CHROMATOGRAPHIC RESOLUTION, PURIFICATION AND CHARACTERIZATION OF H-PPASE AND H+-ATPASE FROM RICINUS COTYLEDONS()

Inorganic pyrophosphatase (PPase) was purified from membrane fractions isolated from Ricinus cotyledons. The non-ionic detergent dodecyl-P-D -maltoside (lauryl maltoside) was used to solubilise the PPase from th e phase-partitioned, upper phase fraction (plasma membrane-enriched), and purification was achieved using a combination of ion exchange chro matography and gel filtration. The PPase was resolved from the plasma membrane ATPase by exploiting the greater phospholipid dependency of e lution of the PPase from a 300 SW gel filtration column. When the phos pholipid concentration in the elution buffer was 0.5 mg/mL the PPase a nd ATPase eluted together but when it was lowered ten-fold the enzymes were resolved. The purification procedure resulted in an approximatel y 30-fold purification of the PPase from the original upper phase memb ranes with a yield of 20-25 %. The final purified fraction was enriche d in a protein with an apparent molecular mass of 68 kDa which cross-r eacted with an antibody raised to the mung bean tonoplast PPase. The p urified PPase activity was markedly stimulated by potassium salts and inhibited by sodium fluoride, methylene diphosphonate, N;N'-dicyclohex ylcarbodiimide and N-ethylmaleimide with no significant inhibition by azide.