> 



< 



X 



< 



10-" 10"' 10"^ 



[pyruvate] 



Fig, 15. 



Pyruvate Inhibition curve for horse LDH-1 and -5 and 

 fluke LDH-1 and-5. Experiments were performed at 23° C 

 In 0.1 M sodium phosphate buffer, pH (apparent) 7.0. The 

 optimal pyruvate concentration for horse LDH- 5 Is higher 

 than that for horse LDH-1, Fluke heart and skeletal 

 muscle LDH, which are electrophoretlcally indistinguish- 

 able, have identical pyruvate optima. These optima are 

 similar to that of horse LDH- 5, 



Krebs cycle so that lactate cannot accumulate 

 in heart muscle. 



As previously mentioned, this correlation 

 extends to embryonic life. The tissues of mam- 

 malian embryos which have a relatively poor 

 oxygen supply contain large amounts of LDH- 5 

 whereas the well- oxygenated tissues of avian 

 embryos contain mainly LDH-1. 



A fundamental aspect of theinterconversion 

 of pyruvate and lactate as catalyzed by LDH is 

 the oxidation- reduction of nicotinamide adenine 

 dinucleotide. And this may be the most important 

 function of LDH, The maintenance of the proper 

 ratio of oxidized to reduced NAD is of con- 

 siderable importance in that NAD is involved 

 in numerous metabolic ractions. 



In conclusion then, since the intracellular 



environment must surely vary from place to 

 place from time to time within the cell, the 

 existence of a spectrum of functionally distinct 

 types of a particular enzyme would allow for a 

 more efficient and precise control of a metabolic 

 step. Since the discovery of the isozymes of LDH 

 more than 100 other enzymes have been shown, 

 at least tentatively, to exist in isozymic form. 



DEERING: Do the isozymes of LDH always 

 exist with four subcomponents? 



MASSARO: Yes, So far as we know. 



DEERING: Is this also true of some of the 

 enzyme systems other than LDH? Are there 

 always four or do you, perhaps, get combina- 

 tions of three subunits there? Is there any 

 reason to expect that they can't exist as dimers 

 or trimers in some systems? 



MASSARO: Isozyme systems other than the 

 LDH system may be constructed on a dimer or 

 trimer basis. The isozymes of MDH, malate 

 dehydrogenase, for example, are dimers. 



DEERING: You mentioned when you went 

 through it the first time that the whiting pattern 

 was very complex. Can you explain it in terms 

 of ^, B, and C subunits? 



MASSARO: This is quite possible. However, 

 we have not yet attempted the analysis. The 

 banding pattern in this fish may be related to the 

 subbanding in rabbit LDH. The multiple banding 

 may have something to do with permutations of 

 the tetramic structure of the individual iso- 

 zymes. Such permutations could conceivably 

 change the electrophoretic mobility of the ios- 

 zymes resulting in the very complex pattern 

 that we find. 



CANTINO: I have a question about the fish 

 story in general. Do you work exclusively with 

 frozen fish or freshly caught fish or mixtures 

 of the two? 



MASSARO: We use both fresh and frozen 

 fish and never mix them unless we are certain 

 that freezing has had no effect on the LDH iso- 

 zyme patterns. 



CANTINO: You stressed the importance of 

 freezing and thawing upon recombination. 



MASSARO: For tne most part, in intact 

 tissues, and let me stress intact tissues, not 

 homogenates, LDH is quite stable. Intact tis- 

 sues can usually be frozen and thawed without 

 altering their LDH patterns. In a very few 

 cases, however, we have seen entirely dif- 

 ferent patterns between frozen fish and fresh 

 fish and, I am sure, this can also occur with 

 tissues from other animal species. Some tis- 

 sues we have studied could not possibly have 

 been obtained fresh. For example, we have ob- 



88 



