Identification of the Reacting Form of Pyruvate Carboxylase

A modification of the reacting enzyme sedimentation technique of Cohen et al. ((1967) Biopolymers 5, 203) has been used to investigate the sedimentation coefficients and probable quaternary structures of the reacting form of pyruvate carboxylase from several different sources. In this technique, the...

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Veröffentlicht in:The Journal of biological chemistry 1972-11, Vol.247 (22), p.7383-7390
Hauptverfasser: Taylor, Barry L., Barden, Roland E., Utter, Merton F.
Format: Artikel
Sprache:eng
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Zusammenfassung:A modification of the reacting enzyme sedimentation technique of Cohen et al. ((1967) Biopolymers 5, 203) has been used to investigate the sedimentation coefficients and probable quaternary structures of the reacting form of pyruvate carboxylase from several different sources. In this technique, the sedimentation of a lamellar layer of enzyme through a reaction mixture is measured by changes in absorption that are due to the appearance of a product or the disappearance of a substrate. In the present studies the method of Cohen et al. has been modified by the addition of malate dehydrogenase as a coupling enzyme, by the inclusion of 50% D2O in the reaction mixture to stabilize the sedimenting band of enzyme, and by the use of the photoelectric scanner accessory to measure the optical changes. The s20,w values for the reacting forms of pyruvate carboxylase from Pseudomonas citronellolis, yeast, and chicken liver have been determined by this procedure to be 12.9 S, 16.3 S, and 15.9 S, respectively. The validity of these results obtained with the coupled enzyme assay were confirmed by experiments in which the direct formation of oxalacetate was observed. The s020,w values which were obtained by sedimentation velocity studies of the protein of the same three varieties of the enzyme were 12.3 S, 16.2 S, and 16.4 S, respectively, and thus are in good agreement with the results of the reacting form studies. Molecular weight determinations of the bacterial enzyme by the principle of Archibald gave values of about 260,000 or approximately one-half of those of the enzymes from yeast and liver. Also, the biotin content of the bacterial enzyme is approximately 2 moles per mole of enzyme compared with 4 moles per mole for the enzyme from yeast or liver. Thus, the cumulative evidence strongly suggests that the predominant reacting form of the bacterial enzyme is a dimer, whereas those of the enzymes from yeast and liver are tetramers.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)44640-1