H+-ATPase Activity from Storage Tissue of Beta vulgaris

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Abstract
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 0°C, a single 97,000 dalton protein was visualized on a fluorograph 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.