l‐α‐Hydroxyacid Oxidase Isozymes

Abstract

l ‐α‐Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 ± 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000. The total amino acid contents of the two rat isozymes were very similar and had identical tyrosine (9 residues per 40000) and tryptophan (5 per 40000) contents. No free suiphydryl groups were found following suiphydryl titration experiments using the purified native rat isozymes. Following denaturation with sodium dodecylsulphate, 2 (A subunit) and 3 (B subunit) suiphydryl residues were titratable. The apoenzyme form of rat isozyme B exhibited 2 of the 3 titratable suiphydryl groups in the absence of sodium dodecylsulphate which suggests an involvement of these residues in coenzyme binding. Flavin mononucleotide content was estimated from spectral studies to be I residue per subunit (40000 molecular weight) of native enzyme. Following storage in ammonium sulphate at 4°C for several months, flavin mononucleotide was irreversibly removed such that the rat A and B isozymes contained 0.3 and 0.7 residue of coenzyme per subunit. This accounts for the more pronounced loss of specific activity following storage for the A isozyme than the B isozyme as well as the comparatively high minimal molecular weights based upon flavin mononucleotide values previously reported by other workers. This evidence for chemical homology together with the previously reported immunochemical crossreactivity for these isozymes provide evidence that the A and B polypeptides are products of recently duplicated genes during evolution.