illustrated by comparing Fig. 5, the isozyme 

 patterns of various tissues of the Rhesus 

 monkey, with Fig. 3, the isozyme patterns of 

 the heart tissues of several other vertebrates. 

 The predominance of LDH-1 and LDH-2 in 

 heart muscle and LDH-5 and LDH-4 in skeletal 

 muscle suggests that the different isozymes have 

 different physiological roles. In general, tissues 

 possessing a highly aerobic metabolism (e.g. 

 heart, brain, kidney cortex) contain mainly the 

 most negatively charged isozymes, LDH-1 and 

 LDH-2, while those tissues possessing a high 

 anaerobic metabolism contain mainly LDH-5 

 and LDH-4. These observations seem to indi- 

 cate that the net charge on an isozyme mole- 

 cule may be important in determining its intra- 

 cellular localization and may thus be a reflec- 

 tion of its metabolic role. 



The stable isozyme pattern of adult tissues 

 must have arisen at some time during develop- 

 ment through a sequence of orderly changes. 

 This has been clearly demonstrated by the 

 direct analysis of tissues of the mouse at dif- 

 ferent stages of development (3) and is illus- 

 trated in Fig. 6 and 7. LDH-5 is the predomi- 

 nant isozyme in embryonic mouse tissues (Fig. 

 7). As development progresses the isozyme 

 pattern migrates, in effect, toward the anodal 

 end of the electrophoretic spectrum so that in 

 most tissues an increasing proportion of enzyme 

 activity becomes localized at the LDH-1 end 

 of the spectrum. Only in those adult tissues, 

 such as liver and skeletal muscle (Fig. 6), in 

 which LDH-5 is the predominant isozyme, is 

 the redistribution of enzyme activity during 

 development relatively insignificant. 



From these studies there was no indication 

 that the isozyme patterns of the different tis- 

 sues shift synchronously, and it is quite obvious 

 that all do not shift to the same extent. How- 

 ever, the direction of the shift, when it occurs 

 at all, is the same for all tissues. In some tis- 

 sues, such as mouse heart muscle, the change 

 in isozyme pattern was sufficiently rapid to 

 exclude a corresponding change in cell popula- 

 tion. Therefore it would seem that isozyme 

 patterns must change within individual cells. 

 It is interesting to note that LDH- 1 is the pre- 

 dominant isozyme in embryonic birds and that 

 during development, in contrast to the situation 

 in mammals, the isozyme patterns shift toward 

 the LDH-5 end of the spectrum so that in adults 

 the LDH patterns in homologous tissues of 

 birds and mammals are reasonably similar. 



From the standpoint of the one gene-one 

 enzyme hypothesis, the discovery of multiple 



molecular forms of enzymes presented per- 

 plexing problems concerning the genetic con- 

 trol of protein biosynthesis. To reconcile the 

 phenomenon of isozymes with this hypothesis, 

 it was proposed that a single gene controlled the 

 synthesis of a single protein which could be 

 folded into five alternative configurations each 

 possessing a different net charge. In order to 

 test this hypothesis an attempt was made in our 

 laboratory to reversibly unfold and refold the 

 LDH molecule. Beef heart LDH was treated 

 with urea or guanidine-HCl to disrupt the hydro- 

 gen bonds maintaining the characteristic tertiary 

 structure of the molecule. LDH is readily de- 

 natured and inactivated by these reagents but all 

 attempts to reactivate the molecule by removal 

 of the denaturing reagents were unsuccessful. 

 A study of the nature of the products of de- 

 naturation was then undertaken. Denaturation of 

 a preparation containing all five isozymes re- 

 sulted in the appearance of only two protein 

 bands following electrophoresis in acrylamide 

 gel. During the denaturation procedure, three 

 bands had disappeared. This unanticipated re- 

 sult combined with sedimentation data opened 

 the door to our present understanding of the 

 structure of LDH. From previous ultracentri- 

 fugal and other studies the molecular weight of 

 native LDH had been calculated to be about 

 135,000 and the 5 major isozymes were all 

 shown to possess identical molecular weights. 

 However, when the guanidine denatured prepa- 

 ration was analyzed in the ultracentrifuge, the 

 molecular weight was shown to be about 35,000 

 or approximately one-fourth that of the native 

 protein. This data is summarized in Table I. 

 The conclusion drawn from these results was 

 that LDH exists in the native state as a tetramer 

 composed of four equal sized (approx. M. W. = 

 35,000) subunits (10). As shown from acryla- 

 mide electrophoretic data these subunits exist 

 as two electrophoretically distinct species 

 designated A and B (11). It is obvious that ran- 

 dom assortment of the two kinds of subunits 

 into all possible combinations of four yields 

 five isozymes of the composition shown in Fig. 8. 

 Several tests can be performed to verify 

 the subunit hypothesis of LDH isozyme struc- 

 ture. This hypothesis assumes that LDH-1 con- 

 sists entirely of B subunits, while LDH-5 con- 

 sists of only A subunits. It follows that LDH- 1 

 and LDH-5 must be distinct protein species. 

 This could easily be verified by a complete 

 amino acid analysis of each of these isozymes. 

 Accordingly, both LDH-1 and LDH-5 were pre- 

 pared in pure form by electrophoresis of crys- 



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