H + -ATPase Activity from Storage Tissue of Beta vulgaris

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 0C, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between 14CDCCD 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. Images: Fig. 2: Fig. 4: Fig. 5: