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rich in wax esters (Sargent el al., 1979). The enzymic activity in the intestines of four fish from each group was measured at weekly intervals over the next month. The results showed that after the first week the total activity of NAD+-dependent acetaldehyde dehydrogenase in intestines of fish fed on zooplankton was 150-200% of that in their control counterparts, and that the percentage increase in activity was similar in all three subcellular fractions (P= 0.07 that the increases were due to chance; binomial distribution). We conclude that NAD+-dependent acetaldehyde dehydrogenases are involved in the intestinal metabolism of dietary fatty alcohols in fish. We thank the Natural Environment Research Council for financial support. Atkins, G. L., Nimmo, I. A. & Bauermeister, A. (1978) Biochem. SOC.Trans. 6, 209-21 1 Bauermeister, A. E. M. (1978) Ph.D Thesis, University of Aberdeen Bauermeister, A. & Sargent, J. (1978) Biochenz. Soc. Trans. 6,222-224 Benson, A. A., Lee, R. F. & Nevenzel, J. C. (1972) Biochem. SOC.Symp. 35, 175-187 Crow, K. E., Kitson, T. M., MacGibbon, A. K. H. & Bett, R. D. (1974) Biochim. Biophys. Acts 350, 121-128 Horton, A. A. & Barrett, M. C. (1975) Arch. Biochem. Biophys. 167,426-436 Patton, J. S., Nevenzel, J. C. & Benson, A. A. (1975) Lipids 10,575-583 Rahn, C. H., Sand, D. M & Schlenk, (1973)J. Nutr. 103,1441-1447 Sand, D. M., Rahn, C. H. & Schlenk, H. ( I 973) J. Nurr. 103,600-607 Sargent,J. R., Lee, R. F. & Nevenzel, J. C. (1976) ChemistryandBiochemistryof Natural Wales (Kolattukudy, P. E., ed.), pp. 50-91, Elsevier, Amsterdam Sargent, J. R., Mclntosh, R., Bauermeister, A. E. M. & Blaxter, J. H. S . (1979) Mar. Biol. in the press

Enzymes of Serine Metabolism in Normal and Neoplastic Tissues KEITH SNELL* and W. EUGENE KNOX Cancer Research Institute, New Englund Deaconess Hospital and Department of’ Biological Chemistry, Harvard University Medical School, Boston, M A 02215, U S A .

The identification of alterations in enzymic composition in neoplastic tissues is relevant to the rational design of selective anti-cancer chemotherapeutic agents and may also bear on diagnosis and therapeutic evaluation in neoplastic diseases. Considerable progress has been made in defining alterations in enzyme patterns in neoplastic tissues of the rat (Knox, 1976;Goldfarb &Pitot, 1976;Weber, 1974).For aminoacid metabolism the relevant gross changes are an increase in the incorporation of amino acids into proteins and a decrease in enzymes involved in catabolic pathways (Weber & Lea, 1966;Goodlad, 1964;Wagle et al., 1963), a situation compatible with the demands of a continuously growing tissue. Serine, an important non-essential amino acid in normal tissues, is formed endogenously from carbohydrate and can then be utilized in alternative pathways for biosynthetic purposes (see Snell & Walker, 1973),as well as for direct incorporation into proteins. Serine has been shown to enhance the growth rate of a number of tumour tissues cultured in uitro (Goodlad, 1964). No systematic studies on the pattern of serine metabolism in neoplastic tissues have been made, and the present work describes variations in tissues of an enzyme involved in serine biosynthesis (phosphoserine aminoand the initiating enzymes of each of the alternative pathways transferase, EC 2.6.1.52) of serine utilization (serine dehydratase, EC 4.2.1.13; serine aminotransferase, EC 2.6.1.51; serine hydroxymethyltransferase, EC 2.1.2.1). Serine dehydratase forms pyruvate for oxidation, lipogenesis or gluconeogenesis; serine aminotransferase initiates gluconeogenesis in certain situations ; serine hydroxymethyltransferase forms 5,lO-methylenetetrahydrofolate(a source of C1 units) and glycine.

* Permanent address: Department of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH, U.K.; to whom reprint requests should be addressed. 1979

583rd MEETING, CAMBRIDGE

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Serine dehydratase activity in normal tissues was confined almost entirely to the liver, with some very low activity also associated with the kidney. Serine aminotransferase activity was mainly associated with the liver, but low activities were found in some other normal tissues. Serine hydroxymethyltransferase activity was distributed in a number of normal tissues, but highest activity was found in liver and kidney. Similarly, phosphoserine aminotransferase activity was also widely distributed, but in this case kidney activity exceeded liver activity. Phosphoserine phosphatase, another enzyme on the pathway of serine biosynthesis, has previously been shown to have a similar distribution in normal tissues (Knox et at., 1969). In the transplantable neoplastic tissues examined (Morris hepatomas of differing growth rates, renal carcinomas, lymphomas, salivary-gland tumours and a mammary tumour), serine dehydratase was almost totally absent, exccpt for the striking exception of the Morris hepatoma 7800. Previous work with ten transplanted hepatomas of differing growth rates has shown a total absence of, or very low, serine dehydratase activity (Davis et at., 1970; Potter et at., 1969; Kizer & Lacey, 1961), although in three hepatomas there were contradictory findings (see Davis et al., 1970; cf. Potter et nl., 1969). For the other enzymes in the survey no previous work has been published on activities in neoplastic tissues. In the present study, serine aminotransferase activity was very low in all the tumours studied. In contrast with serine dehydratase and serine aminotransferase, serine hydroxymethyltransferase was present in all the tumours at activities comparable with those found in normal adult liver tissue. Phosphoserine aminotransferase, involved in serine biosynthesis, was found at activities equal to or greater than adult liver activity. This finding is consistent with the enhanced capacity for serine formation found in hepatoma 5123TC and the elevated activity of 3-phosphoglycerate dehydrogenase, another enzyme on the biosynthetic pathway, found in this and two other hepatomas (Davis et a/., 1970). An increased capacity for serine biosynthesis in neoplastic tissues was also suggested by Knox et ( I / . (1969) on the basis of high activities of phosphoserine phosphatase, the final enzyme in the biosynthetic pathway, in primary and transplanted tumours of differing origins. It appears that, as a generalization, neoplastic tissues are equipped with an enhanced capacity for serine biosynthesis which is compatible with the increased demands for amino acid utilization for protein synthesis found in these tissues. The direction of serine towards protein synthesis is assisted by the low activities of the initiating enzymes of alternative pathways of serine utilization shown in the present study. However, the finding that the pathway of serine utilization initiated by serine hydroxymethyltransferasepersists in neoplastic tissues suggests that, inaddition to its use in protein synthesis, the increased formation of serine may also be involved in the provision of precursors for DNA synthesis. Serine hydroxymethyltransferase not only converts serine into glycine, which is utilized directly for purine nucleotide biosynthesis, but also generates 5,lO-methylenetetrahydrofolate,a cofactor which provides carbon stoicheiometrically for thymidylate synthesis (see Huennekens et al., 1976). It is suggested that neoplastic tissues have the potential to synthesize serine and to make use of the serine carbon for DNA synthesis, for which there is an increased demand in tumours for continuous cell replication. K. S. was the recipient of a Biochemical Society-Boehringer Travelling Fellowship during the course of this work. Davis, J . L., Fallon, H. J . & Morris, H. P. (1970) Cancer Rcs. 30,2917--2920 Goldfarb, S. & Pitot, H. C. (1976) Front. Castruintest. Res. 2, 194-242 Goodlad, G . A. J. (1964) in Man?rna/ianProtein Metabolism (Munro, H. N., ed.), vol. 2, pp. 415-444, Academic Press, New York Huennekens, F. M., Vitols, K . S., Whiteley, J. M. & Neef, V. G . (1976) Methods Cancer Res. 13, 199-225

Kizer, D. E. & Lacey, D. E. (1961) Proc. Soc. Exp. Biol. Med. 106, 790-794 Knox, W. E. (1976) Enzyme Patterns in Fetal Adult and Neoplasric Rat Tissues, 2nd edn., S. Karger, Basel Knox, W. E. Herzfeld, A. & Hudson, J. (1969) Arch. Biochern. Biophys. 132, 397-403

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Potter, V. R., Watanabe, M., Pitot, H . C. & Morris, H. P. (1969) Cancer Rrs. 29, 55-76 Snell, K. &Walker, D. G. (1973) Enzyme 15,40-81 Wagle, S. R., Morris, H. P. & Weber, G. (1963) Cancer Rrs. 23, 1003-1007 Weber, G. (1974) in The Molecular Biology of Cancer (Busch, H., ed.), pp. 487-521, Academic Press, New York Weber, G . & Lea, M. A. (1966) Adv. Enzyme Regul. 4, 115-145

Characterization of the Phosphorylase Phosphatase associated with Dog Liver Particulate Glycogen as a Manganese Metalloenzyme GHISLAIN DEFREYN,* JOZEF VERPLAETSE,? JOZEF GORIS,* RENE LONTIEt and WILFRIED MERLEVEDE* *Afdeling Biochemie, Departement Humane Biologie, Faculteit der Geneeskunde, and ?Afdeling Biochemie, Departement Scheikunde, Faculteit der Wetenschappen, Katholieke Universiteit te Leuven, B-3OOO Leuven, Belgium

Several studies on the regulation of phosphorylase phosphatase in different tissues by nucleotides and bivalent ions have appeared in recent years (Goris et al., 1977; Shimazu et al., 1978; Khandelwal, 1978; Burchell & Cohen, 1978; Hsiao et al., 1978). The role of these factors often appeared ambiguous and even contradictory, probably because of the existence of multiple molecular forms of the enzyme. There is of course a risk that some of the forms of the enzyme may well be artefacts of the purification methods, since several of the phosphatase preparations were obtained by harsh purification procedures. However, some observations point to the fact that manganese might play an important role in determining the activity of phosphorylase phosphatase. Manganese could act as a ligand affecting the structure and activity of the enzyme, or phosphorylase phosphatase could be a manganese metalloenzyme. The preparation of liver phosphorylase (EC 2.4.1 .l) and the assay of phosphorylase phosphatase have been described (Kalala et al., 1977). Phosphorylase phosphatase (EC 3.1.3.17) associated with dog liver particulate glycogen was prepared as described by Goris et al. (1977). Phosphorylase phosphatase was then separated from the glycogen complex by DEAE-cellulose chromatography with a NaCl gradient (Defreyn et al., 1977), and those fractions containing phosphorylase phosphatase free of the protein deinhibitor were pooled, chromatographed on Sephacryl S-200 and concentrated by ultrafiltration. The phosphatase thus collected is characterized by a mol.wt. of 45 OOO as estimated by sucrose-density-gradient centrifugation and Sephacryl S-200 gel filtration. This purified preparation of phosphorylase phosphatase, when examined with sodium dodecyl sulphate/polyacrylamide-disc-gelelectrophoresis, showed a single protein-staining band also corresponding to a mol.wt. of 45000. E.p.r. measurements were carried out with an E-109 Varian Spectrometer at room temperature, microwave frequency 9.48 GHz, field modulation amplitude 1 mT, microwave power 1OmW. The spectrometer was equipped with a Varian E-901 Interface Module, a Hewlett-Packard 9825 A Calculator, a 1350 Graphics Translator with a 1311 A display and a E-900 Varian Software Package. A flat aqueous-solution cell was used and each sample (diluted in 10mM-Tris/HC1,pH7.4) was run six times. The average was plotted and the second integral calculated to give the amount of detected MnZ+, which was shown to be linear with the concentration from 5 to 500p~-MnC1~. With 1mM-EDTA the e.p.r. signal vanished completely, and addition of 0.2~-2-mercaptoethanol decreased the intensity of the signal by 75 %. A typical e.p.r. signal for Mn2+ was observed in a 1mg/ml solution (determined by AZ8,,, bovine serum albumin as standard) of phosphorylase phosphatase ( 2 2 ~ ~ ) . Because of the line-broadening resulting from the interaction of protein (Reed & Cohn, 1970), the phosphatase preparation was denatured with ~ M - H Cand I from the magnitude of the e.p.r. signal the concentration of released Mn2+was calculated to be 1 5 , ~ Complexing ~. agents such as EDTA and 2-mercaptoethanol did not affect the 1979

Enzymes of serine metabolism in normal and neoplastic tissues [proceedings].

1048 BIOCHEMICAL SOCIETY TRANSACTIONS rich in wax esters (Sargent el al., 1979). The enzymic activity in the intestines of four fish from each group...
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