Abstract
Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.
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Selected References
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