Isolation of Chloroplastic Phosphoglycerate Kinase : Kinetics of the Two-Enzyme Phosphoglycerate Kinase/Glyceraldehyde-3-Phosphate Dehydrogenase Couple

Plant Physiol. 1990 September; 94(1): 291–296.

PMCID: PMC1077223

Isolation of Chloroplastic Phosphoglycerate Kinase ¹

Kinetics of the Two-Enzyme Phosphoglycerate Kinase/Glyceraldehyde-3-Phosphate Dehydrogenase Couple

Jerzy Macioszek,² James B. Anderson, and Louise E. Anderson

Department of Biological Sciences (m/c 066), University of Illinois at Chicago, Box 4348, Chicago, Illinois 60680

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

² Present address: Abbott Laboratories, Abbott Park, IL 60064.

¹ This research was supported by grants from the U.S. Department of Energy (DE-FG02-85-ER 60367) and from the University of Illinois, Chicago Research Board.

This article has been cited by other articles in PMC.

Abstract

We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts (J Macioszek, LE Anderson [1987] Biochim Biophys Acta 892: 185-190). Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

McMorrow, Eileen M; Bradbeer, J William. Separation, Purification, and Comparative Properties of Chloroplast and Cytoplasmic Phosphoglycerate Kinase from Barley Leaves. Plant Physiol. 1990 Jun;93(2):374–383. [PubMed]
Anderson, Louise E; Avron, Mordhay. Light Modulation of Enzyme Activity in Chloroplasts: Generation of Membrane-bound Vicinal-Dithiol Groups by Photosynthetic Electron Transport. Plant Physiol. 1976 Feb;57(2):209–213. [PubMed]
Ashton, AR; Polya, GM. The specific interaction of cibacron and related dyes with cyclic nucleotide phosphodiesterase and lactate dehydrogenase. Biochem J. 1978 Nov 1;175(2):501–506. [PubMed]
Bowen, D; Littlechild, JA; Fothergill, JE; Watson, HC; Hall, L. Nucleotide sequence of the phosphoglycerate kinase gene from the extreme thermophile Thermus thermophilus. Comparison of the deduced amino acid sequence with that of the mesophilic yeast phosphoglycerate kinase. Biochem J. 1988 Sep 1;254(2):509–517. [PubMed]
Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
Cavell, S; Scopes, K. Isolation and characterization of the 'photosynthetic' phosphoglycerate kinase from Beta vulgaris. Eur J Biochem. 1976 Apr 1;63(2):483–490. [PubMed]
Cerff, R. Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases. Eur J Biochem. 1979 Feb 15;94(1):243–247. [PubMed]
Ferri, G; Comerio, G; Iadarola, P; Zapponi, MC; Speranza, ML. Subunit structure and activity of glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts. Biochim Biophys Acta. 1978 Jan 12;522(1):19–31. [PubMed]
Fifis, T; Scopes, RK. Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography. Biochem J. 1978 Oct 1;175(1):311–319. [PubMed]
Gontero, B; Cárdenas, ML; Ricard, J. A functional five-enzyme complex of chloroplasts involved in the Calvin cycle. Eur J Biochem. 1988 Apr 15;173(2):437–443. [PubMed]
Hanson, KR; Ling, R; Havir, E. A computer program for fitting data to the Michaelis-Menten equation. Biochem Biophys Res Commun. 1967 Oct 26;29(2):194–197. [PubMed]
Kuntz, GW; Eber, S; Kessler, W; Krietsch, H; Krietsch, WK. Isolation of phosphoglycerate kinases by affinity chromatography. Eur J Biochem. 1978 Apr 17;85(2):493–501. [PubMed]
Kvassman, J; Pettersson, G. Evidence that 1,3-bisphosphoglycerate dissociation from phosphoglycerate kinase is an intrinsically rapid reaction step. Eur J Biochem. 1989 Dec 8;186(1-2):261–264. [PubMed]
Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
Longstaff, M; Raines, CA; McMorrow, EM; Bradbeer, JW; Dyer, TA. Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes. Nucleic Acids Res. 1989 Aug 25;17(16):6569–6580. [PubMed]
Marques, IA; Ford, DM; Muschinek, G; Anderson, LE. Photosynthetic carbon metabolism in isolated pea chloroplasts: metabolite levels and enzyme activities. Arch Biochem Biophys. 1987 Feb 1;252(2):458–466. [PubMed]
Merrett, M. Primary structure of 3-phosphoglycerate kinase from horse muscle. II. Amino acid sequence of cyanogen bromide peptides CB1-CB4 and CB6-CB14, sequence of methionine-containing regions, and complete sequence of the enzyme. J Biol Chem. 1981 Oct 25;256(20):10293–10305. [PubMed]
Pacold, Ivan; Anderson, Louise E. Chloroplast and Cytoplasmic Enzymes: VI. Pea Leaf 3-Phosphoglycerate Kinases. Plant Physiol. 1975 Feb;55(2):168–171. [PubMed]
Perkins, RE; Conroy, SC; Dunbar, B; Fothergill, LA; Tuite, MF; Dobson, MJ; Kingsman, SM; Kingsman, AJ. The complete amino acid sequence of yeast phosphoglycerate kinase. Biochem J. 1983 Apr 1;211(1):199–218. [PubMed]
Weber, JP; Bernhard, SA. Transfer of 1,3-diphosphoglycerate between glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase via an enzyme-substrate-enzyme complex. Biochemistry. 1982 Aug 17;21(17):4189–4194. [PubMed]