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. 1988 Mar 1;250(2):429–434. doi: 10.1042/bj2500429

Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase.

J S Nishimura 1, J Ybarra 1, T Mitchell 1, P M Horowitz 1
PMCID: PMC1148874  PMID: 3281664

Abstract

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between 'strong' and 'weak'. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

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Selected References

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