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© 1992 Oxford University Press

RESEARCH-ARTICLE

Maize Root Plasma Membranes Isolated by Aqueous Polymer Two-phase Partitioning: Assessment of Residual Tonoplast ATPase and Pyrophosphatase Activities

CHRISTOPHER D. FARADAY and ROGER M. SPANSWICK

Section of Plant Biology, Division of Biological Sciences, Plant Science Building, Cornell University Ithaca, NY 14853–5908, USA

To whom correspondence should be addressed.

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 {small tilde}3 mmol mminusb3). 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.

Key words: Bafilomycin, ATPase, pyrophosphatase, plasma membrane, aqueous polymer two-phase partitioning


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