H isozyme

The DNA sequence around the initiation ATG codon of the type-H isozyme cDNA is rather dissimilar to that of the type-L isozyme cDNA and the consensus sequence for plant genes proposed by Heidecker and Messing.69 The difference in the sequences of this region may be correlated with the difference in the expression levels of each isozyme in potato tuber. 

Amino acid sequence comparison of a-glucan phosphorylases. H, the potato type-H isozyme L, the potato type-L isozyme and R, rabbit muscle phosphotylase.791 The type-H isozyme sequence is used as the reference sequence only the amino acid residues that are nonidentical in the type-L isozyme and rabbit muscle enzyme are indicated. (From Biol. Chem., 266 (28), 18453 (1991)). 

Fig. 6.2 Spatial structure of the active site of rabbit muscle phosphorylase85 (A) and sequence comparison of the active site residues (B). A, Hydrogen bonds of less than 3.3 A (dotted lines) and water molecules (crosses) are also indicated. B, Sequences constituting the active-site region are compared among potato type-H isozyme (H), type-L isozyme (L), and rabbit muscle enzyme (R). Residues making van der Waals contact with the pyridoxal moiety and the 5 -phosphate group are boxed. (From J. Biol. Chem., 261 (18), 8233 (1986)).

Previously, it has been shown that most of the residues directly interacting with AMP as well as the phosphorylatable Ser14 and its surroundings in the rabbit muscle enzyme are far less conserved in the potato type-L isozyme sequence.63 Likewise, the amino-terminal region of the potato type-H isozyme is completely different from that of the rabbit muscle enzyme over the first 80 amino acid residues, in which the sites of covalent phosphorylation and of allosteric regulation by AMP are all included. These variances in sequence are compatible with the lack of regulation in the plant phosphorylase isozymes. 

Fig. 6.4 Affinity electrophoresis of the recombinant phosphorylases from potato tuber.,3) A crude bacterial cell extract containing the type-L isozyme (lane 1), the chimeric enzyme (lane2), or the type-H isozyme (lane 3) was electrophoresed in 5% polyacrylamide gels supplemented with 0 (A), 50 (B), and 500 pg/ml (C) glycogen. After electrophoresis, the gels were stained for enzyme activity in KI-b solution. (Reproduced with permission from Goldsmith and Fletterick, Pure and Appl. Chern., 55(4), 583 (1983)).

The plant type-L isozyme is the only phosphorylase that has a large insertion in the middle of the polypeptide chain the type-H isozyme has no such insertion, like other enzymes with or without regulatory properties. The sequence containing the insertion and... 

Figure 10.27. Isozymes of Lactate Dehydrogenase. (A) The rat heart LDH isozyme profile changes in the course of development. The H isozyme is represented by squares and the M isozyme by circles. The negative and positive numbers denote the days before and after birth, respectively. (B) LDH isozyme content varies by tissue. [(A) After W.-H. Li, Molecular Evolution (Sinauer, 1997), p. 283 (B) After K. Urich, Comparative Animal Biochemistry (S nngQrVQrlag, 1990), p. 542.]...

Eszes, R. B. Sessions, R. L. Brady, Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase, Proteins Struct., Puna., Genet., 43, 175-185 (2001). 

Hybridization studies between combinations of H and M subunits in vitro produce a binomial distribution of the five possible isozymes thus, for equal quantities of H and M subunits isozymes are formed in the proportion 1 4 6 4 1 (51,52). Subunit interchange can be induced by repeated freezing and thawing, dilution, or by mild denaturation. With increasing evolutionary distance the hybridization process becomes more difficult and the results more variable. It has been concluded (52) that the M chains from different species are more like each other than they are to the H chains of the same species. These conclusions are supported by chemical and immunological results (20,39,40,53,54). An antiserum against the M4 isozyme of one species will cross react with M4 isozyme from several species but not with its own H isozyme (36,54). The H isozyme is a much poorer immunogen than the M4 isozyme (55), and in some cases anti-H4 serum will cross react with M4 isozymes of other species (53,56,57). 

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