MAIZE ROOT PLASMA-MEMBRANES ISOLATED BY AQUEOUS POLYMER 2-PHASE PARTITIONING - ASSESSMENT OF RESIDUAL TONOPLAST ATPASE AND PYROPHOSPHATASE ACTIVITIES

Plasma membrane-enriched fractions were obtained from maize roots by differential centrifugation and aqueous polymer two-phase partitioning. Plasma membrane enrichment and tonoplast depletion were assessed from comparisons of marker ATPase specific activities among starting microsomal, intermediate, and final phase-partitioned fractions. ATPase assays were conducted in the presence of the membrane permeabilizing detergent Brij 58. The plasma membrane fraction was enriched in ATPase specific activity at pH 6-5. This activity was strongly inhibited by vanadate (50% inhibition at approximately 3 mmol m-3). Two criteria for tonoplast contamination, bafilomycin- and nitrate-inhibited ATPase specific activities at pH 8-0, indicated that tonoplast contamination diminished with successive phase partitions. The existence of a plasma membrane-associated pyrophosphatase was examined by comparing the distribution of pyrophosphatase and ATPase specific activities among fractions. Pyrophosphatase specific activity decreased from starting to intermediate to final fractions, but not in proportion to the decrease in tonoplast ATPase specific activity. Whereas the existence of a plasma membrane pyrophosphatase could not be ruled out, the pyrophosphatase activity associated with the plasma membrane fraction was 46-fold less than the vanadate-sensitive ATPase activity.