Phosphoglycolate phosphatase. Purification and properties.

Abstract

Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 mumol of phosphate released/min/mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating the P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8 to 3.9 pH units by isoelectric focusing. Phosphosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentration, while other phosphate esters of the photosynthetic carbon cycle were without effect.