Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei

John P. Barnard; Peter L. Pedersen

doi:10.1016/0166-6851(88)90165-X

Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei

John P. Barnard, Peter L. Pedersen

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 μmol min^-1 (mg protein)^-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent K_m for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S_0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent K_m for ADP and the S_0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

Original language	English (US)
Pages (from-to)	141-147
Number of pages	7
Journal	Molecular and Biochemical Parasitology
Volume	31
Issue number	2
DOIs	https://doi.org/10.1016/0166-6851(88)90165-X
State	Published - Nov 1988
Externally published	Yes

Keywords

Affinity elution
Bioenergetics
Enzyme kinetics
Enzyme purification
Pyruvate kinase
Trypanosoma brucei

ASJC Scopus subject areas

Parasitology
Molecular Biology

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10.1016/0166-6851(88)90165-X

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title = "Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei",

abstract = "The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 μmol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.",

keywords = "Affinity elution, Bioenergetics, Enzyme kinetics, Enzyme purification, Pyruvate kinase, Trypanosoma brucei",

author = "Barnard, {John P.} and Pedersen, {Peter L.}",

note = "Funding Information: We wish to thank Dr. David Bickar and Dr. Jay Bangs for instruction concerning the growth and isolation of trypanosomes. We also want to thank Dale Hereld and members of the laboratory of Dr. Paul Englund for gifts of trypanosomes, fractioned trypanosomes, and advice. Finally, we thank Dr. Richard Nakashima for his assistance during the initial phases of this project. Financial support for his investigation was provided to J.B. by a grant from the MacArthur Foundation.",

year = "1988",

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doi = "10.1016/0166-6851(88)90165-X",

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AU - Barnard, John P.

AU - Pedersen, Peter L.

N1 - Funding Information: We wish to thank Dr. David Bickar and Dr. Jay Bangs for instruction concerning the growth and isolation of trypanosomes. We also want to thank Dale Hereld and members of the laboratory of Dr. Paul Englund for gifts of trypanosomes, fractioned trypanosomes, and advice. Finally, we thank Dr. Richard Nakashima for his assistance during the initial phases of this project. Financial support for his investigation was provided to J.B. by a grant from the MacArthur Foundation.

PY - 1988/11

Y1 - 1988/11

N2 - The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 μmol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

AB - The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 μmol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

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KW - Bioenergetics

KW - Enzyme kinetics

KW - Enzyme purification

KW - Pyruvate kinase

KW - Trypanosoma brucei

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Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei

Abstract

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ASJC Scopus subject areas

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