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PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF PHOSPHOGLYCERATE KINASE FROM THE GREEN ALGA SELENASTRUM MINUTUM 1
Author(s) -
Lin Min,
Turpin David H.
Publication year - 1993
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.0022-3646.1993.00777.x
Subject(s) - phosphoglycerate kinase , biology , biochemistry , microbiology and biotechnology , enzyme
We report the first purification and characterization of a eukaryotic algal phosphoglycerate kinase (PGK), Two forms of PGK (PGK 1 and PGK 2 ) from the green alga Selenastrum minutum (Naeg.) Collins were purified to electrophoretic homogeneity with specific activities of 1100 and 1069 units · mg −1 protein, respectively. The portion of PGK 1 and PGK 2 (probably the cytosolic and chloroplastic forms, respectively) in this organism was estimated as 32 and 68%, respectively. PGK 1 was more heat‐stable than PGK 2 . The M r estimation for PGK 1 and PGK 2 by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and gel filtration indicated that they both were monomeric with a similar M r of approximately 44 kDa. Antibodies raised against S. minutum PGK 1 cross‐reacted with PGK 2 as well as PGKs from prokaryotic and eukaryotic sources, suggesting that PGK 1 was structurally and immunologically closely related to PGK 2 and other PGKs, which was consistent with NH 2 ‐terminal sequence analysis. Comparative kinetic and regulatory properties of PGK 1 and PGK 2 from S. minutum were investigated, Both forms exhibited hyperbolic kinetics with respect to both 3‐phosphoglycerate (3‐PGA) and Mg‐adenosine triphosphate 2‐ (MgATP 2‐ ) under the conditions tested and had similar K m values for each substrate (PGK 1 ; K m (MgATP 2‐ ) = 0.37 mM, K m (3‐PGA) = 0.59 mM; PGK 2 ; K m (MgATP 2‐ ) = 0.32 mM, K m (3‐PGA) = 0.46 mM). PGK 1 and PGK 2 , however, differed significantly in several other kinetic properties. PGK 2 had a broad pH optimum between 7.3 and 7.8, as compared to PGK 1 , with a pH optimum of 7.3 Mg 2+ was the most efficient cofactor for both forms; it inhibited PGK 1 but not PGK 2 at higher concentrations (>10 mM). Other divalent cations (Mn 2+ , Zn 2+ , Co 2+ , Cd 2+ , and Ca 2+ ) only partially replaced Mg 2+ and were more effective for PGK 1 than for PGK 2 , A wide range of metabolites was examined for regulatory properties. Energy charge was the most important factor in regulating the two forms of S. minutum PGK. These results were interpreted in light of the regulation of this kinase in response to the cell energy requirement and the need for glycolytic carbon flow to provide carbon skeletons for amino acid biosynthesis.