Electrophoretic Characterization of a Detergent-Treated Plasma Membrane Fraction from Corn Roots

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-l 14-extracted membranes from corn, oat, barley, and tomato.The transport of most of the essential mineral nutrients into root cells involves ion carriers in the plasma membrane which are most likely integral proteins or protein complexes which span the membrane (23). Purification and characterization of carrier proteins requires the ability to extract plasma membrane vesicles with detergents without significant denaturation of integral proteins and the ability to follow purification of plasma membrane polypeptides by electrophoresis (17,24,35).The selection ofdetergents and the determination ofextraction conditions for purification of integral proteins cannot be based strictly on theoretical grounds, but must be determined empirically (16, 17). There are many detergents available for use in removing proteins from the membrane, and there are several types of strategies which can be used to purify integral membrane proteins (17,35,36). The two approaches most often used involve removal of peripheral proteins by treatment with low concentrations of detergent followed by recovery of the integral protein still associated with the extracted membrane (negative purification) and/or solubilization of the membrane at a detergent concentration near its critical micelle concentration and ' Supported in part by National Science Foundation grant PCM 8025004.2 Present address: Biological Sciences Department, Stanford University, Stanford, CA 94305. separation of integral proteins by, for example, rate zonal centrifugation or affinity chromatography (positive purification). The success of these approaches is greatly influenced by the initial proportion of the protein of interest in the membrane and the extent of purification...

Potassium Stimulation of Corn Root Plasmalemma ATPase

“…6). Such biochemical heterogeneity has been first reported for native corn root plasmalemma ATPase by Gallagher and Leonard (8). According to these authors, it does not result from a protein degradation but from the existence of two different proteins.…”

“…After glycerol gradient centrifugation of lysoPC solubilized ATPase, the peak of purified enzyme (fractions [8][9][10][11][12] occuffed at about 35% glycerol, as classically reported (2,16,22). Only 12% ofthe proteins layered were recovered in these high density fractions, as a result of the aggregation of the partially delipidated (and reversibly inactivated) enzyme (22).…”

“…On the other hand, Gallagher and Leonard (8) have reported that in SDS-PAGE of sucrose gradient plasmalemma, the ATPase appears as two closely spaced bands in the 100 kD region, even in the presence of antiproteasic agents. According to these authors, "it is likely that this region of the one-dimension gel contains several polypeptides of similar size, but from different proteins."…”

“…The absolute K+ requirement has been observed with a purified ATPase from phase-partitioned plasmalemma (16), whereas the H+-pumping in the absence of K+ have been demonstrated on ATPase purified from plasmalemma isolated on sucrose gradient (28). Such considerations led to the proposal that there are two distinct ATPases, a H+ and/or a H+-K+ transporting species, possibly related to the two bands observed on 8), the phasepartitioned plasmalemma being enriched in the H+/K+ pump.One difficulty in studying ATPase stimulation by K+ is to distinguish between the so-called direct and indirect effect of the cat: n (4). First, indirect potassium stimulation of the proton pump may result from the dissipation of both membrane potential and pH gradient by potassium channels and H+/K+ carriers (15).…”

“…The absolute K+ requirement has been observed with a purified ATPase from phase-partitioned plasmalemma (16), whereas the H+-pumping in the absence of K+ have been demonstrated on ATPase purified from plasmalemma isolated on sucrose gradient (28). Such considerations led to the proposal that there are two distinct ATPases, a H+ and/or a H+-K+ transporting species, possibly related to the two bands observed on SDS-PAGE gel (7,8), the phasepartitioned plasmalemma being enriched in the H+/K+ pump.…”