Chromatographie resolution, purification and characterization of H+-PPase and H+-ATPase from Ricinus cotyledons

Long, AR, Hall, J.L. and Williams, LE (1997) Chromatographie resolution, purification and characterization of H+-PPase and H+-ATPase from Ricinus cotyledons. Journal of Plant Physiology, 151 (1), 16-24. (doi:10.1016/S0176-1617(97)80031-4).

Abstract

Inorganic pyrophosphatase (PPase) was purified from membrane fractions isolated from Ricinus cotyledons. The non-ionic detergent dodecyl-ß-D-maltoside (lauryl maltoside) was used to solubilise the PPase from the phase-partitioned, upper phase fraction (plasma membrane-enriched), and purification was achieved using a combination of ion exchange chromatography and gel filtration. The PPase was resolved from the plasma membrane ATPase by exploiting the greater phospholipid dependency of elution of the PPase from a 300 SW gel filtration column. When the phospholipid concentration in the elution buffer was 0.5 mg/mL the PPase and ATPase eluted together but when it was lowered ten-fold the enzymes were resolved. The purification procedure resulted in an approximately 30-fold purification of the PPase from the original upper phase membranes with a yield of 20–25 %. The final purified fraction was enriched in a protein with an apparent molecular mass of 68 kDa which cross-reacted with an antibody raised to the mung bean tonoplast PPase. The purified PPase activity was markedly stimulated by potassium salts and inhibited by sodium fluoride, methylene diphosphonate, N,N′-dicyclohexylcarbodiimide and N-ethyl-maleimide with no significant inhibition by azide

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