2 K-M'S FOR ATP OF CORN-ROOT H-ATPASE AND THE USE OF GLUCOSE-6-PHOSPHATE AND HEXOKINASE AS AN ATP-REGENERATING SYSTEM()

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 t he vesicle membranes. The activity of this ATPase is enhanced 2- to 3- fold when Triton X-100 or lysophosphatidylcholine is added to the medi um at a protein:detergent ratio of 2:1 (w/w). In the absence of deterg ent, the ATPase exhibits only one K-m for ATP (0.1-0.2 mM), which is t he same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two K-m'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 V-max of the second K-m, for ATP increases as the temperature of the assay medium is raised from 15 degrees C to 38 degrees C. The Arr henius plot reveals a single break at 30 degrees C, both in the absenc e and in the presence of detergents. In the presence of Triton X-100 t he H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both h exokinase and ADP are included in the assay medium. There is no measur able cleavage when the apparent affinity for ATP of the H+-ATPase is n ot enhanced by Triton X-100 or when 1 mM glucose is included in the as say medium. These data indicate that when the high-affinity K-m for AT P is unmasked with the use of detergent, the ATPase can use glucose-6- phosphate and hexokinase as an ATP-regenerating system.