Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP

doi:10.1016/0003-9861(74)90076-9

Archives of Biochemistry and Biophysics

Volume 164, Issue 2, October 1974, Pages 641-654

https://doi.org/10.1016/0003-9861(74)90076-9 Get rights and content

Abstract

Pyruvate carboxylase has been detected in, and partially purified from, cell-free extracts of Azotobacter vinelandii OP. The best preparations obtained have specific activities in the range of 4 units/mg and appear approximately 15% pure when analyzed by polyacrylamide gel electrophoresis. The partially purified enzyme is activated by both univalent and divalent cations, contains one or more functional biotinyl residues, and exhibits apparent Michaelis constants for the substrates (pyruvate, Mg-ATP²⁻, and HCO₃⁻) which are in the same range as those observed for other pyruvate carboxylases. However, A. vinelandii pyruvate carboxylase is fully active in the absence of added acetyl-coenzyme A and is insensitive to inhibition by dicarboxylic acids such as l-aspartate, l-glutamate, and α-ketoglutarate. The molecular weight of the catalytically active species is obtained as 296,000.

The level of pyruvate carboxylase is highest in extracts of A. vinelandii grown on pyruvate or l-lactate as sole carbon source and this level is further enhanced on addition of succinate to the medium. The enzyme is absent from cells grown on succinate and is present at intermediate levels in cells grown on sucrose, glucose, glycerol, or acetate. In contrast, the level of phosphoenolypyruvate carboxylase in these extracts is essentially independent of the carbon source. These data suggest that pyruvate carboxylase in A. vinelandii is induced by pyruvate or some closely related metabolite.

References (47)

R.S. Bandurski et al.
J. Biol. Chem
(1953)
M.F. Utter et al.
J. Biol. Chem
(1963)
M.C. Scrutton et al.
Arch. Biochem. Biophys
(1972)
M.C. Scrutton et al.
Methods Enzymol
(1969)
E. Layne
Methods Enzymol
(1957)
B.L. Taylor et al.
J. Biol. Chem
(1972)
G.W.E. Plaut et al.
J. Biol. Chem
(1949)
H.L. Nadler et al.
Blood
(1969)
E.A. Peterson et al.
Methods Enzymol
(1962)
M.C. Scrutton et al.

M.C. Scrutton et al.

J. Biol. Chem

(1965)

W.R. McClure et al.

J. Biol. Chem

(1971)

J.J. Cazzulo et al.

Arch. Biochem. Biophys

(1968)

A.S. Mildvan et al.

J. Biol. Chem

(1966)

M.C. Scrutton et al.

J. Biol. Chem

(1973)

L.K. Ashman et al.

Biochem. Biophys. Res. Commun

(1973)

S.J. Bloom et al.

J. Biol. Chem

(1962)

M. Ruiz-Amil et al.

J. Mol. Chem

(1965)

C.L. Liao et al.

J. Bacteriol

(1971)

H.L. Kornberg

Essays Biochem

(1966)

M.C. Scrutton

R.W. O'Brien et al.

B.L. Taylor

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^☆: Supported in part by NSF grant No. GB 30223, USPHS grant No. AM11712 and AEC Contract No. 80-(11-1)-1242.

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Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP☆

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