to produce about 128 peptides. The actual num- 

 ber of peptides found by this technique was about 

 thirty or one-fourth the expected number. This 

 result certainly reinforces the proposal that 

 LDH- 1 is a tetramer composed of four identical 



TABLE I 



Molecular Weights of Lactic Dehydrogenases Determined 

 in the Multichannel Short Column Equilibrium Cell, Using 

 Schlleren Optics 



LDH-1 

 i 



aOb* 



LDH- 2 



I 

 A^b3 



LDH- 3 



J. 

 A2b2 



Fig. 8. 



LDH-i* 

 i 



LDH- 5 

 1 



a'*bo 



Proposed subunit composition of the five major isozymes 

 of LDH. LDH-1 consists entirely of B subunitsandLDH-5 

 consists entirely of A subunits. The intervening iso- 

 zymes, LDH-2, -3, and -4, are the various combinations of 

 the A and B subunits. 



* a V of 0. 740 has been assumed in all calculations. 



TABLE II 



Amino acid composition of LDH isozymes from 

 beef muscle. 



Amino Acids 



Lysine 



Hist idine 



Arginine 



Aspartic Acid 



Threonine 



Serine 



Glutamic Acid 



Proline 



Glycine 



Alanine 



Valine 



Methionine 



Isoleucine 



Leucine 



Tyrosine 



Phenylalanine 



Number of amino acid residues per molecule of enzyme 

 LDH-1 LDH- 5 



94 

 25 



34 



123 

 56 

 92 



124 

 42 

 91 

 72 



135 

 32 

 86 



130 

 26 

 19 



95 

 34 

 52 



104 

 62 

 61 



135 

 63 



100 



122 

 82 

 20 

 73 



118 

 35 

 26 



Based upon a molecular weight of 135,000 (assuming 12 residues of cysteine 

 and 30 residues of tryptophan in each isozyme). 



subunits. Beef LDH-5 was subjected to the same 

 type of analysis and also yielded about thirty 

 peptides. A comparison of the peptide maps of 

 LDH-1 and LDH-5 of beef revealed that some 

 of the peptides were common to both of these 

 ioszymic forms, but most were clearly dif- 

 ferent. It may be concluded from these observa- 

 tions that the A and B polypeptides are related, 

 but long stretches of the primary structure must 

 be quite different. 



Perhaps the best test of a subunit hypo- 

 thesis of isozyme structure is the dissociation 

 of the active polymers into their constituent 

 monomers and reassociation of the monomers 

 into new active configurations. It was discov- 

 ered in our laboratory that this can be readily 

 achieved by freezing and thawing equal quan- 

 tities of LDH-1 and -5 in neutral phosphate buf- 

 fer which is one molar in NaCl (15). After 

 thawing, an aliquot of the preparation is analyzed 

 by electrophoresis in starch gel and subsequent 

 staining of the gel slab for LDH activity. Prepa- 

 rations treated in this manner show all five 

 isozymes in the proportions of 1:4:6:4:1, the 

 expected binomial distribution of isozymes as- 

 suming the A and B subnits associated in a 

 random manner. 



In contrast to the irreversible denaturation 

 obtained by treatment of LDH with urea or 

 guanidine, the salt-freezing technique is quite 

 mild. It seems possible that the subunits main- 

 tain their tertiary configurations essentially 

 intact during this mild dissociative procedure. 

 Although the salt-freezing technique is by far 

 the most efficient method for recombining iso- 

 zymes, saturated salt solutions in the absence 

 of freezing as well as repeated freezing and 

 thawing in buffer alone will gradually produce 

 recombination. The recombination of LDH is not 

 influenced by NAD and NADH, lactate, or 

 pyruvate, and is independent, within wide limits, 

 of the concentration of LDH, The optimum salt 



82 



