Functional non-identity of subunits and isolation of active dimers of D-Glyceraldehyde-3-phosphate Dehydrogenase

Ovádi, Judit and Telegdi, Marianna and Batke, József and Keleti, Tamás (1971) Functional non-identity of subunits and isolation of active dimers of D-Glyceraldehyde-3-phosphate Dehydrogenase. EUROPEAN JOURNAL OF BIOCHEMISTRY, 22. pp. 430-438. ISSN 0014-2956

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Abstract

Maleylation of about four groups per tetramer of NAD-free glyceraldehyde-3-phosphate dehydrogenase or of enzyme - NAD, complex inactivates the enzyme. However, enzyme - NAD, complex is inactivated only by maleylation of about 24 groups per tetramer. This shows that the presence of two moles of the relatively loosely bound coenzyme in addition to the firmly bound ones protects the tetramer against inactivation. Up to the maleybtion of about 12 groups per tetramer NAD-free enzyme, the reactive SH-groups do not react with maleic anhydride. Maleylation of the enzyme - NAD, complex up to the same extent does not affect the Racker band characteristic of the enzyme - coenzyme complex and the coenzyme remains firmly bound to the enzyme. However, after maleylation of NAD-free tetrameric enzyme only two NAD molecules can be rebound to the inactive enzyme. Four moles of ATP can be bound tightly to the NAD-free enzyme. Two of them can be bound within a few minutes, whereas the further two only after prolonged incubation. Binding of two moles of ATP to the NAD-free tetrameric enzyme in the absence of 2-mercaptoethanol results in the irreversible inactivation of the enzyme. In the presence of 2-mercaptoethanol the inhibition is reversible and of mixed type between ATP and NAD. The above data provide evidence for the functional non-identity and interaction of subunits. The dissociation of tetrameric enzyme is increased by ATP or maleic anhydride and this process is irreversible in the absence of 2-mercaptoethanol. An enzymically active dimeric form of the ATP-treated enzyme has been isolated in the presence of 2-mercaptoethanol. ATP treatment causes only a slight change in the steric structure of the protein as measured by difference spectrophotometry, showing that the dissociation into dimers is not accompanied necessarily by the unfolding of the molecule. Interaction was observed between active and inactive enzyme molecules, which suggests the dissociation of oligomer and recombination of subunits. In solution, the enzyme exists as a tetramer-dimer equilibrium mixture and both forms are enzymically active.

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