Isoelectric Focusing of Plant Plasma Membrane Proteins

Abstract

Detergent-solubilized plasma membrane proteins from pea (Pisum sativum L.) stem tissue were separated by isoelectric focusing (IEF) using a Bio-Rad Rotofor cell, with the goal of identifying protein(s) involved in beta-1,3-glucan synthase (GS-II) activity. Ordinary IEF procedures result in membrane protein precipitation. Inclusion of 10% glycerol mitigates this problem in digitonin-solubilized preparations, but not in those solubilized in 3-[(-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Loss of GS-II activity during IEF is minimized by improved cooling of the Rotofor cell. GS-II focuses at pH 5.1. Antiserum against a 55 kilodalton (kD) polypeptide that was recognized from other evidence as involved in GS-II activity, detects this polypeptide in exact correspondence with the GS-II activity peak. A presumptive P-type ATPase, detected using an antibody against corn root plasma membrane 97 kD ATPase, focuses at pH 5.3. In this digitonin/glycerol medium, most of the membrane proteins focus within the relatively narrow pH range of 4.5 to 6, compared to pH 5.5 to 8.5 for IEF in the presence of 9 molar urea, 2% Nonidet P-40 (NP-40), and 5% mercaptoethanol, a medium that inactivates GS-II. This latter medium increases the apparent isoelectric point (pl) values of the abovementioned 55 and 97 kD polypeptides to 5.8 and 7.3, respectively. In the digitonin/glycerol medium, membrane polypeptides apparently focus at pH values lower than their true pls, because of adhering negatively charged phospholipids, which can be at least partially removed by the detergent NP-40 in the presence of urea. These results provide independent evidence that the 55 kD polypeptide is associated with the GS-II activity and indicate that inclusion of urea and a strong nonionic detergent such as NP-40 is necessary if membrane proteins are to be focused at pH values near their true pls.