H+-ATPase activity from storage tissue of Beta vulgaris. IV. N,N'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane H+-ATPase

The molecular weight and isoelectric point of the plasma membrane H+-ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H+-ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [14C]-DCCD at 00C, a single 97,000 dalton protein was visualized on a fluorography of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [14C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H+-ATPase. An antibody raised against the plasma membrane H+-ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H+-ATPase to be 6.5