Biochemical Genetics, Vol. 13, Nos. 9/10, 1975
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase: Heterogeneity of the Isoenzymes A4 and X, Joachim Klose 1 and Horst Spielmann 1
Received 1 Apr. 1975--Final29 Apr. 1975
L D H of mouse organs (including testis) was investigated by isoelectric focusing in polyaerylamide gels. The number of L D H bands in this pattern considerably exceeds the five (six in testis) of the standard electrophoretie pattern. An attempt was made to identify these bands as tetramerie isoenzymes formed by random association of different subunits. This included isoelectrie focusing of purifiedLDH A, B, and X, two-dimensional separation of LDH, urea treatment of LDH, staining with specific substrates, and comparison of different organs. Further experiments were performed to exclude artifacts possibly produced by the isoelectrie focusing technique. Different strains of mice were also investigated. The results demonstrate that in addition to the common five L D H bands (A4-B4) one set of five bands is formed by L D H A (A~-A~) and another one by L D H X (X~-X~). Moreover, an unusual band was found which has a lower molecular weight and no affinity to the other isoenzymes. The data suggest that the heterogeneity of the L D H pattern revealed by isoelectric focusing arises from post-transcriptional events rather than from a number of additional genes. KEY WORDS: LDH A, B; LDH X; isoenzymes; mouse; isoelectric focusing.
INTRODUCTION
The development of the isoelectric focusing technique in polyacrylamide gel (Wrigley, 1968; Fawcett, 1968) led to reinvestigations of isoenzymes and This work was supported by grants of the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29 "Embryonale Entwicklung und Differenzierung." Pharmakologisches Institut der Freien Universit~it Berlin, Abteilung Embryonal-Pharmakologie, Berlin, West Germany. 707 © 1975 Plenum Publishing Corporation, 227 West 17th Street, N e w York, N.Y. 10011. N o part o f this publication m a y be reproduced, stored in a retrieval system, or transmitted, in any f o r m or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher.
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proteins with known electrophoretic patterns because in several instances this technique yielded a higher number of bands as a result of its improved resolving power compared to polyacrylamide or starch gel electrophoresis (Dale and Lather, 1968; Kohnert et al., 1973; Wellner and Hayes, 1973). The lactate dehydrogenase (LDH, E.C. 1.1.1.27) isoenzymes are widely used in genetics to elucidate problems in different fields, e.g., in developmental genetics (Markert and Ursprung, 1962; Klose et al., 1969; Spielmann et al., 1973) and in the field of phylogenesis (Ohno et al., 1968; Klose et al., 1968; Whitt et al., 1973). However, only little information exists on the pattern of LDH produced by the gel isoelectric focusing technique. More than five bands have been demonstrated (Dale and Latner, 1968; Chamoles and Karcher, 1970a,b), but no attempts have been made to explain this pattern on the basis of tetrameric isoenzymes formed by random association of different subunits. In this study, the gel isoelectric focusing patterns of LDH of several mouse organs are presented and an attempt is made to identify the isoenzyme bands that were found. MATERIALS AND METHODS Experimental Animals
In most experiments, mice of the NMRI strain (Schwenke & Co., Nauheim, Germany) were used and several organs (heart, muscle, liver, kidney, brain, eye, testis) were investigated. In addition, organs (heart, liver, testis) of mice of the following strains were investigated: C57BL/6J, DBA/2J, AKR/J (Jackson Laboratory, Bar Harbor, Maine), and BALB/c (Zentralinstitut ftir Versuchstierkunde, Hannover, West Germany). Isoelectric Focusing
Isoelectric focusing was performed in polyacrylamide gel rods or slab gels. The preparation of the gel rods and the focusing procedure were carried out as described previously (Klose, 1975). Slab gels were cast in glass cells purchased from Ortec GmbH (Munich, West Germany) and the same gel solution was prepared as for gel rods with the exception that 6% (w/v) acrylamide, 0.15% (w/v) bisacrylamide, and 0.04% (w/v) ammonium persulfate were used. With the Ortec 4100 pulsed constant power supply, the power was regulated for focusing in slab gels as follows: 30 rain 30 V, 2 hr, 60 V, 4 hr 120 V; at a pulse rate of 100 pps and at 1/~farad. At the end of the run, the power was raised to 200 V, 200 pps for 2 rain and 300 V, 300 pps for 2 rain.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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When rough tissue extracts were used as sources of LDH, the samples were prepared as described by Klose (1975). Purified LDH was applied to the gels in 0.03 M phosphate buffer, pH 7.2, containing 20% (w/v) sucrose and 1% (w/v) Ampholine (LKB, Bromma, Sweden). The gels were stained for LDH activity with the following solution: 1.0 g sodium lactate, 0.014 g NAD, 0.006 g nitroblue tetrazolium, and 0.004 g phenazine methosulfate in 100 ml 0.05 M tris-HCt buffer, pH 7.5. Sodium lactate was replaced by 1.0 g sodium valerate when the testis-specific isoenzyme (LDH X) was investigated and by 0.5 ml ethanol for the investigation of alcohol dehydrogenase (ADH, E.C. 1.1.1.1). The gels were stained for protein with Amido Black after washing in 12% (w/v) trichloroacetic acid. Two-Dimensional Separation of L D H
Isoelectric focusing followed by electrophoresis was carried out according to Klose (1975). Dissociation of LDH into Subunits
The dissociation of purified LDH A was performed with 12 M urea according to Appella and Markert (1961). The enzyme was dissolved in 0.01 Mphosphate buffer, pH 7.2, containing 12 M urea and 0.1 M mercaptoethanol, and this solution was applied to the gels for isoelectric focusing after a 1-hr incubation period at 37 C. The gels used for isoelectric focusing contained 6 M urea and the electrode solutions 6 M urea and 0.1 M mercaptoethanol. Isolation of the Mouse LDH Isoenzymes A, B, and X
The first step of the isolation of LDH A and B was the separation of total LDH from all other proteins of mouse liver and heart, respectively, by affinity chromatography according to O'Carra and Barry (1972) in the variation described by Spielmann et al. (1973). LDH A was isolated from total mouse liver LDH by chromatography on DEAE-Sephadex A50 in 10 m~ tris-HCl, pH 8.6, according to Ryan and Vestling (1974). LDH B was isolated from total mouse heart LDH by ion exchange chromatography on DEAE-Sephadex A50 in 0.05 M tris-HC1, pH 7.5, by a linear salt gradient up to 0.3 M NaCI. Pure LDH B is eluted in the fractions containing 0.20-0.23 M NaC1 as described by Lindy (1974). LDH X was separated from the other LDH isoenzymes of mouse testis by affinity chromatography as previously described (Spielmann et al., 1973). The fractions containing LDH X are contaminated by other proteins after this step. Purified LDH X was isolated from these fractions by chromatography on DEAE-Sephadex under the
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conditions outlined above for LDH A. The contaminating proteins are adsorbed by the column, whereas LDH X is not bound by the ion exchanger. Purified mouse LDH A, B, and X were not contaminated by other LDH isoenzymes or proteins as revealed by the appearance of only one band after polyacrylamide gel electrophoresis of the isoenzymes and staining for both LDH activity and proteins (Spielmann et al., 1973). Isolated LDH isoenzymes of such properties are designated as pure. RESULTS
Figure 1 shows the LDH banding patterns of different mouse organs after isoelectric focusing and includes a scheme that summarizes the different bands which were found. It is obvious that the number of bands demonstrated considerably exceeds the five usually obtained in the electrophoretic patterns of mouse LDH. According to the scheme in Fig. 1, the focusing pattern of LDH shows the two regular homomeric isoenzymes A4 ( = A 4~) and B4, creating the three heteromers, A~B, A2~B2, A1Ba, and in addition a third homomeric isoenzyme, called A~, which recombines with A~. An additional series of five bands was found in mouse testis and designated as X 4--1 X 4"2 Furthermore, two main bands are visible which do not recombine
2
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b
c
d
e
f
g
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3
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A : 54 ; 3
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Fig. 1. Lactate dehydrogenase of mouse organs as revealed by gel isoelectric focusing of rough extracts (4.8~ polyacrylamide gel, Ampholine p H 3.5-10); 5 ttl undiluted tissue sap was applied to each gel. The gels were stained for lactate dehydrogenase
activity. (a) Brain, (b) eye, (c) testis, (d) muscle, (e)liver, (f) kidney, (g) heart. (h) Schematic recapitulation of all different bands found in the mouse organs; dotted lines indicate irregular bands.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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with any other bands. One of these bands is present only in liver and more weakly in kidney. It is occupying the most basic position of all bands and could be identified as ADH. The other band lies between LDH A~ and B4 and seems to be present in all organs. This enzyme shows high activity in kidney and it was therefore called K. In addition, some weak bands appear regularly (see Fig. 1, muscle, below A4z) or less permanently (see Fig. 1, dotted lines). We performed a number of experiments to verify the classification of LDH bands indicated in Fig. 1. The results are presented in the following sections. LDH
B4
Pure LDH B from mouse heart appears as a single band in the gel after isoelectric focusing. The position of this band coincides with the top band of the heart pattern (Fig. 2). Comparing different organs, this band shows the highest activity in heart extracts like LDH B4. The five isoenzymes resulting A4~
A~
B4
A; A~
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ADH
a
b
c
d
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Fig. 2. Purified mouse lactate dehydrogenase after isoelectric focusing. Samples of equal lactate dehydrogenase activity were applied to each gel. For further information, see caption of Fig. 1. (a) Purified lactate dehydrogenasc A. (b) Lactate dehydrogenase pattern from rough heart extract. (c) Purified lactate dehydrogenase B. (d) Purified lactate dehyrogenase A was focused in the usual manner and thereafter refocused in the second dimension in a 6 ~ polyacrylamide slab gel, AmpholinepH 3.5-10. (e) Isoelectric focusing of rough liver extract and staining of the gel for alcohol dehydrogenase (ADH) activity.
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Klose and Spielmann
o ~
A'
B~e
a
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b
Fig. 3. Two-dimensional lactate dehydrogenase pattern obtained by isoelectric focusing (see caption of Fig. 1) followed by disc electrophoresis (standard procedure, Davis, cited by Maurer, 1971) and staining for lactate dehydrogenase activity. (a) Photograph of the lactate dehydrogenase pattern of mouse heart. (b) Filled circles, schematic drawing of the pattern shown in Fig. 3a; open circles, positions of the lactate dehydrogenase X-spots and the spots of the lactate dehydrogenase bands below A] (see muscle) are indicated in the same scheme, although the photograph presents only the heart pattern.
from the recombination of LDH B4 and A] subunits are indicated in Fig. 1 and are most clearly demonstrable after two-dimensional separation of heart LDH (Fig. 3).
LDH A~-A~ When pure mouse LDH A was subjected to isoelectric focusing, five bands were found. These bands correspond to five bands which can be identified in the pattern of all mouse organs and which consist of LDH A[ and four additional bands that are located in direction to the basic pole of the gel (Fig. 2). Five bands were also found when commercial rabbit LDH A (Boehringer, Mannheim, West Germany) was investigated by the same procedure. The five mouse LDH A bands could not be clearly separated by the DEAE-Sephadex chromatography described above, but when several successive fractions of the column were investigated separately by isoelectric focusing a shift of the LDH A pattern from the lowest band to the top band occurred (Fig. 4). According to Appella and Markert (1961), LDH dissociates into its subunits when exposed to urea and mercaptoethanol. The degree of dissociation depends on the concentration of the detergent and the duration of incubation. If the five bands of LDH A in our experiments result from the recombination of two different types of LDH A subunits, only two bands
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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o.3j
Fig. 4. Lactate dehydrogenase A was isolated from mouse liver by affinity chromatography and DEAE-Sephadex chroma- o.D. tography. Different fractions from the DEAE-Sephadex column were subjected to isoelectric focusing (see caption of Fig. 1.) Aliquots of equal lactate dehydrogenase activity were applied to the gels. The banding patterns were scanned with the Gilford spectrophotometer 2400S at a wavelength of 550 nm. Unbroken line, scanning curve of lactate dehydrogenase pattern from fraction 8; dotted line, scanning curve of lactate dehydrogenase pattern from fraction 7.
i~
t A2
f AI
MIGRATION DISTANCE
s h o u l d be f o u n d in the focusing p a t t e r n after dissociation o f purified L D H A. F i g u r e 5 shows the result o f this experiment. U n t r e a t e d L D H A has five b a n d s ; L D H An is present in a high c o n c e n t r a t i o n (staining intensity), L D H An in a low concentration. A f t e r dissociation with urea, the same L D H A shows high concentrations for L D H A~ a n d A~ a n d between these b a n d s two b a n d s (A~ A~ a n d A I A ~ ) with relatively low concentrations. W h e n the t r e a t m e n t o f L D H A was carried o u t at higher t e m p e r a t u r e s a n d over longer p e r i o d s o f time, the two h e t e r o m e r i c isoenzymes d i d n o t d i s a p p e a r completely. C o m p l e t e dissociation o f L D H A into two different subunits therefore was n o t achieved u n d e r the conditions used. The results indicate, however, t h a t after t r e a t m e n t with urea a n d m e r c a p t o e t h a n o l two different types o f L D H A h o m o m e r s have a c c u m u l a t e d while the concentrations o f the h e t e r o m e r s have decreased. In conclusion, the isoelectric focusing p a t t e r n o f mouse L D H A creates
0.4-
Fig. 5. Purified lactate dehydrogenase A was treated with 12 M urea and 0.1 M mercaptoethanol and subjected to isoelectric focusing (see caption of Fig. 1) in the presence of 6 M urea in the polyacrylamide gel. The lactate dehydrogenase bands were stained with Amido Black and scanned (see caption of Fig. 4) at a wavelength of 620 nm. An aliquot of the same lactate dehydrogenase sample was focused without treatment and stained and scan.ned, as indicated above. Unbroken line, scanning curve of focused, urea-treated lactate dehydrogenase A; dotted line, scanning curve of focused, untreated lactate dehydrogenase A.
O.D.
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MIGRATION DISTANCE
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Kiose and Spieimann
an additional LDH isoenzyme which is considered as a homomer and situated close to LDH A~ and therefore is designated A]. After two-dimensional separation of total heart LDH, LDH A41 and A~ appear on the same diagonal line as LDH B4 (Fig. 3). This can be expected if under the given experimental conditions three proteins differing in their isoelectric points have similar molecular sizes. The LDH bands A~-A] show high activity in muscle and liver, as expected for LDH A and relatively low activity, e.g., in heart. Comparing the activity of LDH A~ and A~ in several organs, LDH A42 generally has higher activities than A4~, but the relation between A] and A42 activity changes from one organ to another (Fig. 1). The introduction of artifactual bands into the protein pattern by the isoelectric focusing technique seems to be rather unlikely for a number of proteins according to previous investigators (Alpert et aI., 1973; Roy and Ruddle, 1973; Urushizaki et al., 1973; Wellner and Hayes, 1973). In a few cases, however, evidence for artifacts was found (Kaplan and Foster, 1971). The fact that the DEAE-Sephadex chromatography of LDH A generates fractions containing LDH A] and A 2 in different relative amounts clearly shows that the heterogeneity of LDH A already exists before isoelectric focusing is performed. The result of urea treatment of LDH A also is not consistent with the existence of artifactual LDH A bands. The occurrence of several bands also with purified LDH A excludes the possibility that these bands are due to complexing with tissue proteins during focusing (Lather, 1973). Three additional experiments were carried out to elucidate the possible production of artifacts by the focusing technique. First, purified LDH A and total heart LDH were subjected to isoelectric focusing and thereafter the gels were put on slab gels and refocusing was performed in the second dimension. No further LDH spots were observed compared with the onedimensional focusing pattern (Fig. 2). Second, rough heart LDH was separated in the presence of increased amounts of Ampholine in the separation gel (1%, 2%, and 3% Ampholine). The number and position of the bands did not change under these conditions, indicating that complexing of LDH isoenzymes with Ampholine (Latner, 1973) is unlikely. Finally, when the sample (heart extract) to be investigated for LDH was mixed with the total separation gel solution or was applied to the gel on the basic end, the same pattern was obtained as by the regular procedure of sample application. Apparently, the migration of the enzyme through either the aniodic or the cathodic part of the pH gradient did not affect the focusing pattern of the isoenzymes. L D H X 41- - X 42
When LDH of rough testis homogenate was investigated by isoelectric
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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focusing, the highest activity occurred in five bands which are situated below L D H A z and which could only be found in testis (Figs. 1 and 6). Purified L D H X shows five bands at the same position as the five main bands of total testis L D H (Fig. 6). Furthermore, only these five bands of the total testis L D H pattern appear when valerate is used as substrate (Fig. 6), which is characteristic of mouse L D H X (Allen, 1961). Therefore, mouse L D H X can be separated from L D H A by the gel isoelectric focusing technique, and it consists of five bands. Total testis L D H was separated by isoelectric focusing followed by electrophoresis in the second dimension. The pattern shows that the five spots characteristic of L D H X are situated somewhat below the line derived from the spots of L D H B4, A~, and A4z (Fig. 3). This may indicate a difference in molecular size between the testis-specific L D H and the common LDH, but it also demonstrates that in the mouse the five testis-specific spots do not result from a recombination of L D H X4 and A~ subunits. Apparently, mouse L D H X is formed by two homomeric isoenzymes X[ and X4z. The L D H patterns of muscle and liver also show a few bands below L D H A ,2 (Fig. 1). These bands are weak and do not occupy the same position as the L D H X bands when run side by side in a slab gel or when compared after two-dimensional separation (Fig. 3). In addition, these bands do not
B4
B4
A,~ A~
A~ X,~
xJ x~
X~
a
b
x~
c
d
e
f
g
h
i
Fig. 6. Mouse lactate dehydrogenase X after isoelectric focusing in gel rods (a-c) (see caption of Fig. 1) or slab gels (d-i) (see caption of Fig. 2d). (a) Lactate dehydrogenase pattern from rough testis extract. (b) Purified lactate dehydrogenase X. (c) Lactate dehydrogenase pattern from rough testis extract, staining for enzyme activity with sodium valerate as substrate. (d-i) Lactate dehydrogenase pattern from testis and heart extracts of different strains of mice: (d) heart DBA/2J, (e) testis DBA/2J, (f) testis C57BL/6J, (g) testis AKR/J, (h) testis BALB/c, (i) heart BALB/c.
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react with valerate. Thus by use of the isoelectric focusing technique LDH X could not be found in organs other than in testis. A D H Band and Enzyme Band K
The most basic enzyme band in liver and kidney homogenates (Fig. 1) had a much higher activity when lactate was replaced by ethanol in the reaction mixture and was therefore identified as ADH (Fig. 2). ADH reacts with the LDH staining mixture when acrylamide is contaminated with alcohol (Hitzeroth et al., 1968) during the recrystallization procedure. Close to the A~ Bz isoenzyme band, another single band was found (band K) (Fig. 1) which shows an unusual behavior when separated by electrophoresis after isoelectric focusing. It migrates much faster than all other spots (Fig. 3), indicating a comparatively low molecular weight. This enzyme could therefore be either a variant LDH isoenzyme like LDH X or a derivative of LDH B4, since it could be observed only in the presence of LDH B4. Additional L D H Bands
Some of the bands demonstrated by isoelectric focusing of mouse LDH tend to split into double bands. This was sometimes observed for B4 after the procedure of purification and may be the explanation for double bands like A~ B2 or A~ B. One of the two bands may become inactive since in a few instances double bands could be detected after protein staining of purified LDH isoenzymes but not when the same sample was investigated for LDH activity. However, the weak bands between A4z and B4 could in part arise from association of the subunits of these two isoenzymes. Four weak bands appear regularly below the isoenzyme A] in the pattern of muscle and liver LDH. Isoeleetric Focusing Patterns of L D H Isoenzymes of Different Strains of Mice
Heart, liver, and testis LDH of the four strains of mice indicated above was investigated by isoelectric focusing in slab gels. No variant LDH isoenzyme bands were found. The detection of a variant would show whether bands like A42 and X4z are genetically determined or the result of posttranscriptional alterations. DISCUSSION
The electrophoretic pattern of five LDH isoenzymes in mammalian tissues
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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results from the random association of the subunits of the two homomeric isoenzymes A4 and B4 governed by two genes (Markert, 1963; Lebherz, 1974). In several species, a testis-specific isoenzyme C4 (LDH X) was found which gives rise to one additional LDH band in mouse testis and to a third locus for LDH (Zinkham et al., 1963). Besides these major bands, however, under certain electrophoretic conditions multiple subbands were observed by several authors. In some cases, a total number of 15 bands was found in the electrophoretic pattern of mouse LDH from somatic tissues (Costello and Kaplan, 1963; Fritz and Jacobsen, 1965). This pattern was explained by the assumption of three homomeric isoenzymes, suggesting the existence of three cistrons for LDH (besides LDH X) (Costello and Kaplan, 1963). However, the total number of bands and their relative position observed by other investigators could not be explained on the basis of three homomeric LDH isoenzymes. These findings were interpreted by the existence of several conformations of the enzyme (Houssais, 1966), by LDH tetramers formed by hybrid subunits (Stambaugh and Buckley, 1967), or, as another possibility, by the binding of metabolites of low molecular weight to the enzyme (Dudman and Zerner, 1973). In our study, the heterogeneity of mouse LDH was investigated by the gel isoelectric focusing technique and the results show that in addition to the common LDH isoenzymes two distinct sets of five LDH isoenzyme bands can be demonstrated. One of these sets was found in all organs and was interpreted on the assumption that LDH A appears in two homomeric forms A~ and A 2 generating five isoenzymes by recombination of its subunits. The other group of five bands could be found only in testis homogenates and was interpreted as derived from two isoenzymes X~ and X~. A number of experiments such as focusing of pure LDH A, B, and X, urea treatment of LDH A, and two-dimensional separations of LDH according to different biochemical properties of the isoenzymes (isoelectric point and molecular size) are in good agreement with our interpretation. The observation that treatment of LDH A with urea did not result in a complete separation into two bands may demonstrate that even the highest concentration of urea which can be used is not strong enough to reach complete dissociation of the subunits. However, the relatively weak bands between A~ and A~ could also be explained as artifacts resulting from the urea treatment of LDH A. The possibility that artifactual bands are introduced by the focusing technique could be excluded to a high degree by additional experiments. However, the tendency of certain bands to split into double bands may indicate an artifactual effect. An instability of LDH B after isoelectric focusing was reported by Weller et al. (1973). This finding explains our observation that LDH B4 shows comparatively low activity in the gel focusing pattern (see heart, Fig. 1).
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Our results partly support the results of other investigators, that three homomeric LDH isoenzymes exist in the somatic tissues of mammals resulting in 15 bands (Costello and Kaplan, 1963; Fritz and Jacobsen, 1965). The isoelectric focusing pattern also shows three homomers; however, the third isoenzyme, designated as A 2 (Fig. 1), does not react (or only in a very restricted way) with B4 to form the heteromeric isoenzymes, whereas three distinct bands appear between A~ and B4 and between A 2 and A~. Assuming an extra gene locus for A 2, this gene may have become too different from the B gene during phylogenesis to allow an interaction between the two primary gene products. A diminished affinity between LDH A and B subunits has been reported for other organisms (Klose et al., 1968). However, to explain the complete focusing pattern of LDH on a genetic basis at least three additional genes have to be postulated, one for A 2, one for X 4, 2 and one to interpret the four bands below A 2 in the pattern of muscle LDH. A simpler explanation, in our view, interprets the heterogeneity of LDH as a result of post-transcriptional events. Such mechanisms were recently reviewed by Williamson et al. (1973). A secondary modification of LDH AL resulting in LDH A 2, may restrict or exclude interactions with LDH B4. Isoelectric focusing of LDH X has not been performed so far by other investigators, and electrophoretic studies of mouse LDH X have revealed only one band. Therefore, the demonstration of five distinct bands for mouse LDH X by isoelectric focusing is an unexpected finding. It may be interpreted in the same way as the heterogeneity of LDH A. The results of our investigations on different inbred strains of mice exclude the possibility of allelic cistrons but not of nonallelic cistrons causing the heterogeneity of LDH A and X. It is interesting to note that in certain lower vertebrates genetic evidence exists for the occurrence of at least five gene loci for LDH (Klose et al., 1968; Shaklee et al., 1973). The presence of one ADH band in the focusing pattern of mouse liver and kidney is consistent with the electrophoretic pattern of ADH (Ohno et al., 1970). However, another single band (K), which has not been previously described, could be demonstrated by the isoelectric focusing technique and especially by its combination with electrophoresis. This band is not identical with the kidney ~-hydroxy acid oxidase which produces an additional band in the LDH isoenzyme pattern of rats (Domenech and Blanco, 1967), since mouse kidney homogenates exhibit band K only in the presence of the coenzyme NAD. The enzyme band K shows no reassociation with other isoenzymes in the LDH pattern and its molecular weight seems to be much lower than that of normal LDH isoenzymes. We investigated the LDH of mouse preimplantation embryos by isoelectric focusing, and unexpectedly the only isoenzyme band which could be found in oocytes is situated at the same position as band K (Spielmann and Klose, unpublished observations). This observation is still under investigation.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
719
W e have shown t h a t m o u s e L D H X is clearly separable f r o m L D H A b y isoelectric focusing. F u r t h e r experiments should show whether L D H X can be detected b y this technique in animals in which this enzyme c o u l d n o t be f o u n d b y electrophoresis ( M a r k e r t , 1971). F u r t h e r m o r e , i m m u n o c h e m i c a l studies a n d genetic investigations o f wild p o p u l a t i o n s o f mice will be p e r f o r m e d to elucidate the origin o f the L D H isoenzymes A4z a n d X 2 detected by gel isoelectric focusing o f mouse tissues.
REFERENCES Allen, J. M. (1961). Multiple forms of lactic dehydrogenase in tissues of the mouse: Their specificity, cellular localisation and response to altered physiological conditions. Ann. N.Y. Acad. Sci. 94:937. Alpert, E., Drysdale, J. W., and Isselbacher, K. J. (1973). Isoelectric focusing of human c~-fetoprotein: An aid in purification and characterization of microheterogeneity. Ann. N. Y. Acad. Sci. 209:387. Appella, E., and Markert, C. L. (1961). Dissociation of lactate dehydrogenase into subunits with guanidine hydrochloride. Bioehem. Biophys. Res. Commun. 6:171. Chamoles, N., and Karcher, D. (1970a). Corr61ation entre l°enzymogramme classique de la lacticoddshydrogdnase et son fractionnement par isoelectrofocusing en gel d'acrylamide. Clin. Chim. Acta 30:337. Chamoles, N., and Karcher, D. (1970b). Isoelectro-focusing en acrylamide de la lacticod6shydrog6nase hydrosoluble de tissus humains. Clin. Chim. Acta 30:359. Costello, L. A., and Kaplan, N. O. (1963). Evidence for two forms of M-type lactate dehydrogenase in the mouse. Biochim. Biophys. Acta 73:658. Dale, G., and Latner, A. L. (1968). Isoelectric focusing in polyacrylamide gels. Lancet 1:847. Domenech, C. E., and Blanco, A. (1967). Alpha-hydroxy acid oxidases in subcellular fractions from rat kidney. Biochem. Biophys. Res. Commun. 28:209. Dudman, N. P. B., and Zerner, B. (1973). Lactate dehydrogenase: Electrophoretic behaviour, electron microscopy and structure. Biochim. Biophys. Acta 3!0:248. Fawcett, J. S. (1968). Isoelectric fractionation of proteins on polyacrylamide gels. FEBS Letters 1:81. Fritz, P. J., and Jacobsen, K. B. (1965). Multiple forms of lactate dehydrogenase. Biochemistry 4:282. Hitzeroth, H., Klose, J., Ohno, S., and Wolf, U. (1968). Asynchronous activation of parental alleles at the tissue-specific gene loci observed in hybrid during early development. Biochem. Genet. 1:287. Houssais, J. F. (1966). Transformations molecutaires au niveau des isoenzymes de la lacticodehydrogenase de la souris, raises en evidence par electrophorese en gel d'amidon. Biochim. Biophys. Acta 128:239. Kaplan, L. J., and Foster, J. F. (1971). Isoelectric focusing behaviour of bovine plasma albumin, mercaptalbumin, and fl-lactoglobulins A and B. Biochemistry 10:630. Klose, J. (1975). Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissue: A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231. Klose, J., Wolf, U., Hitzeroth, H., Ritter, H., Atkin, N. B., and Ohno, S. (1968). Duplication of the LDH gene loci by polyploidization in the fish order Clupeiformes. Humangenetik 5:190. Klose, J., Hitzeroth, H., Ritter, H., Schmidt, E., and Wolf, U. (1969). Persistence of maternal isoenzyme patterns of the lactate dehydrogenase and phosphoglucomutase system during early development of hybrid trout. Biochem. Genet. 3:91.
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Kohnert, K. D., Schmidt, E., Ziihlke, H., and Fiedler, H. (1973). Isoelectric focusing of insulins in polyacrylamide gel. J. Chromatog. 76:263. Lather, A. L. (1973). Some clinical biochemical aspects of isoelectric focusing. Ann. N. Y. Acad. Sci. 209:281. Lebherz, H. G. (1974). On the regulation of lactate dehydrogenase isoenzyme concentrations in mammalian cells. Experientia 30:655. Lindy, S. (1974). Synthesis and degradation of rat liver lactate dehydrogenase M4. J. Biol. Chem. 249:4961. Markert, C. L. (1963). Lactate dehydrogenase isozymes: Dissociation and recombination of subunits. Science 140:1329. Markert, C. L. (1971). Isozymes and cellular differentiation. In Rasp6, G. (ed.), Advances in the Biosc&nces, Vol. 6, Pergamon Press, New York, pp. 511-526. Markert, C. L., and Ursprung H. (1962). The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Develop. Biol. 5:363. Maurer, H. R., ed. (1971). Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, 2nd ed., Walter de Gruyter, New York, pp. 44-45. O'Carra, P., and Barry, S. (1972) Affinity chromatography of lactate dehydrogenase. FEBS Letters 21:281. Ohno, S., Wolf, U., and Atkin, N. B. (1968). Evolution from fish to mammals by gene duplication. Hereditas 59:169. Ohno, S., Stenius, C., and Christian, L. C. (1970). Sex differences in alcohol metabolism; androgenic steroid as an inducer of kidney alcohol dehydrogenase. Clin. Genet. 1:35. Roy, K. L., and Ruddle, F. H. (1973). Microscale isoelectric focusing studies of mouse and human hypoxanthine-guanine phosphoribosyl transferases. Biochem. Genet. 9:175. Ryan, L. D., and Vestling, C. S. (1974). Rapid purification of lactate dehydrogenase from rat liver hepatoma: A new approach. Arch. Biochem. Biophys. 160:279. Shaklee, J. B., Kepes, K. L., and Whitt, G. S. (1973). Specialized lactate dehydrogenase isoenzymes: The molecular and genetic basis for the unique eye and liver LDHs of teleost fishes. J. Exp. Zool. 185:217. Spielmann, H., Erickson, R. P., and Epstein, C. J. (1973). The separation of lactate dehydrogenase X from other lactate dehydrogenase isozymes of mouse testes by affinity chromatography. FEBS Letters 35:19. Spielmann, H., Erickson, R. P., and Epstein, C. J. (1974). Immunochemical studies of lactate dehydrogenase and glucose-6-phosphate dehydrogenase in preimplantation mouse embryos. J. Reprod. Fert. 40:367. Stambaugh, R., and Buckley J. (1967). The enzymic and molecular nature of lactic dehydrogenase subbands and X4 isoenzyme. J. Biol. Chem. 242:4053.. Urushizaki, I., Ishitani, K., and Niitsu, Y. (1973). Microheterogeneity of rat liver ferritin: Comparison of electrofocusing and chromatographic fractions. Biochim. Biophys. Acta 328:95. Weller, D. L., Heaney, A., Franceschi, R. T., Boudreau, R. E., and Shaw, D. E. (1973). Isoelectric focusing and study of ribosomal proteins and lactate dehydrogenase. Ann. N. Y. Acad. Sci. 209:258. Wellner, D., and Hayes, M. B. (1973). Isoelectric focusing in polyacrylamide gels. Ann. N. Y. Acad. Sci. 209:34. Whitt, G. S., Miller, E. T., and Shaklee, J. B. (1973). Developmental and biochemical genetics of lactate dehydrogenase isozymes in fishes. In Schr6der, J. H. (ed.), Genetics and Mutagenesis offish, Springer, New York. Williamson, A. R., Salaman, M. R., and Kreth, H. W. (1973). Microheterogeneity and allomorphism of proteins. Ann. N. Y. Acad. Sci. 209:210. Wrigley, C. (1968). Gel electrofocusing--A technique for analyzing multiple protein samples by isoelectric focusing. Sci. Tools 15:17. Zinkham, W. H., Blanco, A., and Kupchyk, L. (1963). Lactate dehydrogenase in testis: Dissociation and recombination. Science 142:1303.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase: Heterogeneity of the Isoenzymes A4 and X, Joachim Klose 1 and Horst Spielmann 1
Received 1 Apr. 1975--Final29 Apr. 1975
L D H of mouse organs (including testis) was investigated by isoelectric focusing in polyaerylamide gels. The number of L D H bands in this pattern considerably exceeds the five (six in testis) of the standard electrophoretie pattern. An attempt was made to identify these bands as tetramerie isoenzymes formed by random association of different subunits. This included isoelectrie focusing of purifiedLDH A, B, and X, two-dimensional separation of LDH, urea treatment of LDH, staining with specific substrates, and comparison of different organs. Further experiments were performed to exclude artifacts possibly produced by the isoelectrie focusing technique. Different strains of mice were also investigated. The results demonstrate that in addition to the common five L D H bands (A4-B4) one set of five bands is formed by L D H A (A~-A~) and another one by L D H X (X~-X~). Moreover, an unusual band was found which has a lower molecular weight and no affinity to the other isoenzymes. The data suggest that the heterogeneity of the L D H pattern revealed by isoelectric focusing arises from post-transcriptional events rather than from a number of additional genes. KEY WORDS: LDH A, B; LDH X; isoenzymes; mouse; isoelectric focusing.
INTRODUCTION
The development of the isoelectric focusing technique in polyacrylamide gel (Wrigley, 1968; Fawcett, 1968) led to reinvestigations of isoenzymes and This work was supported by grants of the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29 "Embryonale Entwicklung und Differenzierung." Pharmakologisches Institut der Freien Universit~it Berlin, Abteilung Embryonal-Pharmakologie, Berlin, West Germany. 707 © 1975 Plenum Publishing Corporation, 227 West 17th Street, N e w York, N.Y. 10011. N o part o f this publication m a y be reproduced, stored in a retrieval system, or transmitted, in any f o r m or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher.
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proteins with known electrophoretic patterns because in several instances this technique yielded a higher number of bands as a result of its improved resolving power compared to polyacrylamide or starch gel electrophoresis (Dale and Lather, 1968; Kohnert et al., 1973; Wellner and Hayes, 1973). The lactate dehydrogenase (LDH, E.C. 1.1.1.27) isoenzymes are widely used in genetics to elucidate problems in different fields, e.g., in developmental genetics (Markert and Ursprung, 1962; Klose et al., 1969; Spielmann et al., 1973) and in the field of phylogenesis (Ohno et al., 1968; Klose et al., 1968; Whitt et al., 1973). However, only little information exists on the pattern of LDH produced by the gel isoelectric focusing technique. More than five bands have been demonstrated (Dale and Latner, 1968; Chamoles and Karcher, 1970a,b), but no attempts have been made to explain this pattern on the basis of tetrameric isoenzymes formed by random association of different subunits. In this study, the gel isoelectric focusing patterns of LDH of several mouse organs are presented and an attempt is made to identify the isoenzyme bands that were found. MATERIALS AND METHODS Experimental Animals
In most experiments, mice of the NMRI strain (Schwenke & Co., Nauheim, Germany) were used and several organs (heart, muscle, liver, kidney, brain, eye, testis) were investigated. In addition, organs (heart, liver, testis) of mice of the following strains were investigated: C57BL/6J, DBA/2J, AKR/J (Jackson Laboratory, Bar Harbor, Maine), and BALB/c (Zentralinstitut ftir Versuchstierkunde, Hannover, West Germany). Isoelectric Focusing
Isoelectric focusing was performed in polyacrylamide gel rods or slab gels. The preparation of the gel rods and the focusing procedure were carried out as described previously (Klose, 1975). Slab gels were cast in glass cells purchased from Ortec GmbH (Munich, West Germany) and the same gel solution was prepared as for gel rods with the exception that 6% (w/v) acrylamide, 0.15% (w/v) bisacrylamide, and 0.04% (w/v) ammonium persulfate were used. With the Ortec 4100 pulsed constant power supply, the power was regulated for focusing in slab gels as follows: 30 rain 30 V, 2 hr, 60 V, 4 hr 120 V; at a pulse rate of 100 pps and at 1/~farad. At the end of the run, the power was raised to 200 V, 200 pps for 2 rain and 300 V, 300 pps for 2 rain.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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When rough tissue extracts were used as sources of LDH, the samples were prepared as described by Klose (1975). Purified LDH was applied to the gels in 0.03 M phosphate buffer, pH 7.2, containing 20% (w/v) sucrose and 1% (w/v) Ampholine (LKB, Bromma, Sweden). The gels were stained for LDH activity with the following solution: 1.0 g sodium lactate, 0.014 g NAD, 0.006 g nitroblue tetrazolium, and 0.004 g phenazine methosulfate in 100 ml 0.05 M tris-HCt buffer, pH 7.5. Sodium lactate was replaced by 1.0 g sodium valerate when the testis-specific isoenzyme (LDH X) was investigated and by 0.5 ml ethanol for the investigation of alcohol dehydrogenase (ADH, E.C. 1.1.1.1). The gels were stained for protein with Amido Black after washing in 12% (w/v) trichloroacetic acid. Two-Dimensional Separation of L D H
Isoelectric focusing followed by electrophoresis was carried out according to Klose (1975). Dissociation of LDH into Subunits
The dissociation of purified LDH A was performed with 12 M urea according to Appella and Markert (1961). The enzyme was dissolved in 0.01 Mphosphate buffer, pH 7.2, containing 12 M urea and 0.1 M mercaptoethanol, and this solution was applied to the gels for isoelectric focusing after a 1-hr incubation period at 37 C. The gels used for isoelectric focusing contained 6 M urea and the electrode solutions 6 M urea and 0.1 M mercaptoethanol. Isolation of the Mouse LDH Isoenzymes A, B, and X
The first step of the isolation of LDH A and B was the separation of total LDH from all other proteins of mouse liver and heart, respectively, by affinity chromatography according to O'Carra and Barry (1972) in the variation described by Spielmann et al. (1973). LDH A was isolated from total mouse liver LDH by chromatography on DEAE-Sephadex A50 in 10 m~ tris-HCl, pH 8.6, according to Ryan and Vestling (1974). LDH B was isolated from total mouse heart LDH by ion exchange chromatography on DEAE-Sephadex A50 in 0.05 M tris-HC1, pH 7.5, by a linear salt gradient up to 0.3 M NaCI. Pure LDH B is eluted in the fractions containing 0.20-0.23 M NaC1 as described by Lindy (1974). LDH X was separated from the other LDH isoenzymes of mouse testis by affinity chromatography as previously described (Spielmann et al., 1973). The fractions containing LDH X are contaminated by other proteins after this step. Purified LDH X was isolated from these fractions by chromatography on DEAE-Sephadex under the
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conditions outlined above for LDH A. The contaminating proteins are adsorbed by the column, whereas LDH X is not bound by the ion exchanger. Purified mouse LDH A, B, and X were not contaminated by other LDH isoenzymes or proteins as revealed by the appearance of only one band after polyacrylamide gel electrophoresis of the isoenzymes and staining for both LDH activity and proteins (Spielmann et al., 1973). Isolated LDH isoenzymes of such properties are designated as pure. RESULTS
Figure 1 shows the LDH banding patterns of different mouse organs after isoelectric focusing and includes a scheme that summarizes the different bands which were found. It is obvious that the number of bands demonstrated considerably exceeds the five usually obtained in the electrophoretic patterns of mouse LDH. According to the scheme in Fig. 1, the focusing pattern of LDH shows the two regular homomeric isoenzymes A4 ( = A 4~) and B4, creating the three heteromers, A~B, A2~B2, A1Ba, and in addition a third homomeric isoenzyme, called A~, which recombines with A~. An additional series of five bands was found in mouse testis and designated as X 4--1 X 4"2 Furthermore, two main bands are visible which do not recombine
2
a
b
c
d
e
f
g
I
! i
3
Ii
A : 54 ; 3
I
h
Fig. 1. Lactate dehydrogenase of mouse organs as revealed by gel isoelectric focusing of rough extracts (4.8~ polyacrylamide gel, Ampholine p H 3.5-10); 5 ttl undiluted tissue sap was applied to each gel. The gels were stained for lactate dehydrogenase
activity. (a) Brain, (b) eye, (c) testis, (d) muscle, (e)liver, (f) kidney, (g) heart. (h) Schematic recapitulation of all different bands found in the mouse organs; dotted lines indicate irregular bands.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
711
with any other bands. One of these bands is present only in liver and more weakly in kidney. It is occupying the most basic position of all bands and could be identified as ADH. The other band lies between LDH A~ and B4 and seems to be present in all organs. This enzyme shows high activity in kidney and it was therefore called K. In addition, some weak bands appear regularly (see Fig. 1, muscle, below A4z) or less permanently (see Fig. 1, dotted lines). We performed a number of experiments to verify the classification of LDH bands indicated in Fig. 1. The results are presented in the following sections. LDH
B4
Pure LDH B from mouse heart appears as a single band in the gel after isoelectric focusing. The position of this band coincides with the top band of the heart pattern (Fig. 2). Comparing different organs, this band shows the highest activity in heart extracts like LDH B4. The five isoenzymes resulting A4~
A~
B4
A; A~
i iijili!il ¸¸i¸¸ !i
ADH
a
b
c
d
e
Fig. 2. Purified mouse lactate dehydrogenase after isoelectric focusing. Samples of equal lactate dehydrogenase activity were applied to each gel. For further information, see caption of Fig. 1. (a) Purified lactate dehydrogenasc A. (b) Lactate dehydrogenase pattern from rough heart extract. (c) Purified lactate dehydrogenase B. (d) Purified lactate dehyrogenase A was focused in the usual manner and thereafter refocused in the second dimension in a 6 ~ polyacrylamide slab gel, AmpholinepH 3.5-10. (e) Isoelectric focusing of rough liver extract and staining of the gel for alcohol dehydrogenase (ADH) activity.
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Klose and Spielmann
o ~
A'
B~e
a
__e
o0
,
eK
b
Fig. 3. Two-dimensional lactate dehydrogenase pattern obtained by isoelectric focusing (see caption of Fig. 1) followed by disc electrophoresis (standard procedure, Davis, cited by Maurer, 1971) and staining for lactate dehydrogenase activity. (a) Photograph of the lactate dehydrogenase pattern of mouse heart. (b) Filled circles, schematic drawing of the pattern shown in Fig. 3a; open circles, positions of the lactate dehydrogenase X-spots and the spots of the lactate dehydrogenase bands below A] (see muscle) are indicated in the same scheme, although the photograph presents only the heart pattern.
from the recombination of LDH B4 and A] subunits are indicated in Fig. 1 and are most clearly demonstrable after two-dimensional separation of heart LDH (Fig. 3).
LDH A~-A~ When pure mouse LDH A was subjected to isoelectric focusing, five bands were found. These bands correspond to five bands which can be identified in the pattern of all mouse organs and which consist of LDH A[ and four additional bands that are located in direction to the basic pole of the gel (Fig. 2). Five bands were also found when commercial rabbit LDH A (Boehringer, Mannheim, West Germany) was investigated by the same procedure. The five mouse LDH A bands could not be clearly separated by the DEAE-Sephadex chromatography described above, but when several successive fractions of the column were investigated separately by isoelectric focusing a shift of the LDH A pattern from the lowest band to the top band occurred (Fig. 4). According to Appella and Markert (1961), LDH dissociates into its subunits when exposed to urea and mercaptoethanol. The degree of dissociation depends on the concentration of the detergent and the duration of incubation. If the five bands of LDH A in our experiments result from the recombination of two different types of LDH A subunits, only two bands
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
713
o.3j
Fig. 4. Lactate dehydrogenase A was isolated from mouse liver by affinity chromatography and DEAE-Sephadex chroma- o.D. tography. Different fractions from the DEAE-Sephadex column were subjected to isoelectric focusing (see caption of Fig. 1.) Aliquots of equal lactate dehydrogenase activity were applied to the gels. The banding patterns were scanned with the Gilford spectrophotometer 2400S at a wavelength of 550 nm. Unbroken line, scanning curve of lactate dehydrogenase pattern from fraction 8; dotted line, scanning curve of lactate dehydrogenase pattern from fraction 7.
i~
t A2
f AI
MIGRATION DISTANCE
s h o u l d be f o u n d in the focusing p a t t e r n after dissociation o f purified L D H A. F i g u r e 5 shows the result o f this experiment. U n t r e a t e d L D H A has five b a n d s ; L D H An is present in a high c o n c e n t r a t i o n (staining intensity), L D H An in a low concentration. A f t e r dissociation with urea, the same L D H A shows high concentrations for L D H A~ a n d A~ a n d between these b a n d s two b a n d s (A~ A~ a n d A I A ~ ) with relatively low concentrations. W h e n the t r e a t m e n t o f L D H A was carried o u t at higher t e m p e r a t u r e s a n d over longer p e r i o d s o f time, the two h e t e r o m e r i c isoenzymes d i d n o t d i s a p p e a r completely. C o m p l e t e dissociation o f L D H A into two different subunits therefore was n o t achieved u n d e r the conditions used. The results indicate, however, t h a t after t r e a t m e n t with urea a n d m e r c a p t o e t h a n o l two different types o f L D H A h o m o m e r s have a c c u m u l a t e d while the concentrations o f the h e t e r o m e r s have decreased. In conclusion, the isoelectric focusing p a t t e r n o f mouse L D H A creates
0.4-
Fig. 5. Purified lactate dehydrogenase A was treated with 12 M urea and 0.1 M mercaptoethanol and subjected to isoelectric focusing (see caption of Fig. 1) in the presence of 6 M urea in the polyacrylamide gel. The lactate dehydrogenase bands were stained with Amido Black and scanned (see caption of Fig. 4) at a wavelength of 620 nm. An aliquot of the same lactate dehydrogenase sample was focused without treatment and stained and scan.ned, as indicated above. Unbroken line, scanning curve of focused, urea-treated lactate dehydrogenase A; dotted line, scanning curve of focused, untreated lactate dehydrogenase A.
O.D.
/
/ i
I' A~
i
t
~
tl
1' A,~
MIGRATION DISTANCE
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Kiose and Spieimann
an additional LDH isoenzyme which is considered as a homomer and situated close to LDH A~ and therefore is designated A]. After two-dimensional separation of total heart LDH, LDH A41 and A~ appear on the same diagonal line as LDH B4 (Fig. 3). This can be expected if under the given experimental conditions three proteins differing in their isoelectric points have similar molecular sizes. The LDH bands A~-A] show high activity in muscle and liver, as expected for LDH A and relatively low activity, e.g., in heart. Comparing the activity of LDH A~ and A~ in several organs, LDH A42 generally has higher activities than A4~, but the relation between A] and A42 activity changes from one organ to another (Fig. 1). The introduction of artifactual bands into the protein pattern by the isoelectric focusing technique seems to be rather unlikely for a number of proteins according to previous investigators (Alpert et aI., 1973; Roy and Ruddle, 1973; Urushizaki et al., 1973; Wellner and Hayes, 1973). In a few cases, however, evidence for artifacts was found (Kaplan and Foster, 1971). The fact that the DEAE-Sephadex chromatography of LDH A generates fractions containing LDH A] and A 2 in different relative amounts clearly shows that the heterogeneity of LDH A already exists before isoelectric focusing is performed. The result of urea treatment of LDH A also is not consistent with the existence of artifactual LDH A bands. The occurrence of several bands also with purified LDH A excludes the possibility that these bands are due to complexing with tissue proteins during focusing (Lather, 1973). Three additional experiments were carried out to elucidate the possible production of artifacts by the focusing technique. First, purified LDH A and total heart LDH were subjected to isoelectric focusing and thereafter the gels were put on slab gels and refocusing was performed in the second dimension. No further LDH spots were observed compared with the onedimensional focusing pattern (Fig. 2). Second, rough heart LDH was separated in the presence of increased amounts of Ampholine in the separation gel (1%, 2%, and 3% Ampholine). The number and position of the bands did not change under these conditions, indicating that complexing of LDH isoenzymes with Ampholine (Latner, 1973) is unlikely. Finally, when the sample (heart extract) to be investigated for LDH was mixed with the total separation gel solution or was applied to the gel on the basic end, the same pattern was obtained as by the regular procedure of sample application. Apparently, the migration of the enzyme through either the aniodic or the cathodic part of the pH gradient did not affect the focusing pattern of the isoenzymes. L D H X 41- - X 42
When LDH of rough testis homogenate was investigated by isoelectric
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
715
focusing, the highest activity occurred in five bands which are situated below L D H A z and which could only be found in testis (Figs. 1 and 6). Purified L D H X shows five bands at the same position as the five main bands of total testis L D H (Fig. 6). Furthermore, only these five bands of the total testis L D H pattern appear when valerate is used as substrate (Fig. 6), which is characteristic of mouse L D H X (Allen, 1961). Therefore, mouse L D H X can be separated from L D H A by the gel isoelectric focusing technique, and it consists of five bands. Total testis L D H was separated by isoelectric focusing followed by electrophoresis in the second dimension. The pattern shows that the five spots characteristic of L D H X are situated somewhat below the line derived from the spots of L D H B4, A~, and A4z (Fig. 3). This may indicate a difference in molecular size between the testis-specific L D H and the common LDH, but it also demonstrates that in the mouse the five testis-specific spots do not result from a recombination of L D H X4 and A~ subunits. Apparently, mouse L D H X is formed by two homomeric isoenzymes X[ and X4z. The L D H patterns of muscle and liver also show a few bands below L D H A ,2 (Fig. 1). These bands are weak and do not occupy the same position as the L D H X bands when run side by side in a slab gel or when compared after two-dimensional separation (Fig. 3). In addition, these bands do not
B4
B4
A,~ A~
A~ X,~
xJ x~
X~
a
b
x~
c
d
e
f
g
h
i
Fig. 6. Mouse lactate dehydrogenase X after isoelectric focusing in gel rods (a-c) (see caption of Fig. 1) or slab gels (d-i) (see caption of Fig. 2d). (a) Lactate dehydrogenase pattern from rough testis extract. (b) Purified lactate dehydrogenase X. (c) Lactate dehydrogenase pattern from rough testis extract, staining for enzyme activity with sodium valerate as substrate. (d-i) Lactate dehydrogenase pattern from testis and heart extracts of different strains of mice: (d) heart DBA/2J, (e) testis DBA/2J, (f) testis C57BL/6J, (g) testis AKR/J, (h) testis BALB/c, (i) heart BALB/c.
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Klose and Spielmann
react with valerate. Thus by use of the isoelectric focusing technique LDH X could not be found in organs other than in testis. A D H Band and Enzyme Band K
The most basic enzyme band in liver and kidney homogenates (Fig. 1) had a much higher activity when lactate was replaced by ethanol in the reaction mixture and was therefore identified as ADH (Fig. 2). ADH reacts with the LDH staining mixture when acrylamide is contaminated with alcohol (Hitzeroth et al., 1968) during the recrystallization procedure. Close to the A~ Bz isoenzyme band, another single band was found (band K) (Fig. 1) which shows an unusual behavior when separated by electrophoresis after isoelectric focusing. It migrates much faster than all other spots (Fig. 3), indicating a comparatively low molecular weight. This enzyme could therefore be either a variant LDH isoenzyme like LDH X or a derivative of LDH B4, since it could be observed only in the presence of LDH B4. Additional L D H Bands
Some of the bands demonstrated by isoelectric focusing of mouse LDH tend to split into double bands. This was sometimes observed for B4 after the procedure of purification and may be the explanation for double bands like A~ B2 or A~ B. One of the two bands may become inactive since in a few instances double bands could be detected after protein staining of purified LDH isoenzymes but not when the same sample was investigated for LDH activity. However, the weak bands between A4z and B4 could in part arise from association of the subunits of these two isoenzymes. Four weak bands appear regularly below the isoenzyme A] in the pattern of muscle and liver LDH. Isoeleetric Focusing Patterns of L D H Isoenzymes of Different Strains of Mice
Heart, liver, and testis LDH of the four strains of mice indicated above was investigated by isoelectric focusing in slab gels. No variant LDH isoenzyme bands were found. The detection of a variant would show whether bands like A42 and X4z are genetically determined or the result of posttranscriptional alterations. DISCUSSION
The electrophoretic pattern of five LDH isoenzymes in mammalian tissues
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
717
results from the random association of the subunits of the two homomeric isoenzymes A4 and B4 governed by two genes (Markert, 1963; Lebherz, 1974). In several species, a testis-specific isoenzyme C4 (LDH X) was found which gives rise to one additional LDH band in mouse testis and to a third locus for LDH (Zinkham et al., 1963). Besides these major bands, however, under certain electrophoretic conditions multiple subbands were observed by several authors. In some cases, a total number of 15 bands was found in the electrophoretic pattern of mouse LDH from somatic tissues (Costello and Kaplan, 1963; Fritz and Jacobsen, 1965). This pattern was explained by the assumption of three homomeric isoenzymes, suggesting the existence of three cistrons for LDH (besides LDH X) (Costello and Kaplan, 1963). However, the total number of bands and their relative position observed by other investigators could not be explained on the basis of three homomeric LDH isoenzymes. These findings were interpreted by the existence of several conformations of the enzyme (Houssais, 1966), by LDH tetramers formed by hybrid subunits (Stambaugh and Buckley, 1967), or, as another possibility, by the binding of metabolites of low molecular weight to the enzyme (Dudman and Zerner, 1973). In our study, the heterogeneity of mouse LDH was investigated by the gel isoelectric focusing technique and the results show that in addition to the common LDH isoenzymes two distinct sets of five LDH isoenzyme bands can be demonstrated. One of these sets was found in all organs and was interpreted on the assumption that LDH A appears in two homomeric forms A~ and A 2 generating five isoenzymes by recombination of its subunits. The other group of five bands could be found only in testis homogenates and was interpreted as derived from two isoenzymes X~ and X~. A number of experiments such as focusing of pure LDH A, B, and X, urea treatment of LDH A, and two-dimensional separations of LDH according to different biochemical properties of the isoenzymes (isoelectric point and molecular size) are in good agreement with our interpretation. The observation that treatment of LDH A with urea did not result in a complete separation into two bands may demonstrate that even the highest concentration of urea which can be used is not strong enough to reach complete dissociation of the subunits. However, the relatively weak bands between A~ and A~ could also be explained as artifacts resulting from the urea treatment of LDH A. The possibility that artifactual bands are introduced by the focusing technique could be excluded to a high degree by additional experiments. However, the tendency of certain bands to split into double bands may indicate an artifactual effect. An instability of LDH B after isoelectric focusing was reported by Weller et al. (1973). This finding explains our observation that LDH B4 shows comparatively low activity in the gel focusing pattern (see heart, Fig. 1).
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Our results partly support the results of other investigators, that three homomeric LDH isoenzymes exist in the somatic tissues of mammals resulting in 15 bands (Costello and Kaplan, 1963; Fritz and Jacobsen, 1965). The isoelectric focusing pattern also shows three homomers; however, the third isoenzyme, designated as A 2 (Fig. 1), does not react (or only in a very restricted way) with B4 to form the heteromeric isoenzymes, whereas three distinct bands appear between A~ and B4 and between A 2 and A~. Assuming an extra gene locus for A 2, this gene may have become too different from the B gene during phylogenesis to allow an interaction between the two primary gene products. A diminished affinity between LDH A and B subunits has been reported for other organisms (Klose et al., 1968). However, to explain the complete focusing pattern of LDH on a genetic basis at least three additional genes have to be postulated, one for A 2, one for X 4, 2 and one to interpret the four bands below A 2 in the pattern of muscle LDH. A simpler explanation, in our view, interprets the heterogeneity of LDH as a result of post-transcriptional events. Such mechanisms were recently reviewed by Williamson et al. (1973). A secondary modification of LDH AL resulting in LDH A 2, may restrict or exclude interactions with LDH B4. Isoelectric focusing of LDH X has not been performed so far by other investigators, and electrophoretic studies of mouse LDH X have revealed only one band. Therefore, the demonstration of five distinct bands for mouse LDH X by isoelectric focusing is an unexpected finding. It may be interpreted in the same way as the heterogeneity of LDH A. The results of our investigations on different inbred strains of mice exclude the possibility of allelic cistrons but not of nonallelic cistrons causing the heterogeneity of LDH A and X. It is interesting to note that in certain lower vertebrates genetic evidence exists for the occurrence of at least five gene loci for LDH (Klose et al., 1968; Shaklee et al., 1973). The presence of one ADH band in the focusing pattern of mouse liver and kidney is consistent with the electrophoretic pattern of ADH (Ohno et al., 1970). However, another single band (K), which has not been previously described, could be demonstrated by the isoelectric focusing technique and especially by its combination with electrophoresis. This band is not identical with the kidney ~-hydroxy acid oxidase which produces an additional band in the LDH isoenzyme pattern of rats (Domenech and Blanco, 1967), since mouse kidney homogenates exhibit band K only in the presence of the coenzyme NAD. The enzyme band K shows no reassociation with other isoenzymes in the LDH pattern and its molecular weight seems to be much lower than that of normal LDH isoenzymes. We investigated the LDH of mouse preimplantation embryos by isoelectric focusing, and unexpectedly the only isoenzyme band which could be found in oocytes is situated at the same position as band K (Spielmann and Klose, unpublished observations). This observation is still under investigation.
Gel Isoelectric Focusing of Mouse Lactate Dehydrogenase
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W e have shown t h a t m o u s e L D H X is clearly separable f r o m L D H A b y isoelectric focusing. F u r t h e r experiments should show whether L D H X can be detected b y this technique in animals in which this enzyme c o u l d n o t be f o u n d b y electrophoresis ( M a r k e r t , 1971). F u r t h e r m o r e , i m m u n o c h e m i c a l studies a n d genetic investigations o f wild p o p u l a t i o n s o f mice will be p e r f o r m e d to elucidate the origin o f the L D H isoenzymes A4z a n d X 2 detected by gel isoelectric focusing o f mouse tissues.
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