PHOSPHOGLYCOLATE PHOSPHATASE - PURIFICATION AND PROPERTIES

  • 1 January 1978
    • journal article
    • research article
    • Vol. 253  (6) , 1780-1785
Abstract
Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco [Nicotiana tabacum] leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography and preparative polyacrylamide gel electrophoresis. Preparations were judged 90-95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis and by isoelectric focusing. The highest specific activity obtained was 468 .mu.mol of phosphate released/min per mg of protein. The native protein has a MW of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a MW of 20,700, indicating that P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8-3.9 pH units by isoelectric focusing. Phosphoglycolate phosphatase from spinach [Spinacia oleracea] leaves has a MW 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 for isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentrations, while other phosphate esters of the photosynthetic carbon cycle were without effect.

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