Structural investigations into L-type pyruvate kinase from rat liver
Structural investigations into L-type pyruvate kinase from rat liver
The reactive triazine dye, Procion Blue NIX-R behaved as an active site-directed inhibitor of pure rat liver pyruvate kinase. The only ligand able to protect the enzyme from inactivation was magnesium ions. In the unprotected reaction and protected reaction respectively, 1.54 + 0.057 and 0.73 + 0.024 (means + S.E.M) moll dye boundVcovalently per mol enzyme subunit. The DI-type isoenzyme from rabbit muscle did not behave similarly. Binding of L-type pyruvate kinase to Sepharose-immobilized Procion Blue MX-R required 2 - 5 mM-magnesium ionsin the irrigating buffer; elution of the enzyme was effected by omission of the magnesium ions. This procedure was readily exploited in the routine purification of the enzyme in relatively high yield (about 25%). Again, the M-type enzyme did not behave similarly. Pure L-type pyruvate kinase was phosphorylated usingV [32p]-ATP and the catalytic subunit of cAMP-dependent protein kinase. This facilitated identification of which of the enzyme's two major aspartyl-prolyl cleavage fragments contained the phosphorylatable site. Subsequent cleavage of a larger quantity of unphosphorylated enzyme resulted in the purification of this fragment. The determination of an N-terminal prolyl residue showed that it was derived from the C-terminal of the protein. The enzyme responsible for the bulk of L-typepyruvate kinase phosphatase activity in liver extracts was located in the soluble fraction of the liver and was dependent on magnesium ions. It was distinct from that protein phosphatase activity located in the glycogen fraction and that activity revealed by trypsin. Further structural investigations on L-type pyruvate kinase were begun. Several peptides derived from the enzyme were isolated which would be of value in sequence analysis. Gel electrophoresis of the immunocomplexes formed when antiserum to homogeneous L-type .pyruvate kinase was incubated with liver extracts revealed, in conjunction with the use of anti(fructose bisphosphatase), that fructose bisphosphatase was a major species precipitated by anti(L-type pyruvate kinase). This was not due to the a conta-miaiun± in the immunizing antigen. This information enabled an improved antiserum to be produced, which revealed a single immunological and catalytic form of the enzyme present in the liver cytosols.
University of Southampton
Byford, Michael Frederick
1983
Byford, Michael Frederick
Byford, Michael Frederick
(1983)
Structural investigations into L-type pyruvate kinase from rat liver.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The reactive triazine dye, Procion Blue NIX-R behaved as an active site-directed inhibitor of pure rat liver pyruvate kinase. The only ligand able to protect the enzyme from inactivation was magnesium ions. In the unprotected reaction and protected reaction respectively, 1.54 + 0.057 and 0.73 + 0.024 (means + S.E.M) moll dye boundVcovalently per mol enzyme subunit. The DI-type isoenzyme from rabbit muscle did not behave similarly. Binding of L-type pyruvate kinase to Sepharose-immobilized Procion Blue MX-R required 2 - 5 mM-magnesium ionsin the irrigating buffer; elution of the enzyme was effected by omission of the magnesium ions. This procedure was readily exploited in the routine purification of the enzyme in relatively high yield (about 25%). Again, the M-type enzyme did not behave similarly. Pure L-type pyruvate kinase was phosphorylated usingV [32p]-ATP and the catalytic subunit of cAMP-dependent protein kinase. This facilitated identification of which of the enzyme's two major aspartyl-prolyl cleavage fragments contained the phosphorylatable site. Subsequent cleavage of a larger quantity of unphosphorylated enzyme resulted in the purification of this fragment. The determination of an N-terminal prolyl residue showed that it was derived from the C-terminal of the protein. The enzyme responsible for the bulk of L-typepyruvate kinase phosphatase activity in liver extracts was located in the soluble fraction of the liver and was dependent on magnesium ions. It was distinct from that protein phosphatase activity located in the glycogen fraction and that activity revealed by trypsin. Further structural investigations on L-type pyruvate kinase were begun. Several peptides derived from the enzyme were isolated which would be of value in sequence analysis. Gel electrophoresis of the immunocomplexes formed when antiserum to homogeneous L-type .pyruvate kinase was incubated with liver extracts revealed, in conjunction with the use of anti(fructose bisphosphatase), that fructose bisphosphatase was a major species precipitated by anti(L-type pyruvate kinase). This was not due to the a conta-miaiun± in the immunizing antigen. This information enabled an improved antiserum to be produced, which revealed a single immunological and catalytic form of the enzyme present in the liver cytosols.
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Published date: 1983
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Local EPrints ID: 460184
URI: http://eprints.soton.ac.uk/id/eprint/460184
PURE UUID: 1b6c8d53-635b-47ba-a6e2-fb628640d388
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Date deposited: 04 Jul 2022 18:08
Last modified: 04 Jul 2022 18:08
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Author:
Michael Frederick Byford
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