PLANT PHYSIOLOGY , Vol 105, Issue 3 853-859, Copyright © 1994 by American Society of Plant Biologists
The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System
R. S. Ramos, M. T. Caldeira, P. Arruda and L. de Meis
Instituto de Ciencias Biomedicas, Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, CEP 21941-590, Brazil (R.S.R., M.T.C., L.d.M.)
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.
