. Electron Transport and Oxidative Phosphorylation in the NE . C. Are Nuclear Oxidation and Phosphorylation due to Mitochondria1 Contamination? . . . . . . . . . . . . . . . . . IV. The Role of NEs . . . . . . . . . . . . . . . . . . A. Relationship of the NE to Protein and Nucleic Acid Synthesis B. Nucleocytoplasmic Transport and Enzymes of the NE . . . C. Peculiarities of the NE in Relation to Physiological and Pathological Conditions . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . A. Characteristics of the NE Enzyme Profile . . . . . . . . B. Biogenesis of the NE and Related Structures . . . . . . . C. Problems of Enzyme Studies of the NE in Relation to Its Functions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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295 296 296 298 299 299 312 325 329 329 329 331 335 335 338 344 347 347 347 348 349
I. Introduction The nuclear membrane (NM) is a characteristic structure separating the nucleus from the cytoplasm in all eukaryotic cells. In different tissues and organisms the structure of the nuclear envelope (NE) is surprisingly uniform. Its common features are outer and inner NMs, a perinuclear space between these membranes, and rather large (60-70 nm in diameter) nuclear pores usually surrounded by an annulus having octagonal symmetry. NMs interact morphologically and functionally with nuclear and cytoplasmic structures and 295 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364354-6
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therefore can be regarded as part of both the nucleus and the cell membrane system. In cells with intensive metabolism the NE has numerous invaginations and protrusions which increase the nucleocytoplasmic interface and enhance the interaction between nucleus and cytoplasm. In such cells (silk gland cells, neurons, certain glandular and embryonal cells) rupture of the NE and its membranes, especially the outbursts and blebs of the outer NM, is usual, and the formation of vesicles and possibly of membranes of endoplasmic reticulum from the outer NM is often observed, as well as of the annulate lamella-a counterpart of the NE structure (Kessel, 1968; Scharrer and Wurzelmann, 1969). A. HISTORICAL ASPECTS As far back as the end of the last century (Schwartz, 1892) the existence of a membrane surrounding the nucleus was suspected. Later this envelope was thought to be a double-membrane structure (Robyns, 1924; Scarth, 1927; Cohen, 1937; Dangeard, 1942; Policard and Bessis, 1956). The existence of the NE was proved by means of electron microscopy which enabled investigators to see for the first time the NMs and their special substructures-the nuclear pores (Callan and Tomlin, 1950; Watson, 1955, 1959). In the fifties and sixties, the morphology of NMs and nuclear pores was studied and reviewed by several workers (Haguenau and Bernhard, 1955; Policard and Baud, 1958; Baud, 1959; David, 1964; Gall, 1964; Feldherr and Harding, 1964; Goldstein, 1964; Loewenstein, 1964; Zagorski, 1965). B. PROGRESS IN STUDIES ON
THE
NE
While the first data on the chemical components and enzymes of the NE were obtained by electron microscope histochemistry, the isolation of NMs (Franke, 1966; Zbarsky et al., 1967) was the principal achievement in the understanding of their biochemical and enzymic properties (Pokrovsky et al., 1968; Zbarsky et al., 1968, 1969; Kashnig and Kasper, 1969; Frankeet al., 1970). With the use of isolated preparations, the morphological structure, chemical composition, and enzyme profile of the NE could be studied in more detail. Nevertheless, the results obtained were often contradictory, and many aspects of the problem remain unsolved. The present state of our knowledge of the structure, function, biochemical properties, methods of isolation, and other aspects of NE research is represented in numerous reviews published during the last 10 years (Gouranton, 1969; Stevens and Andrk, 1969; Zbarsky, 1969, 1972a,b,c, 1973, 1975b; Franke, 1970a, 1974a,b; Feldherr, 1972; Kay and Johnston, 1973; Kessel, 1973; Wishnitzer, 1973; Berezney, 1974; Franke and Scheer, 1974a; Kasper, 1974; Fry, 1976,
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1977; Harris and Agutter, 1976; Wunderlich et a l . , 1976). Therefore only some basic data on the morphology of the NE (Fig. 1) are mentioned in this article. Apart from the outer and inner NMs and pore complexes the NE also contains a “fibrous lamina” (Fawcett, 1966), “internal dense lamella” (Stevens and Andre, 1969), or “zonula nucleum limitans” (Patrizi and Poger, 1967) closely associated with the inner NM. This lamella is pronounced in amebas, where it was previously described as the “honeycomb layer” (Pappas, 1956). The inner membrane or the fibrous lamina is associated with the peripheral chromatin (Davies and Small, 1968). This association may involve special peripheral granules about 25 nm in diameter (Barton et a l . , 1971; Onishchenko and Chentsov, 1973, 1974a,b). The outer NM is directly connected with the endoplasmic reticulum and is frequently associated with mitochondria as well. Many cell types contain in the cytoplasm and sometimes in the nucleus annulate lamellae structurally similar to the NE and probably originating from it. The chemical composition of the NE is typical of membrane structures with relatively high protein and low cholesterol content; the presence of RNA and DNA has also been established. Certain peculiarities of NE composition may be due to the fact that it comprises not only the inner and outer NMs but also the pore complexes and fibrous lamina consisting mostly of proteins. During the cell cycle the latter structures degenerate and are reconstructed anew, apparently independently of the NMs (Zatsepina ef al., 1976a,b; 1977). Thus the nuclear pores appear to have some autonomy and can be separated from the NMs. As already mentioned, the pores are usually surrounded by octagonal annuli. The octagonal symmetry is due to usually eight peripheral granules interconnected with filaments. Inside the pore a central granule bound to other parts of the pore complex with filaments is often seen. Apart from the central granule amorphous material may fill the pore orifice. Although the details of the pore 3
J
6
\7 \2
FIG. 1. General scheme of a NE fragment (cytoplasm side up). 1, Outer nuclear membrane; 2 , inner nuclear membrane; 3, endoplasmic reticulum; 4, ribosomes; 5, fibrous lamina; 6, peripheral granules associated with chromosome telomeres; 7, peripheral chromatin adhering to the NE; 8, pore complexes.
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structure are still unclear, a tentative scheme of a nuclear pore is presented in Fig. 2.
c.
PROBABLE FUNCTIONS OF THE NE
IN
RELATION TO ITS ENZYME PROFILE
The functions of the NE are not well known, but its location at the interface between the nucleus and the cytoplasm implies a role in maintenance of the shape and composition of the nucleus. Electron microscope observations and the presence of the pores indicate that the NE may participate in the nucleocytoplasmic transport of various substances, especially RNAs and ribonucleoproteins. A wide spectrum of enzyme activity in the NE and its specialized structure suggests its participation in the control of nuclear metabolism. The presence of oxidative enzymes and oxidative phosphorylation may enable the NE to supply the energy for nucleocytoplasmic transport and intranuclear biosynthetic reactions. The main body of our knowledge of the enzymes of the NE was obtained from the biochemical study of isolated preparations. Histochemical studies, mainly by means of electron microscopy, have provided additional important data. Indirect information comes from the enzymic analysis of isolated nuclei, autoradiography, and studies on biogenesis, turnover, and reconstruction of NMs and nuclear pores, as well as on nucleocytoplasmic transport. Most reliable quantitative results were obtained by enzymic analysis of isolated structures. However, certain enzymes and other components may be extracted and lost during the isolation of nuclei, and especially of NEs present in these structures in vivo. The adsorption of nuclear and cytoplasmic enzymes by the NE is also possible. Treatment with salt or enzyme preparations, as well as all kinds of contamination, may inactivate or alter the properties of its enzymes. It is 100-120 nm
4
-4
\
60-70 nmD
--. P 25nm
3
FIG.2. Schematic drawing of a pore complex (cytoplasm side up). 1, Peripheral dense layer connected to the fibrous lamina; 2, fibrous lamina; 3, peripheral granules; 4, central granule; 5, amorphous material filling up the pore interior; 6, filaments projecting inside the nucleus.
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obvious that the purity of isolated NE preparations is a prerequisite for an adequate analysis of their enzyme profile. The localization of enzyme activity is usually better determined by histochemical methods, though they are as a rule less specific and less reliable than biochemical analyses and can give only semiquantitative data. However, histochemical methods may reveal soluble enzymes which are extracted from the NEs during isolation. Thus it is not surprising that available data on the enzymes of NEs are contradictory and highly dependent on the methods used for the isolation of nuclei and NEs, as well as on the procedures for determining enzyme activity. Therefore a thorough comparison of results obtained by different methods for each enzyme is necessary. Despite the uniformity of the NE structure in different cells, its enzymic activity may depend on the function, physiological state, and metabolic activity of a given cell or tissue, as well as on pathological or damaging conditions. Finally, different substructures of the NE may differ considerably from each other in enzyme profile and function. Thus we have a general idea of the enzyme profile of the NE, but many aspects remain unresolved and are a matter of discussion. We are almost unaware of peculiarities of NE enzymes in different tissues and organisms and their dependence on physiologically active and damaging agents. We are unable also to locate certain enzymes on definite substructures of the NE such as the inner and outer NMs and the nuclear pores.
11. Enzyme Profile of NEs
A. HYDROLYTIC ENZYMESA N D TRANSFERASES Hydrolytic enzyme activities in the NE were primarily detected by histochemical methods. Afterward, quantitative data on the isolated preparations became available. Table I lists quantitative data on hydrolytic and other nonoxidative enzymes in the NE in comparison with those in the corresponding tissue and other cell components. Apart from the above-mentioned reasons for the discrepancies between the results obtained by different investigators, they do not always correspond to histochemical data. These discrepancies may be due to localization of some enzymes in the perinuclear space, to their lipophilic nature, or to their weak association with the membrane structure and loss of easily extractable enzymes in the course of isolation.
TABLE I HYDROLYTIC A N D OTHER NONOXIDATIVE ENZYMEACTIVITIES OF THE NE Enzyme Alkaline phosphatase
Acid phosphataqe
W g
Glucose-6-phosphatase
Tissue Rat live@ Rat liverb Rat liver Pig liverb Rat liverb Rat liverb Rat liver Pig liverb Rooster liver Onion stems Onion roots Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Pig liver Bovine liver Bovine liver Rooster liver Rat thymus CHO
IN
COMPARISON TO THOSE OF OTHER CELL STRUCTURES"
NE
NElnuclei
NE/microsomes
Reference
28 2.5 1.6 35 53 13 4.1 167 13.2 0.5 lo00 11.5 22k 4.1
NWmicrosomes 0.18O 0.03O 5.8 1.0
-
0.11
-
4.5
-
Reference Pokrovsky ef al. (1970) Pokrovsky ef al. (1970) Pokrovsky ef al. (1%8) Pokrovsky et d.(1968) Pokrovsky ef al. (1%8) Kasper ( 1971) Jarasch (1973) Zentgraf ef al. (1971) Fukushima et al. (1976) Skridonenko and Gorchakova (1973) Skridonenko and Gorchakova (1973) Skridonenko ef ul. (1975) Skridonenko et al. (1975) Skridonenko et ul. (1975) Skridonenko er al. (1975) Koen ef al. (1976) Koen ef al. (1976)
"Nanomoles of metabolized substrate or product per minute per milligram protein. Other measurements reported in the literature are recalculated *p-Nitrophenyl as substrate. 'Crude nuclear fraction. dHeavy (h) and light (1) NE subfractions. 'NEs were in addition purified in sucrose gradient. I n relation to the homogenate. @Inrelation to the mitochondria. hMethylbutyrin as substrate. 'Tributyrin as substrate. 'The outer nuclear membrane was isolated by extraction with 0.5% Triton X-100. M p-chloromercuribenzoate. "In the presence of 3 X
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305
1. Phosphohydrolases a. Glycerophosphatases. This group comprises phosphomonoesterases characterized by a broad specificity, which are usually called acid and alkaline phosphatases and have pH optima of 4.5-5.5 and 8.5-9.5, respectively. According to Enzyme Nomenclature they are called orthophosphoric-monoester phosphohydrolases (EC 3.1.3.1, alkaline phosphatase, and EC 3.1.3.2, acid phosphatase). Since the individual characters of these enzymes are still not established and their affinities for different substrates vary, it is convenient to call them glycerophosphatases, since P-glycerophosphate is usually employed as a substrate. Earlier histochemical data on high nuclear phosphatase activity turned out to be erroneous, being due to diffusion and adsorption of the reaction products, that is, calcium phosphate, by the nucleus (Pearse, 1968; Vorbrodt, 1974). Quantitative determinations in the isolated liver NE canied out with P-glycerophosphate and p-nitrophenyl phosphate showed negligible activity which could practically be regarded as the absence of both alkaline and acid phosphatase (Franke et al., 1970; Kartenbeck et al., 1973; Buchwalow et al., 1974). These data correlate well with the earlier finding of major alkaline and acid phosphatase activity in the salt-soluble deoxyribonucleoprotein fraction of isolated nuclei, while only traces of acid phosphatase were detected in insoluble fractions which probably contained the envelope proteins (Karuzina, 1953). However, in some cases acid phosphatase was found histochemically in the NE of embryonic blood-forming cells (Vorbrodt, 1967; Shamsuddin et al., 1976), eosinophilic leukocytes (Bainton and Farquhar, 1970), rat liver (Kessel and Decker, 1972), and calf thymocytes (Monneron, 1974b). Recently alkaline phosphatase was detected histochemically in the pore complexes of the human parotid salivary gland. A positive reaction in the NE was found in 30% of mixed parotid tumor cells and only in 1-2% of normal cells (Cutler et al., 1974). It is possible that positive staining in a minority of nuclei was due only to a special phosphatase with a pH optimum in a nearly neutral zone. This P-glycerophosphatase is clearly detected histochemically in rat liver nuclear pore complexes only at pH 6.4; a shift of the pH to 6.2 or 6.7 strongly diminished the staining, and a further change in the pH caused the reaction to become negative (Buchwalow and Unger, 1977; Buchwalow et al., 1977; Unger et al. 1978). A similar type of phosphatase had been described previously in other cell structures (Nelson, 1966; Borgers and Thone, 1976). Thus a very low activity of both kinds of phosphatase was detected in the NE. However, histochemical detection of an enzyme may be an indication of its presence in the perinuclear space, or the presence of a special phosphatase associated with the nuclear pores. b. Glucosed-Phosphatase. Glucose-6-phosphatase (~-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) is a tissue-specific enzyme characteristic of liver
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and kidney; its activity is negligible or absent in the majority of other mammalian organs. In addition to glucose-6-phosphate hydrolysis, mannose-6-phosphatase activity and some phosphotransferase activity are associated with this enzyme (Nordlie and Arion, 1964, 1965; Gunderson and Nordlie, 1973; Kartenbeck et al., 1973). Glucose-6-phosphatase is usually regarded as a marker enzyme for the microsome fraction; it is bound to the endoplasmic reticulum, partially in a latent state, and is activated by detergents (Stetten and Burnett, 1966). Histochemically, glucose-6-phosphatase was reported in the rat liver NE by several workers (Tice and Barnett, 1962; Goldfischer et al., 1964; Ericsson, 1966; Rosen et al., 1966; Saito, 1966; Leskes and Siekevitz, 1969; Rosen, 1969; Kanamura, 1971a,b; Kartenbeck et al., 1973; Buchwalow et al., 1974, 1977; Sikstrom et al., 1976). It was also found in mouse intestinal epithelium (Hugon et al., 1971, 1972), dog coronary blood vessels and heart muscle (Borgers et al., 1971), and chick embryo heart cell cultures (Schafer and Hundgen, 1971). However, the results obtained for isolated NEs are contradictory. At first only negligible activity was found in isolated rat liver NEs (Pokrovsky et al., 1968; Zbarsky et al., 1969; Franke et al., 1970; Agutter, 1972). Other workers found pronounced activity amounting to one-third to two-thirds of that present in the isolated microsomes of the same tissue (Kashnig and Kasper, 1969; Berezney et al., 1970b, 1972; Kay et al., 1972). Similar values were found in the NEs isolated from pea sprouts (Stavy et al., 1973). However, sometimes activity markedly higher than that in isolated microsomes has been reported for glucose-6-phosphatase (Gunderson and Nordlie, 1973; Sikstrom et al., 1975, 1976; Wilson and Chytyl, 1976) and associated enzymes (carbamyl phosphate: glucose phosphotransferase, mannose-6-phosphate:glucosephosphotransferase, and mannose-6-phosphate phosphohydrolase). Contrary to the situation in microsomes, in the NE the total activity could be measured without the presence of a detergent (deoxycholate) or other agent to destroy the membrane structure (Gunderson and Nordlie, 1973). It appears that the first data on the absence of glucose-6-phosphatase from the NE were erroneous, since (1) they were disproved by us (Zbarsky, 1975a; Koen et al., 1976), as well as by Franke (Kartenbeck et al., 1973), and (2) the enzyme has been invariably revealed histochemically. In histochemical procedures the reaction product is usually deposited on the perinuclear cisternal faces of both NMs (Kartenbeck et al., 1973; Buchwalow et al., 1974) and appears not to be tightly bound to the membranes (Gunderson and Nordlie, 1973). It is possible that in the process of a thorough washing it may be removed from isolated membranes. Glucose-6-phosphatase activity in the rat liver NE probably amounts to half that in microsomes, and data on its higher activity require confirmation. Several other phosphatase and phosphotransferase activities appear to accompany the glucose-6-phosphatase activity (Gunderson and Nordlie, 1973). Apart from the
ENZYME PROFILE OF NUCLEAR ENVELOPE
307
above mentioned activities they include also pyrophosphatase, pyrophosphateglucose phosphotransferase, and a variety of nuc1eosidetriphosphate:glucose phosphotransferases. In this connection it is noteworthy that the enzymic hydrolysis of carbamyl phosphate studied histochemically was found to occur at the same sites as the action of glucose-6-phosphatase (Mizutani and Fujita, 1968). Therefore it is possible that in fact the carbamyl phosphate:glucose phosphotransferase activity was measured, as this enzyme accompanies glucose-6phosphatase (Gunderson and Nordlie, 1973). However, where glucose-6-phosphatase activity is very low or absent, as in fowl erythrocytes (Zentgraf et al., 1971), rat thymus (Jarasch et al., 1973), and rat hepatoma 27 (Zbarsky, 1975a; Koen et al., 1976) the NEs are practically free of this activity. c. 5'-Nucleotidase. 5'-Nucleotidase has a rather broad specificity toward different 5'-nucleotides. The enzyme (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) is usually regarded as a marker for the plasma membrane, although it is found in the endoplasmic reticulum as well (Widnell, 1972). Only traces of 5'-nucleotidase were found in isolated rat liver NEs (Agutter, 1972; Jarasch, 1973). Enzyme activity was found to be two to three times lower than in total liver homogenate and nearly equaled that in isolated nuclei (Zbarsky, 1975a; Koen et al., 1976). Contrary to the above observations, considerable enzyme activity in isolated rat NEs and a positive histochemical reaction in NMs were reported by other investigators (Sikstrom et al., 1975, 1976). For tissues other than liver the results are not uniform. In hepatoma 27, as in the liver, NE activity is much lower than in the homogenate (Koen et al., 1976); it is negligible also in nuclei and NEs of chicken erythrocytes (Zentgraf et al., 1971). However, in the NEs of lymphoid cells, especially thymocytes, distinct 5'-nucleotidase activity was detected histochemically (Monneron, 1974a,b; Buchwalow et al., 1977). In thymocytes, in addition to 5'-nucleotidase, 3'nucleotidase activity was found, the rate of 3'-TMP and 5'-TMP hydrolysis being higher than that of the corresponding adenine mononucleotides (Monneron, 1974a,b). Rather high activity in pea sprout NEs was reported (Stavy et al., 1973). d. Nucleosidediphosphatses. Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) hydrolyzes various ribonucleoside diphosphates and is usually regarded as a marker enzyme for the endoplasmic reticulum where its activity is relatively high. Inosinediphosphate is often used as a substrate, and the total IDPase activity is revealed in the presence of a detergent (0.1% Triton X-loo), for the bulk of it is tightly bound to membranes. Nucleosidediphosphatase (sometimes accompanied by thiamine pyrophosphatase) activity was detected histochemically in the NE of various cells (Novikoff et al., 1962; Novikoff and Heus, 1963; Kessel and Decker, 1971) including liver (Rubin, 1969; Goldfischer et al., 1971; Pelletier and Novikoff,
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I. B. ZBARSKY
1972). Recently IDPase was demonstrated also in isolated rat liver and hepatoma NEs (Zbarsky, 1975a; Koen et a f . , 1976). The activity of the enzyme in the NE was five times (liver) and three times (hepatoma), respectively, higher than in isolated nuclei, but in both tissues several times lower than in their mierosomes or homogenates. Thus IDPase (nucleosidediphosphatase) is probably a proper NM component and cannot be attributed only to microsomal contamination. e. Nucleosidetriphosphatases. It appears that the enzymes hydrolyzing nucleosidetriphosphates and reported as nucleosidetriphosphatases represent a multicomponent group of enzymes. ATP is usually employed as a substrate, but the enzyme activity measured in this way may be actually due to phosphate transfer and utilization of ATP energy. ATPase activity is usually measured in the presence of M g + at neutral pH and is described as Mg+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.4). Rather high ATPase activity is found in the NE by the use of quantitative as well as histochemical methods. M g +-ATPase revealed by light microscope histochemistry was reported to be localized at the rat liver cell nuclear periphery (Baiikowski, 1963). Similar results were reported for NEs from electron microscopy of the liver (Vorbrodt, 1967; Buchwalow et al., 1971, 1977), chick embryo myocardium cells (Klein and Afzelius, 1966), mouse chorioid plexus (Yasuzumi and Tsubo, 1966), human Leydig cells (Yasuzumi et a f . , 1967), pond snail spermatids (Yasuzumi et al., 1969), newt oocytes (Scheer and Franke, 1969), and mouse hepatomas (Buchwalow et al., 1972). High ATPase activity exceeding that of other cell structures was found in isolated NMs, especially in the lighter fraction, presumably originating from the outer NM of rat liver and mouse Ehrlich ascites cells; in rat Zajdela hepatoma it was markedly lower (Delektorskaya and Perevoshchikova, 1969; Zbarsky et a f . , 1969). A considerable amount of ATPase activity was found in isolated preparations of the NE by several workers, but the activities were lower than those in the microsomes. This discrepancy appears to depend on the method of NE isolation, especially when treatment with enzymic preparations and/or salt solutions is employed (Kashnig and Kasper, 1969; Franke et ul., 1970; Berezney et a f . , 1972; Kartenbecketaf., 1973; Pierietal., 1973; Koenetal., 1976; Sikstrom et al., 1976). Indeed, it was shown that the ATPase activity in the NE, but not in other cell structures, decreased upon RNase treatment or 1.5 M KCl extraction and was stimulated by the addition of RNA (Agutter et al., 1977). In solid liver tumors, particularly in poorly differentiated outgrowths, ATPase activity in the NE is usually lower, while in ascites tumors it may be higher than in the liver (Delektorskaya and Perevoschchikova, 1969; Buchwalow et a f . , 1972; Koen er al., 1976). The ATPase activity in isolated NEs of rat brain (Rapava et al., 1973), thymus (Reilly, 1971; Jarasch et al., 1973), and chicken erythrocytes (Zentgrafetaf., 1971; Blanchet, 1974) was also reported. The activity appears to be enhanced by the stimulation of erythropoiesis in rabbit bone marrow cells
ENZYME PROFILE OF NUCLEAR ENVELOPE
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(Jarasch, 1973; Zbarsky et al., 1975a). Mainly Mgl+-dependent ATPase with a neutral pH optimum has been reported in NE. C$+-activated ATPase with a slightly alkaline pH optimum (ATPase B) was reported to be present in the nucleus (Bafikowski, 1963) or to be partially associated with the inner NM (Buchwalow et al., 1971). The absence of Naf , K+-dependentATPase from the NE is unanimously reported (Delectorskaya and Perevoshchikova, 1969; Franke et al., 1970; Reilly, 1971; Jarasch, 1973; Kartenbeck et al., 1973; Blanchet, 1974). However, NM ATPase can be activated by 2,4-dinitrophenol and inhibited by gramicidin S (Delektorskaya and Perevoshchikova, 1969). Association of ATPase with the nuclear pores was reported (Yasuzumi and Tsubo, 1966; Yasuzumi et al., 1967; Chardonnet and Dales, 1972), but these results were obtained mainly by a modified histochemical method (lead nitrate was added at the end of the reaction) and therefore require confirmation. A presumed difference in ATPase activity in the inner and outer NMs (Delektorskaya and Perevoshchikova, 1969; Buchwalow et al., 1971) may also be of importance but cannot be regarded as definitely established. 2. Esterases The biochemical data on esterases of the NE are confined to carboxylesterase, acetylesterase, and arylesterase, while histochemical studies include only acetylcholinesterase and arylsulfatase. a. Carboxylesterase. Carboxylesterase (carbonic-ester hydrolase, EC 3.1.1.1) activity has been determined in isolated NMs with two substrates, tributyrin and methyl butyrate, which gave similar results. The lighter rat liver NM fraction was free of activity, while the activity of the heavier fraction exceeded that of the nuclear fraction by 30-60% and was four times less than that of the mitochondria-lysosome fraction (Pokrovsky et al., 1970). b. Acetylesterase. The relative acetylesterase (acetic-ester hydrolase, EC 3.1.1.6) activity withp-nitrophenylacetate as a substrate was very similar to that of carboxylesterase. The specific enzyme activity of the lighter fraction of rat liver NE was negligible, while the heavier fraction contained twice the activity of the isolated nuclei and five to six times less than the mitochondria-lysosome fraction (Pokrovsky et al., 1970). Thus both the carboxylesterase and the acetylesterase appear to be present only in the heavier fraction (presumably the inner NM), but their low activity suggests that their presence may be the result of contamination. c. Arylsulfatase. Arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was histochemically demonstrated in the NEs of cucumber root meristem cells (Poux, 1965, 1966) and eosinophilic leukocytes (Bainton and Farquhar, 1970). Arylsulfatases A and B, measured with 2-hydroxy-5-nitrophenyl sulfate as substrate, were found to be rather high in the heavier fraction of rat liver NEs where their activity exceeded by a factor of 4 to 5 that of isolated nuclei or microsomes.
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In the lighter NM fraction this activity was practically the same as in isolated nuclei or microsomes (Pokrovsky et a f . , 1968). A high activity in the NE, consistent with histochemical data, indicates that arylsulfatases A and B are integral components of this subcellular structure. d. Acetylcholinesterase. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), studied only histochemically, was found in various nervous cells of the rat (Novikoff et a f . , 1966; Ednko et a f . , 1967), frog (Brzin et a f . , 1966; Majcen and Brzin, 1971), chick embryo (Pannese et a f . , 1971), rabbit (Reale et al., 1971), and starling (Welsch and Haase, 1971), and in chick muscle cell cultures (Golder et a f . , 1976). As the enzyme was not measured quantitatively and data for other tissues are absent, it is premature to draw any definite conclusions concerning its NE activity. 3. Proteinases Although isolated nuclei showed distinct proteolytic activity, no measurable activity was found either in the lighter or the heavier rat liver NE fraction (Pokrovsky et al., 1968). Nevertheless, the study of proteolytic activity in the NE, especially for latent proteinases found in cell nuclei (Carter and Chae, 1976), should continue. 4. Nucleases Nucleases constitute a broad group of enzymes with different mechanisms of action (Shapot, 1968). The study of these enzymes in the NE may be important in relation to the NE’s role in processing pre-rRNA and pre-mRNA, nucleocytoplasmic RNA transport, and initiation of DNA replication (see Sections IV,A and B). Histochemically, RNases and DNases in cell nuclei were detected mostly in the condensed chromatin frequently associated with the NE (Zotikov and Bernhard, 1970; Taperer a f . , 1971a,b; Fortet a f . , 1974). Variable substrate composition, preliminary tissue fixation, and phosphatase treatment may produce artifacts, and the evaluation of the results calls for caution (Vorbrodt, 1974). Considerable RNase activity (two to three times as high as that-in the nuclei) was found in isolated rat liver NEs (Skridonenko and Gorchakova, 1973; Skridonenko et a f . , 1975). It appears that the NE contains a 3’-endoribonuclease related to the pancreatic enzyme (EC 2.7.7.16), and 5’-endoribonucleasepossibly identical to the enzyme reported in guinea pig hepatocyte nuclei (Heppel et a f . , 1956). In the NEs of rat hepatoma 27 the RNase activity is still higher in comparison to that in the nuclei and is strongly stimulated by pchlormercuribenzoate (Koen et al., 1976). The activity of RNase in the NE seems to increase in correspondence with its purity but remains bound to an inhibitor; the latter is removed in the course of polyacrylamide gel elec-
ENZYME PROFILE OF NUCLEAR ENVELOPE
311
trophoresis of the enzyme (Shlyakhovenko, 1973; Skridonenko et al., 1975). No DNase activity has been found in the NE.
5. Other Nonoxidative Enzymes Only occasional data are available on the presence or absence of other nonoxidative enzymes in the NE. P-Glucuronidase (P-D-glucoronide glucuronosohydrolase, EC 3.2.1.31) (Smith and Fishman, 1969), acetyl-CoA carboxylase (acetyl-CoA:carbon-dioxide ligase, EC 6.4.1.2) (Yates et al., 1969), and carbamoyl phosphate hydrolysis (Mitzutani and Fujita, 1968) were detected histochemically in rat and mouse NEs. Glutamic-oxaloacetic transaminotransferase, EC 2.6.1.1) was found in aminase (~-aspartate:2-oxoglutarate NEs of rat liver (Lee and Torrack, 1968) and heart muscle (Lee, 1969). Lipase was detected in NMs of rat acinar pancreatic cells (Murata et al., 1968). Glucose-1-phosphate hydrolysis (D-glucose-1-phosphate phosphohydrolase, EC 3.1.3.10) in rat liver NEs was as intense as in isolated nuclei but three times less active than in microsomes (Kartenbeck et al., 1973). Kasper (1971) claims that N-demethylase (involved in the demethylation of 3-methyl-4-aminobenzene) is 10 times less active in isolated rat liver NEs than in microsomes and cannot be induced by phenobarbital as it can in microsomes. NAD+ pyrophosphorylase (ATP:NMN adenylyltransferase, EC 2.7.7.1) and nucleic acid polymerases (nuc1eosidetriphosphate:RNAnucleotidyltransferase, EC 2.7.7.6; deoxynuc1eosidetriphosphate:DNAdeoxynucleotidyltransferase,EC 2.7.7.7; and others) may be of interest because they exert their action in the cell nucleus, and the first two are thought to be typical nuclear enzymes (Siebert and Humphrey, 1965). NAD pyrophosphorylase was shown to be absent from rat liver NEs (Jarasch, 1973; Kay and Johnston, 1973; Sikstrom et al., 1976), as well as from those of chick erythrocytes (Zentgraf et al., 1971). However, its activity is markedly higher in isolated chromatin and in nucleoli than in nuclei (Siebert et al., 1966; Unger et al., 1975). Similar results were obtained histochemically (Buchwalow and Unger, 1974; Unger et al., 1975). Activity of another enzyme, NAD+ glycohydrolase (NADase, EC 3.2.2.5), in isolated rat liver NE was reported to be 4.5-fold greater than that in nuclei and about one-half of that in microsomes (Table I). NADase in the NE was found to be identical to that in the microsome but was regarded as an integral component of the NE (Fukushima et al., 1976). RNA polymerase also appears to be absent from NEs and to be associated with chromatin (Kay and Johnston, 1973; Sikstrom et al., 1975, 1976). No poly-A polymerase was found by Sikstrom et al. (1976), but Kay and Johnston (1973) claimed that this enzyme was present in the NE in concentrations twice as high as in isolated nonfractionated nuclei. Recently, M~+-dependentbut not Mg2+dependent poly-A polymerase was reported to be localized mainly on the membrane-associated chromatin (Louis et al., 1978).
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I. B. ZBARSKY
Various workers found DNA polymerase activity in isolated NEs (Yoshida et al., 1971; Yoshikawa-Fukada and Ebert, 1971; Deumling and Franke, 1972; Tsuruo et al., 1972). However, the data on the subject are conflicting: There are several types of DNA polymerases, and the role of the NE in eukaryotic DNA replication remains unclear and highly complicated (see Section IV ,A). The important role of cyclic nucleotides and the localization of the enzymes involved in their synthesis and hydrolysis in the membrane suggest the probable presence of adenylate cyclase and CAMP phosphodiesterase in the NE. The presence of adenylate cyclase was reported in isolated nuclei of the liver and other tissues (Liaoer al., 1971; Soifer and Hechter, 1971). However, the enzyme was revealed in the NE only histochemically with the light microscope (Coulson and Kennedy, 1971, 1972), and the lack of sufficient controls makes the results uncertain (Vorbrodt, 1974). Electron cytochemical studies of localization of creatine kinase (EC 2.7.3.2)in heart cells revealed the enzyme in different sites, including the NE (Sharov et al., 1977). B. OXIDOREDUCTASES The problem of oxidative processes in cell nuclei has been discussed for a long time (Conover, 1967). Oxidation and oxidative phosphorylation were formerly ascribed only to the cell nuclei of lymphoid tissues (Betel and Klouwen, 1966). Later oxidative enzymes were found in the cell nuclei of various tissues comprising the liver and were shown to be localized in the NMs (Kuzmina et al., 1969; Zbarsky et al., 1969; Betel, 1972; Zbarsky, 1972b,c). The problem remains unsolved and is considered in more detail in Section 111. Here, quantitative data on oxidoreductase activity in isolated NEs (Table 11) and related histochemical observations are presented. 1 . Succinate Dehydrogenase Succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.11 or, correspondingly, succinate oxidase, succinate-cytochrome c reductase, succinate reductase activity in the presence of another electron acceptor [iodonitrotetrazolium violet(INT), phenazine methosulfate (PMS), dichlorophenolindophenol, ferricyanide] as a rule was not found in isolated nuclei or NEs. Even in crude rat liver nuclei only traces of succinate oxidase could be demonstrated (Rees and Rowland, 1961; Rees et al., 1963). Some claims for the presence of succinate dehydrogenase in thymus (Dancheva, 1965) and liver (Manta et al., 1966) nuclei have not been substantiated. The succinate oxidase or succinate dehydrogenase activity in isolated NMs is negligible and is regarded by many investigators as an indicator of mitochondrial contamination (Zbarsky et al., 1968; Kuzmina et al., 1969; Berezney et al., 1970a,b, 1972; Berezney and Crane, 1971; Zentgrafet al., 1971; Agutter, 1972:
TABLE 11 OXIDATIVE ENZYME ACTIVITIES OF THE NE IN COMPARISON TO THOSEOF OTHER CELL STRUCTURESa Enzyme Cytochrome c oxidase
Tetraclorohydroquinoneoxidase Succinate oxidase
Succinate-cytochrome c reductase
Tissue
NE
NE/nuclei
Wmicrosomes
Reference
Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Bovine liver Bovine liver Bovine liver Rat thymus Rat thymus Calf thymus Chick erythrocytes Rabbit bone marrow cells Onion roots Bovine liver Rat liver Rat thymus Rat liver Bovine liver Bovine liver Rat thymus Rat liver Rat liver
38 (h)b 31 ( I ) b 532 240 372 0.027d
An Enzyme Profile of the Nuclear Envelope I. B. ZBARSKY Biochemistry Laboratory, N . K . Koltzov Institute of Developmental Biology, Academy of Sciences of the USSR. Moscow, USSR
I. Introduction . . . . . . . . . . . . . . . . . . . . A. Historical Aspects . . . . . . . . . . . . . . . . B. Progress in Studies on the NE . . . . . . . . . , . . C. Probable Functions of the NE in Relation to Its Enzyme Profile 11. Enzyme Profile of NEs . . . . . . . . . . . . , . . A. Hydrolytic Enzymes and Transferases . . . . , . . B. Oxidoreductases . . . . . . . . . . . . . . . . . C. Enzyme Profile of the NE in Comparison with That of Other Cell StNCtUES . . . . . . . . . . . . . . . . . . . 111. The Problem of Oxidation and Oxidative Phosphorylation in the NE A. Nuclear Oxidation and Oxidative Phosphorylation . . . . . B. Electron Transport and Oxidative Phosphorylation in the NE . C. Are Nuclear Oxidation and Phosphorylation due to Mitochondria1 Contamination? . . . . . . . . . . . . . . . . . IV. The Role of NEs . . . . . . . . . . . . . . . . . . A. Relationship of the NE to Protein and Nucleic Acid Synthesis B. Nucleocytoplasmic Transport and Enzymes of the NE . . . C. Peculiarities of the NE in Relation to Physiological and Pathological Conditions . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . A. Characteristics of the NE Enzyme Profile . . . . . . . . B. Biogenesis of the NE and Related Structures . . . . . . . C. Problems of Enzyme Studies of the NE in Relation to Its Functions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
. .
.
.
295 296 296 298 299 299 312 325 329 329 329 331 335 335 338 344 347 347 347 348 349
I. Introduction The nuclear membrane (NM) is a characteristic structure separating the nucleus from the cytoplasm in all eukaryotic cells. In different tissues and organisms the structure of the nuclear envelope (NE) is surprisingly uniform. Its common features are outer and inner NMs, a perinuclear space between these membranes, and rather large (60-70 nm in diameter) nuclear pores usually surrounded by an annulus having octagonal symmetry. NMs interact morphologically and functionally with nuclear and cytoplasmic structures and 295 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364354-6
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therefore can be regarded as part of both the nucleus and the cell membrane system. In cells with intensive metabolism the NE has numerous invaginations and protrusions which increase the nucleocytoplasmic interface and enhance the interaction between nucleus and cytoplasm. In such cells (silk gland cells, neurons, certain glandular and embryonal cells) rupture of the NE and its membranes, especially the outbursts and blebs of the outer NM, is usual, and the formation of vesicles and possibly of membranes of endoplasmic reticulum from the outer NM is often observed, as well as of the annulate lamella-a counterpart of the NE structure (Kessel, 1968; Scharrer and Wurzelmann, 1969). A. HISTORICAL ASPECTS As far back as the end of the last century (Schwartz, 1892) the existence of a membrane surrounding the nucleus was suspected. Later this envelope was thought to be a double-membrane structure (Robyns, 1924; Scarth, 1927; Cohen, 1937; Dangeard, 1942; Policard and Bessis, 1956). The existence of the NE was proved by means of electron microscopy which enabled investigators to see for the first time the NMs and their special substructures-the nuclear pores (Callan and Tomlin, 1950; Watson, 1955, 1959). In the fifties and sixties, the morphology of NMs and nuclear pores was studied and reviewed by several workers (Haguenau and Bernhard, 1955; Policard and Baud, 1958; Baud, 1959; David, 1964; Gall, 1964; Feldherr and Harding, 1964; Goldstein, 1964; Loewenstein, 1964; Zagorski, 1965). B. PROGRESS IN STUDIES ON
THE
NE
While the first data on the chemical components and enzymes of the NE were obtained by electron microscope histochemistry, the isolation of NMs (Franke, 1966; Zbarsky et al., 1967) was the principal achievement in the understanding of their biochemical and enzymic properties (Pokrovsky et al., 1968; Zbarsky et al., 1968, 1969; Kashnig and Kasper, 1969; Frankeet al., 1970). With the use of isolated preparations, the morphological structure, chemical composition, and enzyme profile of the NE could be studied in more detail. Nevertheless, the results obtained were often contradictory, and many aspects of the problem remain unsolved. The present state of our knowledge of the structure, function, biochemical properties, methods of isolation, and other aspects of NE research is represented in numerous reviews published during the last 10 years (Gouranton, 1969; Stevens and Andrk, 1969; Zbarsky, 1969, 1972a,b,c, 1973, 1975b; Franke, 1970a, 1974a,b; Feldherr, 1972; Kay and Johnston, 1973; Kessel, 1973; Wishnitzer, 1973; Berezney, 1974; Franke and Scheer, 1974a; Kasper, 1974; Fry, 1976,
ENZYME PROFILE OF NUCLEAR ENVELOPE
297
1977; Harris and Agutter, 1976; Wunderlich et a l . , 1976). Therefore only some basic data on the morphology of the NE (Fig. 1) are mentioned in this article. Apart from the outer and inner NMs and pore complexes the NE also contains a “fibrous lamina” (Fawcett, 1966), “internal dense lamella” (Stevens and Andre, 1969), or “zonula nucleum limitans” (Patrizi and Poger, 1967) closely associated with the inner NM. This lamella is pronounced in amebas, where it was previously described as the “honeycomb layer” (Pappas, 1956). The inner membrane or the fibrous lamina is associated with the peripheral chromatin (Davies and Small, 1968). This association may involve special peripheral granules about 25 nm in diameter (Barton et a l . , 1971; Onishchenko and Chentsov, 1973, 1974a,b). The outer NM is directly connected with the endoplasmic reticulum and is frequently associated with mitochondria as well. Many cell types contain in the cytoplasm and sometimes in the nucleus annulate lamellae structurally similar to the NE and probably originating from it. The chemical composition of the NE is typical of membrane structures with relatively high protein and low cholesterol content; the presence of RNA and DNA has also been established. Certain peculiarities of NE composition may be due to the fact that it comprises not only the inner and outer NMs but also the pore complexes and fibrous lamina consisting mostly of proteins. During the cell cycle the latter structures degenerate and are reconstructed anew, apparently independently of the NMs (Zatsepina ef al., 1976a,b; 1977). Thus the nuclear pores appear to have some autonomy and can be separated from the NMs. As already mentioned, the pores are usually surrounded by octagonal annuli. The octagonal symmetry is due to usually eight peripheral granules interconnected with filaments. Inside the pore a central granule bound to other parts of the pore complex with filaments is often seen. Apart from the central granule amorphous material may fill the pore orifice. Although the details of the pore 3
J
6
\7 \2
FIG. 1. General scheme of a NE fragment (cytoplasm side up). 1, Outer nuclear membrane; 2 , inner nuclear membrane; 3, endoplasmic reticulum; 4, ribosomes; 5, fibrous lamina; 6, peripheral granules associated with chromosome telomeres; 7, peripheral chromatin adhering to the NE; 8, pore complexes.
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structure are still unclear, a tentative scheme of a nuclear pore is presented in Fig. 2.
c.
PROBABLE FUNCTIONS OF THE NE
IN
RELATION TO ITS ENZYME PROFILE
The functions of the NE are not well known, but its location at the interface between the nucleus and the cytoplasm implies a role in maintenance of the shape and composition of the nucleus. Electron microscope observations and the presence of the pores indicate that the NE may participate in the nucleocytoplasmic transport of various substances, especially RNAs and ribonucleoproteins. A wide spectrum of enzyme activity in the NE and its specialized structure suggests its participation in the control of nuclear metabolism. The presence of oxidative enzymes and oxidative phosphorylation may enable the NE to supply the energy for nucleocytoplasmic transport and intranuclear biosynthetic reactions. The main body of our knowledge of the enzymes of the NE was obtained from the biochemical study of isolated preparations. Histochemical studies, mainly by means of electron microscopy, have provided additional important data. Indirect information comes from the enzymic analysis of isolated nuclei, autoradiography, and studies on biogenesis, turnover, and reconstruction of NMs and nuclear pores, as well as on nucleocytoplasmic transport. Most reliable quantitative results were obtained by enzymic analysis of isolated structures. However, certain enzymes and other components may be extracted and lost during the isolation of nuclei, and especially of NEs present in these structures in vivo. The adsorption of nuclear and cytoplasmic enzymes by the NE is also possible. Treatment with salt or enzyme preparations, as well as all kinds of contamination, may inactivate or alter the properties of its enzymes. It is 100-120 nm
4
-4
\
60-70 nmD
--. P 25nm
3
FIG.2. Schematic drawing of a pore complex (cytoplasm side up). 1, Peripheral dense layer connected to the fibrous lamina; 2, fibrous lamina; 3, peripheral granules; 4, central granule; 5, amorphous material filling up the pore interior; 6, filaments projecting inside the nucleus.
ENZYME PROFILE OF NUCLEAR ENVELOPE
299
obvious that the purity of isolated NE preparations is a prerequisite for an adequate analysis of their enzyme profile. The localization of enzyme activity is usually better determined by histochemical methods, though they are as a rule less specific and less reliable than biochemical analyses and can give only semiquantitative data. However, histochemical methods may reveal soluble enzymes which are extracted from the NEs during isolation. Thus it is not surprising that available data on the enzymes of NEs are contradictory and highly dependent on the methods used for the isolation of nuclei and NEs, as well as on the procedures for determining enzyme activity. Therefore a thorough comparison of results obtained by different methods for each enzyme is necessary. Despite the uniformity of the NE structure in different cells, its enzymic activity may depend on the function, physiological state, and metabolic activity of a given cell or tissue, as well as on pathological or damaging conditions. Finally, different substructures of the NE may differ considerably from each other in enzyme profile and function. Thus we have a general idea of the enzyme profile of the NE, but many aspects remain unresolved and are a matter of discussion. We are almost unaware of peculiarities of NE enzymes in different tissues and organisms and their dependence on physiologically active and damaging agents. We are unable also to locate certain enzymes on definite substructures of the NE such as the inner and outer NMs and the nuclear pores.
11. Enzyme Profile of NEs
A. HYDROLYTIC ENZYMESA N D TRANSFERASES Hydrolytic enzyme activities in the NE were primarily detected by histochemical methods. Afterward, quantitative data on the isolated preparations became available. Table I lists quantitative data on hydrolytic and other nonoxidative enzymes in the NE in comparison with those in the corresponding tissue and other cell components. Apart from the above-mentioned reasons for the discrepancies between the results obtained by different investigators, they do not always correspond to histochemical data. These discrepancies may be due to localization of some enzymes in the perinuclear space, to their lipophilic nature, or to their weak association with the membrane structure and loss of easily extractable enzymes in the course of isolation.
TABLE I HYDROLYTIC A N D OTHER NONOXIDATIVE ENZYMEACTIVITIES OF THE NE Enzyme Alkaline phosphatase
Acid phosphataqe
W g
Glucose-6-phosphatase
Tissue Rat live@ Rat liverb Rat liver Pig liverb Rat liverb Rat liverb Rat liver Pig liverb Rooster liver Onion stems Onion roots Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Pig liver Bovine liver Bovine liver Rooster liver Rat thymus CHO
IN
COMPARISON TO THOSE OF OTHER CELL STRUCTURES"
NE
NElnuclei
NE/microsomes
Reference
28 2.5 1.6 35 53 13 4.1 167 13.2 0.5 lo00 11.5 22k 4.1
NWmicrosomes 0.18O 0.03O 5.8 1.0
-
0.11
-
4.5
-
Reference Pokrovsky ef al. (1970) Pokrovsky ef al. (1970) Pokrovsky ef al. (1%8) Pokrovsky et d.(1968) Pokrovsky ef al. (1%8) Kasper ( 1971) Jarasch (1973) Zentgraf ef al. (1971) Fukushima et al. (1976) Skridonenko and Gorchakova (1973) Skridonenko and Gorchakova (1973) Skridonenko ef ul. (1975) Skridonenko et al. (1975) Skridonenko et ul. (1975) Skridonenko er al. (1975) Koen ef al. (1976) Koen ef al. (1976)
"Nanomoles of metabolized substrate or product per minute per milligram protein. Other measurements reported in the literature are recalculated *p-Nitrophenyl as substrate. 'Crude nuclear fraction. dHeavy (h) and light (1) NE subfractions. 'NEs were in addition purified in sucrose gradient. I n relation to the homogenate. @Inrelation to the mitochondria. hMethylbutyrin as substrate. 'Tributyrin as substrate. 'The outer nuclear membrane was isolated by extraction with 0.5% Triton X-100. M p-chloromercuribenzoate. "In the presence of 3 X
ENZYME PROFILE OF NUCLEAR ENVELOPE
305
1. Phosphohydrolases a. Glycerophosphatases. This group comprises phosphomonoesterases characterized by a broad specificity, which are usually called acid and alkaline phosphatases and have pH optima of 4.5-5.5 and 8.5-9.5, respectively. According to Enzyme Nomenclature they are called orthophosphoric-monoester phosphohydrolases (EC 3.1.3.1, alkaline phosphatase, and EC 3.1.3.2, acid phosphatase). Since the individual characters of these enzymes are still not established and their affinities for different substrates vary, it is convenient to call them glycerophosphatases, since P-glycerophosphate is usually employed as a substrate. Earlier histochemical data on high nuclear phosphatase activity turned out to be erroneous, being due to diffusion and adsorption of the reaction products, that is, calcium phosphate, by the nucleus (Pearse, 1968; Vorbrodt, 1974). Quantitative determinations in the isolated liver NE canied out with P-glycerophosphate and p-nitrophenyl phosphate showed negligible activity which could practically be regarded as the absence of both alkaline and acid phosphatase (Franke et al., 1970; Kartenbeck et al., 1973; Buchwalow et al., 1974). These data correlate well with the earlier finding of major alkaline and acid phosphatase activity in the salt-soluble deoxyribonucleoprotein fraction of isolated nuclei, while only traces of acid phosphatase were detected in insoluble fractions which probably contained the envelope proteins (Karuzina, 1953). However, in some cases acid phosphatase was found histochemically in the NE of embryonic blood-forming cells (Vorbrodt, 1967; Shamsuddin et al., 1976), eosinophilic leukocytes (Bainton and Farquhar, 1970), rat liver (Kessel and Decker, 1972), and calf thymocytes (Monneron, 1974b). Recently alkaline phosphatase was detected histochemically in the pore complexes of the human parotid salivary gland. A positive reaction in the NE was found in 30% of mixed parotid tumor cells and only in 1-2% of normal cells (Cutler et al., 1974). It is possible that positive staining in a minority of nuclei was due only to a special phosphatase with a pH optimum in a nearly neutral zone. This P-glycerophosphatase is clearly detected histochemically in rat liver nuclear pore complexes only at pH 6.4; a shift of the pH to 6.2 or 6.7 strongly diminished the staining, and a further change in the pH caused the reaction to become negative (Buchwalow and Unger, 1977; Buchwalow et al., 1977; Unger et al. 1978). A similar type of phosphatase had been described previously in other cell structures (Nelson, 1966; Borgers and Thone, 1976). Thus a very low activity of both kinds of phosphatase was detected in the NE. However, histochemical detection of an enzyme may be an indication of its presence in the perinuclear space, or the presence of a special phosphatase associated with the nuclear pores. b. Glucosed-Phosphatase. Glucose-6-phosphatase (~-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) is a tissue-specific enzyme characteristic of liver
306
I. B. ZBARSKY
and kidney; its activity is negligible or absent in the majority of other mammalian organs. In addition to glucose-6-phosphate hydrolysis, mannose-6-phosphatase activity and some phosphotransferase activity are associated with this enzyme (Nordlie and Arion, 1964, 1965; Gunderson and Nordlie, 1973; Kartenbeck et al., 1973). Glucose-6-phosphatase is usually regarded as a marker enzyme for the microsome fraction; it is bound to the endoplasmic reticulum, partially in a latent state, and is activated by detergents (Stetten and Burnett, 1966). Histochemically, glucose-6-phosphatase was reported in the rat liver NE by several workers (Tice and Barnett, 1962; Goldfischer et al., 1964; Ericsson, 1966; Rosen et al., 1966; Saito, 1966; Leskes and Siekevitz, 1969; Rosen, 1969; Kanamura, 1971a,b; Kartenbeck et al., 1973; Buchwalow et al., 1974, 1977; Sikstrom et al., 1976). It was also found in mouse intestinal epithelium (Hugon et al., 1971, 1972), dog coronary blood vessels and heart muscle (Borgers et al., 1971), and chick embryo heart cell cultures (Schafer and Hundgen, 1971). However, the results obtained for isolated NEs are contradictory. At first only negligible activity was found in isolated rat liver NEs (Pokrovsky et al., 1968; Zbarsky et al., 1969; Franke et al., 1970; Agutter, 1972). Other workers found pronounced activity amounting to one-third to two-thirds of that present in the isolated microsomes of the same tissue (Kashnig and Kasper, 1969; Berezney et al., 1970b, 1972; Kay et al., 1972). Similar values were found in the NEs isolated from pea sprouts (Stavy et al., 1973). However, sometimes activity markedly higher than that in isolated microsomes has been reported for glucose-6-phosphatase (Gunderson and Nordlie, 1973; Sikstrom et al., 1975, 1976; Wilson and Chytyl, 1976) and associated enzymes (carbamyl phosphate: glucose phosphotransferase, mannose-6-phosphate:glucosephosphotransferase, and mannose-6-phosphate phosphohydrolase). Contrary to the situation in microsomes, in the NE the total activity could be measured without the presence of a detergent (deoxycholate) or other agent to destroy the membrane structure (Gunderson and Nordlie, 1973). It appears that the first data on the absence of glucose-6-phosphatase from the NE were erroneous, since (1) they were disproved by us (Zbarsky, 1975a; Koen et al., 1976), as well as by Franke (Kartenbeck et al., 1973), and (2) the enzyme has been invariably revealed histochemically. In histochemical procedures the reaction product is usually deposited on the perinuclear cisternal faces of both NMs (Kartenbeck et al., 1973; Buchwalow et al., 1974) and appears not to be tightly bound to the membranes (Gunderson and Nordlie, 1973). It is possible that in the process of a thorough washing it may be removed from isolated membranes. Glucose-6-phosphatase activity in the rat liver NE probably amounts to half that in microsomes, and data on its higher activity require confirmation. Several other phosphatase and phosphotransferase activities appear to accompany the glucose-6-phosphatase activity (Gunderson and Nordlie, 1973). Apart from the
ENZYME PROFILE OF NUCLEAR ENVELOPE
307
above mentioned activities they include also pyrophosphatase, pyrophosphateglucose phosphotransferase, and a variety of nuc1eosidetriphosphate:glucose phosphotransferases. In this connection it is noteworthy that the enzymic hydrolysis of carbamyl phosphate studied histochemically was found to occur at the same sites as the action of glucose-6-phosphatase (Mizutani and Fujita, 1968). Therefore it is possible that in fact the carbamyl phosphate:glucose phosphotransferase activity was measured, as this enzyme accompanies glucose-6phosphatase (Gunderson and Nordlie, 1973). However, where glucose-6-phosphatase activity is very low or absent, as in fowl erythrocytes (Zentgraf et al., 1971), rat thymus (Jarasch et al., 1973), and rat hepatoma 27 (Zbarsky, 1975a; Koen et al., 1976) the NEs are practically free of this activity. c. 5'-Nucleotidase. 5'-Nucleotidase has a rather broad specificity toward different 5'-nucleotides. The enzyme (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) is usually regarded as a marker for the plasma membrane, although it is found in the endoplasmic reticulum as well (Widnell, 1972). Only traces of 5'-nucleotidase were found in isolated rat liver NEs (Agutter, 1972; Jarasch, 1973). Enzyme activity was found to be two to three times lower than in total liver homogenate and nearly equaled that in isolated nuclei (Zbarsky, 1975a; Koen et al., 1976). Contrary to the above observations, considerable enzyme activity in isolated rat NEs and a positive histochemical reaction in NMs were reported by other investigators (Sikstrom et al., 1975, 1976). For tissues other than liver the results are not uniform. In hepatoma 27, as in the liver, NE activity is much lower than in the homogenate (Koen et al., 1976); it is negligible also in nuclei and NEs of chicken erythrocytes (Zentgraf et al., 1971). However, in the NEs of lymphoid cells, especially thymocytes, distinct 5'-nucleotidase activity was detected histochemically (Monneron, 1974a,b; Buchwalow et al., 1977). In thymocytes, in addition to 5'-nucleotidase, 3'nucleotidase activity was found, the rate of 3'-TMP and 5'-TMP hydrolysis being higher than that of the corresponding adenine mononucleotides (Monneron, 1974a,b). Rather high activity in pea sprout NEs was reported (Stavy et al., 1973). d. Nucleosidediphosphatses. Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) hydrolyzes various ribonucleoside diphosphates and is usually regarded as a marker enzyme for the endoplasmic reticulum where its activity is relatively high. Inosinediphosphate is often used as a substrate, and the total IDPase activity is revealed in the presence of a detergent (0.1% Triton X-loo), for the bulk of it is tightly bound to membranes. Nucleosidediphosphatase (sometimes accompanied by thiamine pyrophosphatase) activity was detected histochemically in the NE of various cells (Novikoff et al., 1962; Novikoff and Heus, 1963; Kessel and Decker, 1971) including liver (Rubin, 1969; Goldfischer et al., 1971; Pelletier and Novikoff,
308
I. B. ZBARSKY
1972). Recently IDPase was demonstrated also in isolated rat liver and hepatoma NEs (Zbarsky, 1975a; Koen et a f . , 1976). The activity of the enzyme in the NE was five times (liver) and three times (hepatoma), respectively, higher than in isolated nuclei, but in both tissues several times lower than in their mierosomes or homogenates. Thus IDPase (nucleosidediphosphatase) is probably a proper NM component and cannot be attributed only to microsomal contamination. e. Nucleosidetriphosphatases. It appears that the enzymes hydrolyzing nucleosidetriphosphates and reported as nucleosidetriphosphatases represent a multicomponent group of enzymes. ATP is usually employed as a substrate, but the enzyme activity measured in this way may be actually due to phosphate transfer and utilization of ATP energy. ATPase activity is usually measured in the presence of M g + at neutral pH and is described as Mg+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.4). Rather high ATPase activity is found in the NE by the use of quantitative as well as histochemical methods. M g +-ATPase revealed by light microscope histochemistry was reported to be localized at the rat liver cell nuclear periphery (Baiikowski, 1963). Similar results were reported for NEs from electron microscopy of the liver (Vorbrodt, 1967; Buchwalow et al., 1971, 1977), chick embryo myocardium cells (Klein and Afzelius, 1966), mouse chorioid plexus (Yasuzumi and Tsubo, 1966), human Leydig cells (Yasuzumi et a f . , 1967), pond snail spermatids (Yasuzumi et al., 1969), newt oocytes (Scheer and Franke, 1969), and mouse hepatomas (Buchwalow et al., 1972). High ATPase activity exceeding that of other cell structures was found in isolated NMs, especially in the lighter fraction, presumably originating from the outer NM of rat liver and mouse Ehrlich ascites cells; in rat Zajdela hepatoma it was markedly lower (Delektorskaya and Perevoshchikova, 1969; Zbarsky et a f . , 1969). A considerable amount of ATPase activity was found in isolated preparations of the NE by several workers, but the activities were lower than those in the microsomes. This discrepancy appears to depend on the method of NE isolation, especially when treatment with enzymic preparations and/or salt solutions is employed (Kashnig and Kasper, 1969; Franke et ul., 1970; Berezney et a f . , 1972; Kartenbecketaf., 1973; Pierietal., 1973; Koenetal., 1976; Sikstrom et al., 1976). Indeed, it was shown that the ATPase activity in the NE, but not in other cell structures, decreased upon RNase treatment or 1.5 M KCl extraction and was stimulated by the addition of RNA (Agutter et al., 1977). In solid liver tumors, particularly in poorly differentiated outgrowths, ATPase activity in the NE is usually lower, while in ascites tumors it may be higher than in the liver (Delektorskaya and Perevoschchikova, 1969; Buchwalow et a f . , 1972; Koen er al., 1976). The ATPase activity in isolated NEs of rat brain (Rapava et al., 1973), thymus (Reilly, 1971; Jarasch et al., 1973), and chicken erythrocytes (Zentgrafetaf., 1971; Blanchet, 1974) was also reported. The activity appears to be enhanced by the stimulation of erythropoiesis in rabbit bone marrow cells
ENZYME PROFILE OF NUCLEAR ENVELOPE
309
(Jarasch, 1973; Zbarsky et al., 1975a). Mainly Mgl+-dependent ATPase with a neutral pH optimum has been reported in NE. C$+-activated ATPase with a slightly alkaline pH optimum (ATPase B) was reported to be present in the nucleus (Bafikowski, 1963) or to be partially associated with the inner NM (Buchwalow et al., 1971). The absence of Naf , K+-dependentATPase from the NE is unanimously reported (Delectorskaya and Perevoshchikova, 1969; Franke et al., 1970; Reilly, 1971; Jarasch, 1973; Kartenbeck et al., 1973; Blanchet, 1974). However, NM ATPase can be activated by 2,4-dinitrophenol and inhibited by gramicidin S (Delektorskaya and Perevoshchikova, 1969). Association of ATPase with the nuclear pores was reported (Yasuzumi and Tsubo, 1966; Yasuzumi et al., 1967; Chardonnet and Dales, 1972), but these results were obtained mainly by a modified histochemical method (lead nitrate was added at the end of the reaction) and therefore require confirmation. A presumed difference in ATPase activity in the inner and outer NMs (Delektorskaya and Perevoshchikova, 1969; Buchwalow et al., 1971) may also be of importance but cannot be regarded as definitely established. 2. Esterases The biochemical data on esterases of the NE are confined to carboxylesterase, acetylesterase, and arylesterase, while histochemical studies include only acetylcholinesterase and arylsulfatase. a. Carboxylesterase. Carboxylesterase (carbonic-ester hydrolase, EC 3.1.1.1) activity has been determined in isolated NMs with two substrates, tributyrin and methyl butyrate, which gave similar results. The lighter rat liver NM fraction was free of activity, while the activity of the heavier fraction exceeded that of the nuclear fraction by 30-60% and was four times less than that of the mitochondria-lysosome fraction (Pokrovsky et al., 1970). b. Acetylesterase. The relative acetylesterase (acetic-ester hydrolase, EC 3.1.1.6) activity withp-nitrophenylacetate as a substrate was very similar to that of carboxylesterase. The specific enzyme activity of the lighter fraction of rat liver NE was negligible, while the heavier fraction contained twice the activity of the isolated nuclei and five to six times less than the mitochondria-lysosome fraction (Pokrovsky et al., 1970). Thus both the carboxylesterase and the acetylesterase appear to be present only in the heavier fraction (presumably the inner NM), but their low activity suggests that their presence may be the result of contamination. c. Arylsulfatase. Arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was histochemically demonstrated in the NEs of cucumber root meristem cells (Poux, 1965, 1966) and eosinophilic leukocytes (Bainton and Farquhar, 1970). Arylsulfatases A and B, measured with 2-hydroxy-5-nitrophenyl sulfate as substrate, were found to be rather high in the heavier fraction of rat liver NEs where their activity exceeded by a factor of 4 to 5 that of isolated nuclei or microsomes.
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I. B. ZBARSKY
In the lighter NM fraction this activity was practically the same as in isolated nuclei or microsomes (Pokrovsky et a f . , 1968). A high activity in the NE, consistent with histochemical data, indicates that arylsulfatases A and B are integral components of this subcellular structure. d. Acetylcholinesterase. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), studied only histochemically, was found in various nervous cells of the rat (Novikoff et a f . , 1966; Ednko et a f . , 1967), frog (Brzin et a f . , 1966; Majcen and Brzin, 1971), chick embryo (Pannese et a f . , 1971), rabbit (Reale et al., 1971), and starling (Welsch and Haase, 1971), and in chick muscle cell cultures (Golder et a f . , 1976). As the enzyme was not measured quantitatively and data for other tissues are absent, it is premature to draw any definite conclusions concerning its NE activity. 3. Proteinases Although isolated nuclei showed distinct proteolytic activity, no measurable activity was found either in the lighter or the heavier rat liver NE fraction (Pokrovsky et al., 1968). Nevertheless, the study of proteolytic activity in the NE, especially for latent proteinases found in cell nuclei (Carter and Chae, 1976), should continue. 4. Nucleases Nucleases constitute a broad group of enzymes with different mechanisms of action (Shapot, 1968). The study of these enzymes in the NE may be important in relation to the NE’s role in processing pre-rRNA and pre-mRNA, nucleocytoplasmic RNA transport, and initiation of DNA replication (see Sections IV,A and B). Histochemically, RNases and DNases in cell nuclei were detected mostly in the condensed chromatin frequently associated with the NE (Zotikov and Bernhard, 1970; Taperer a f . , 1971a,b; Fortet a f . , 1974). Variable substrate composition, preliminary tissue fixation, and phosphatase treatment may produce artifacts, and the evaluation of the results calls for caution (Vorbrodt, 1974). Considerable RNase activity (two to three times as high as that-in the nuclei) was found in isolated rat liver NEs (Skridonenko and Gorchakova, 1973; Skridonenko et a f . , 1975). It appears that the NE contains a 3’-endoribonuclease related to the pancreatic enzyme (EC 2.7.7.16), and 5’-endoribonucleasepossibly identical to the enzyme reported in guinea pig hepatocyte nuclei (Heppel et a f . , 1956). In the NEs of rat hepatoma 27 the RNase activity is still higher in comparison to that in the nuclei and is strongly stimulated by pchlormercuribenzoate (Koen et al., 1976). The activity of RNase in the NE seems to increase in correspondence with its purity but remains bound to an inhibitor; the latter is removed in the course of polyacrylamide gel elec-
ENZYME PROFILE OF NUCLEAR ENVELOPE
311
trophoresis of the enzyme (Shlyakhovenko, 1973; Skridonenko et al., 1975). No DNase activity has been found in the NE.
5. Other Nonoxidative Enzymes Only occasional data are available on the presence or absence of other nonoxidative enzymes in the NE. P-Glucuronidase (P-D-glucoronide glucuronosohydrolase, EC 3.2.1.31) (Smith and Fishman, 1969), acetyl-CoA carboxylase (acetyl-CoA:carbon-dioxide ligase, EC 6.4.1.2) (Yates et al., 1969), and carbamoyl phosphate hydrolysis (Mitzutani and Fujita, 1968) were detected histochemically in rat and mouse NEs. Glutamic-oxaloacetic transaminotransferase, EC 2.6.1.1) was found in aminase (~-aspartate:2-oxoglutarate NEs of rat liver (Lee and Torrack, 1968) and heart muscle (Lee, 1969). Lipase was detected in NMs of rat acinar pancreatic cells (Murata et al., 1968). Glucose-1-phosphate hydrolysis (D-glucose-1-phosphate phosphohydrolase, EC 3.1.3.10) in rat liver NEs was as intense as in isolated nuclei but three times less active than in microsomes (Kartenbeck et al., 1973). Kasper (1971) claims that N-demethylase (involved in the demethylation of 3-methyl-4-aminobenzene) is 10 times less active in isolated rat liver NEs than in microsomes and cannot be induced by phenobarbital as it can in microsomes. NAD+ pyrophosphorylase (ATP:NMN adenylyltransferase, EC 2.7.7.1) and nucleic acid polymerases (nuc1eosidetriphosphate:RNAnucleotidyltransferase, EC 2.7.7.6; deoxynuc1eosidetriphosphate:DNAdeoxynucleotidyltransferase,EC 2.7.7.7; and others) may be of interest because they exert their action in the cell nucleus, and the first two are thought to be typical nuclear enzymes (Siebert and Humphrey, 1965). NAD pyrophosphorylase was shown to be absent from rat liver NEs (Jarasch, 1973; Kay and Johnston, 1973; Sikstrom et al., 1976), as well as from those of chick erythrocytes (Zentgraf et al., 1971). However, its activity is markedly higher in isolated chromatin and in nucleoli than in nuclei (Siebert et al., 1966; Unger et al., 1975). Similar results were obtained histochemically (Buchwalow and Unger, 1974; Unger et al., 1975). Activity of another enzyme, NAD+ glycohydrolase (NADase, EC 3.2.2.5), in isolated rat liver NE was reported to be 4.5-fold greater than that in nuclei and about one-half of that in microsomes (Table I). NADase in the NE was found to be identical to that in the microsome but was regarded as an integral component of the NE (Fukushima et al., 1976). RNA polymerase also appears to be absent from NEs and to be associated with chromatin (Kay and Johnston, 1973; Sikstrom et al., 1975, 1976). No poly-A polymerase was found by Sikstrom et al. (1976), but Kay and Johnston (1973) claimed that this enzyme was present in the NE in concentrations twice as high as in isolated nonfractionated nuclei. Recently, M~+-dependentbut not Mg2+dependent poly-A polymerase was reported to be localized mainly on the membrane-associated chromatin (Louis et al., 1978).
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I. B. ZBARSKY
Various workers found DNA polymerase activity in isolated NEs (Yoshida et al., 1971; Yoshikawa-Fukada and Ebert, 1971; Deumling and Franke, 1972; Tsuruo et al., 1972). However, the data on the subject are conflicting: There are several types of DNA polymerases, and the role of the NE in eukaryotic DNA replication remains unclear and highly complicated (see Section IV ,A). The important role of cyclic nucleotides and the localization of the enzymes involved in their synthesis and hydrolysis in the membrane suggest the probable presence of adenylate cyclase and CAMP phosphodiesterase in the NE. The presence of adenylate cyclase was reported in isolated nuclei of the liver and other tissues (Liaoer al., 1971; Soifer and Hechter, 1971). However, the enzyme was revealed in the NE only histochemically with the light microscope (Coulson and Kennedy, 1971, 1972), and the lack of sufficient controls makes the results uncertain (Vorbrodt, 1974). Electron cytochemical studies of localization of creatine kinase (EC 2.7.3.2)in heart cells revealed the enzyme in different sites, including the NE (Sharov et al., 1977). B. OXIDOREDUCTASES The problem of oxidative processes in cell nuclei has been discussed for a long time (Conover, 1967). Oxidation and oxidative phosphorylation were formerly ascribed only to the cell nuclei of lymphoid tissues (Betel and Klouwen, 1966). Later oxidative enzymes were found in the cell nuclei of various tissues comprising the liver and were shown to be localized in the NMs (Kuzmina et al., 1969; Zbarsky et al., 1969; Betel, 1972; Zbarsky, 1972b,c). The problem remains unsolved and is considered in more detail in Section 111. Here, quantitative data on oxidoreductase activity in isolated NEs (Table 11) and related histochemical observations are presented. 1 . Succinate Dehydrogenase Succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.11 or, correspondingly, succinate oxidase, succinate-cytochrome c reductase, succinate reductase activity in the presence of another electron acceptor [iodonitrotetrazolium violet(INT), phenazine methosulfate (PMS), dichlorophenolindophenol, ferricyanide] as a rule was not found in isolated nuclei or NEs. Even in crude rat liver nuclei only traces of succinate oxidase could be demonstrated (Rees and Rowland, 1961; Rees et al., 1963). Some claims for the presence of succinate dehydrogenase in thymus (Dancheva, 1965) and liver (Manta et al., 1966) nuclei have not been substantiated. The succinate oxidase or succinate dehydrogenase activity in isolated NMs is negligible and is regarded by many investigators as an indicator of mitochondrial contamination (Zbarsky et al., 1968; Kuzmina et al., 1969; Berezney et al., 1970a,b, 1972; Berezney and Crane, 1971; Zentgrafet al., 1971; Agutter, 1972:
TABLE 11 OXIDATIVE ENZYME ACTIVITIES OF THE NE IN COMPARISON TO THOSEOF OTHER CELL STRUCTURESa Enzyme Cytochrome c oxidase
Tetraclorohydroquinoneoxidase Succinate oxidase
Succinate-cytochrome c reductase
Tissue
NE
NE/nuclei
Wmicrosomes
Reference
Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Bovine liver Bovine liver Bovine liver Rat thymus Rat thymus Calf thymus Chick erythrocytes Rabbit bone marrow cells Onion roots Bovine liver Rat liver Rat thymus Rat liver Bovine liver Bovine liver Rat thymus Rat liver Rat liver
38 (h)b 31 ( I ) b 532 240 372 0.027d
