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. 1986 Feb;165(2):483–488. doi: 10.1128/jb.165.2.483-488.1986

Partial purification and characterization of pyruvate, orthophosphate dikinase from Rhodospirillum rubrum.

S M Ernst, R J Budde, R Chollet
PMCID: PMC214444  PMID: 3003027

Abstract

We confirmed an earlier report (B. B. Buchanan, J. Bacteriol. 119:1066-1068, 1974) that the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum contains pyruvate, orthophosphate dikinase (EC 2.7.9.1) activity that is absolutely dependent upon all three substrates by performing enzyme assays in both the forward (phosphoenolpyruvate formation) and reverse (ATP formation) directions. Of the various carbon sources tested, photoheterotrophic growth on DL-lactate plus bicarbonate proved to be best for the production of dikinase activity units. A four-step protocol, which included batch DEAE-cellulose processing, ammonium sulfate fractionation, and chromatography on hydroxylapatite and Blue A Dyematrex gels, was devised for partially purifying the enzyme from such cells. The protein was purified about 80-fold to an apparent electrophoretic purity of about 60% and a final specific activity of 3.6 U/mg of protein, with about a 35% overall recovery of activity units. Estimations of native and monomeric relative molecular weights by sucrose density gradient centrifugation, high-pressure liquid chromatography-based size exclusion chromatography, denaturing electrophoresis, and immunoblotting suggested that the holoenzyme was most likely a homodimer of 92.7-kilodalton subunits. The results are compared with related previous data on the nonphotosynthetic bacterial dikinase and the C4 mesophyll chloroplast enzyme.

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Selected References

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

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