Purification and characterization of human muscle pyruvate kinase

R. N. Harkins, J. A. Black, Marvin Rittenberg

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionally by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (s20,w) of 10.04 S. It contains four subunits with identical molecular weights of 61 000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross- react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.

Original languageEnglish (US)
Pages (from-to)301-307
Number of pages7
JournalCanadian Journal of Biochemistry
Volume55
Issue number4
StatePublished - 1977

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Pyruvate Kinase
Isoenzymes
Muscles
Immunodiffusion
Erythrocytes
Molecular Weight
Psoas Muscles
Amino Acids
Carboxymethylcellulose Sodium
Antibodies
Ammonium Sulfate
Enzymes
Acetone
Cellulose
Sodium Dodecyl Sulfate
Chromatography
Polyacrylamide Gel Electrophoresis
Hot Temperature

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Harkins, R. N., Black, J. A., & Rittenberg, M. (1977). Purification and characterization of human muscle pyruvate kinase. Canadian Journal of Biochemistry, 55(4), 301-307.

Purification and characterization of human muscle pyruvate kinase. / Harkins, R. N.; Black, J. A.; Rittenberg, Marvin.

In: Canadian Journal of Biochemistry, Vol. 55, No. 4, 1977, p. 301-307.

Research output: Contribution to journalArticle

Harkins, RN, Black, JA & Rittenberg, M 1977, 'Purification and characterization of human muscle pyruvate kinase', Canadian Journal of Biochemistry, vol. 55, no. 4, pp. 301-307.
Harkins, R. N. ; Black, J. A. ; Rittenberg, Marvin. / Purification and characterization of human muscle pyruvate kinase. In: Canadian Journal of Biochemistry. 1977 ; Vol. 55, No. 4. pp. 301-307.
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