concentration for attaining equilibrium recom- 

 bination ranges from 0.1 to 4.0 M. Concentra- 

 tions lower than 0. 1 A7 are much less effective. 

 It is of interest to note that only a few salts 

 promote recombination and that both cations 

 and anions play an important role in the process. 

 Among the effective cations are sodium, potas- 

 sium, lithium, magnesium, and zinc. Chloride, 

 bromide, iodide, nitrate and phosphate are 

 effective anions. Certain other ions, for ex- 

 ample, borate, sulfate, and tris, inhibit recom- 

 bination. 



From elementary genetic considerations, 

 since the A and B subunits of LDH are dif- 

 ferent proteins, they must be under the control 

 of different genes. Recent genetic evidence bears 

 this out. An LDH mutant has been discovered in 

 the deer mouse Peromyscus maniculatus (16). 

 In these animals, the mutation occurred at the B 

 locus, and, as theory predicts, the heterozy- 

 gote produced fifteen isozymes. During the 

 screening of several diverse human populations 

 mutants were found at either the ^ or B loci 

 (17). To our knowledge, no double heterozygotes 

 have yet been reported. 



A third gene controlling the synthesis of a 

 third type of LDH subunit, designated the C sub- 

 unit, was discovered by Zinkham and co- 

 workers (18). C polypeptides appear to be 

 formed mainly (perhaps exclusively) in the 

 sperm. Isozymes containing C polypeptides are 

 responsible for the so-called "X-bands" of LDH 

 activity found on zymograms of testis homo- 

 genates. In some mammals only one X-band is 

 observed and it is assumed to be a tetramer of 

 C subunits. Several X-bands have been detected 

 in testis homogenates of other mammals. How- 

 ever, in these cases it has been shown that the 

 additional bands are the result of the polymeri- 

 zation of C subunits with either ^ or B sub- 

 units (19). More recently, Zinkham and co- 

 workers have shown that, in pigeons, the C gene 

 exists in two widely distributed allelic forms 

 designated C and C' (20). From testicular 

 homogenates resolved by the technique of starch 

 gel electrophoresis, they have been able to 

 classify each pigeon into one of three phenotypic 

 classes designated CC, CC, and C'C. 



Although it is theoretically possible to form 

 fifteen isozymes from three different subunits 

 no such number has been observed in sperm 

 homogenates. The following interpretations of 

 this may be brought forth. It is possible that 

 the freedom of combination necessary for the 

 formation of the fifteen isozymes does not exist 

 or that the gene controlling the C polypeptide 



biosynthesis may be turned on only when the 

 A and B genes are turned off. It is also pos- 

 sible that certain hybrid molecules cannot be 

 formed for purely physical reasons or that 

 certain hybrid combinations are inactive. How- 

 ever, a mixture of a, B, and C subunits will 

 readily recombine in vitro to yield the expected 

 fifteen different tetramers. 



LDH zymograms of many different animals, 

 especially the rabbit, show that several of the 

 basic five isozymes exist as two or more distinct 

 bands of enzyme activity (Fig. 9). An entirely 

 satisfactory interpretation of the phenomenon 

 of subbanding is not yet available although 



(+) 



LDH- 





< 



a 



ORIGIN 



(-) 





Fig. 9. 



Zymogram of LDH patterns of various tissues of the 

 rabbit. Note the multiple banding, termed subbanding, of 

 most of the isozymes and the variation in subbanding 

 which exists among homologous isozymes of different 

 tissues. The subbanding Is relatively constant for any 

 particular species but varies considerably among dif- 

 ferent species. {From Markert, in The Harvey Lectures, 

 Series 59, 187, 1965; reproduced with permission of 

 Academic Press.) 



83 



