19] Phosphoglycerate kinase from spinach, blue-green algae, and yeast

Abstract

This chapter describes an assay of phosphoglycerate kinase from spinach, blue-green algae, and yeast. Protein determination of purified plant phosphoglycerate kinases is performed spectrophotometrically. In contaminated phosphoglycerate kinase solutions, protein determination is performed by the biuret method. Preparation of extract from blue-green algae is done either from fresh algae or from spray dried algae. From the slightly green supernatant, phosphoglycerate kinase is precipitated by the addition of another portion of ammonium sulfate. Further purification steps involve affinity chromatography and gel chromatography. Purity testing is performed for phosphoglycerate kinase isolated from vertebrates. Phosphoglycerate kinases isolated from plants are homogeneous in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All phosphoglycerate kinases remain stable for years without loss of specific activity when stored as precipitate in 3.3 M ammonium sulfate. The isoelectric points of plant phosphoglycerate kinases so far studied are in the acidic range.