Immunodetection of a 90 000- Mr polypeptide related to yeast plasma membrane ATPase in plasma membrane from maize shoots

Abstract

Maize shoot plasma membranes were prepared using either polyethyleneglycol (PEG)-dextran phase partition or centrifugation through a 30% sucrose cushion. The ATPase specific activity of membranes obtained with the phase partition method (1.4 μmol P i · min −1 · mg −1 protein) was twice that of those prepared with the sucrose cushion method. After solubilization by lysolecithin and precipitation by ammonium sulfate, ATPase activities of the order of 3.0–3.5 μmol P i · min −1 · mg −1 were obtained. A polypeptide of M r = 90 000 was enriched during ATPase purification. Antibodies against pure plasma membrane ATPase from Saccharomyces cerevisiae inhibited the plant ATPase activity. Immunodetection during purification of the plant enzyme strongly supported the conclusion that the polypeptide of M r = 90 000 belongs to plant plasma membrane ATPase.