Integral and peripheral proteins of the spinach leaf plasma membrane.

Plasma membrane vesicles of high purity were isolated from spinach (Spinacia oleracea) leaves by aqueous polymer 2-phase partition and analyzed with respect to their protein organization. This was done by Triton X-114 fractionation which separates integral, hydrophobic, membrane-spanning proteins from peripheral, hydrophilic membrane proteins. About 80% of the proteins were recovered in the hydrophobic phase of the Triton X-114/H2O phase system and the remaining 20% in the aqueous phase. SDS-PAGE showed that the integral membrane polypeptides of the plasma membrane were dominated by 52, 49, 33, 29, 25 and 17 kilodalton polypeptides, while the major peripheral plasma membrane polypeptide was 41 kilodaltons. Different wash treatments of the right-side-out plasma membrane vesicles suggest that the peripheral proteins are largely localized at the inner, cytoplasmic surface of the plasma membrane. Thus, Triton X-114 fractionation of plasma membranes should be useful for the functional identification of plasma membrane proteins as well as a first step in protein isolation.