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  • Title: Cross-linking of Escherichia coli succinic thiokinase. I. Reaction with diiminoesters and dimaleimides.
    Author: Teherani JA, Nishimura JS.
    Journal: J Biol Chem; 1975 May 25; 250(10):3883-90. PubMed ID: 1092680.
    Abstract:
    Treatment of the tetrameric alpha2 beta2 protein succinic thiokinase of Escherichia coli with dimethylsuberimidate (DMS) yielded five protein species detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These five protein species had estimated molecular weight values of 29,500, 41,000, 73,000, 117,000, and 132,000, and corresponded to alpha monomer, beta monomer, alpha beta dimer, alpha2 beta trimer and alpha2 beta2 tetramer, respectively. In all cases, the cross-linking produced predominantly the 73,000 molecular weight dimer with respectively lesser amounts of the tetramer and trimer. Succinic thiokinase was also cross-linked by reaction with N, N'-o-phynylenedimaleimide or with N, N'-P-phenylenedimaleimide. In these instances, treatment produced the alpha beta dimer as the only oligomeric species. Ammonolysis of isolated tetramer, trimer, and dimer produced by DMS treatment gave the 29,500 molecular weight monomer (alpha monomer) and the 38,500 molecular weight monomer (beta monomer). The absence of dimers of like subunits and the predominance of the dimer of unlike subunits are consistent with a quaternary structure of the native enzyme in which unlike subunits are closely associated but like subunits are not. Under certain conditions, an additional dimer of approximately 60,000 molecular weight was produced. This appeared to result from cleavage of the beta chain of an alpha beta dimer. Phosphorylation of native succinic thiokinase with [gamma-32P]ATP and [gamma-32P]GTP showed radioactivity only in the alpha monomer. Phosphorylation of enzyme before or after cross-linking showed radioactivity in all cross-linked bands except the beta monomer. Experiments in which the enzyme was titrated with [14C]DMS and trinitrobenzenesulfonate revealed that approximately half of the available amino groups reacted with the diiminoester, but that a small fraction of these (smaller than 20%) had reacted bifunctionally
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