Abstract
Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.