Target Molecular Size of the Red Beet Plasma Membrane ATPase

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...

Section: Discussionmentioning

confidence: 99%

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

Gallagher

Leonard²

1987

“…In a previous study from this laboratory (11), radiation inactivation analysis was used to estimate the holoenzyme mol wt of the red beet plasma membrane ATPase when associated with native plasma membrane vesicles. This method involves irradiating protein samples with high energy radiation such as accelerated electrons, x-rays, or '-rays and then analyzing the decrease in enzyme activity with increasing total radiation dose in terms of target theory (4,17 (20).…”

mentioning

confidence: 99%

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution

Briskin

Reynolds-Niesman²

1989

Self Cite

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of y-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.As with other E1/E2 type ATPases, the catalytic mechanism ofthe plant plasma membrane ATPase involves the formation of a covalent phosphorylated intermediate (7,8,21,25) at an active site aspartyl residue (9, 26). When assayed in the presence of [y-32P]ATP as substrate and analyzed by dodecyl sulfate gel electrophoresis, this phosphorylated intermediate has been shown to be associated with a 100 kD peptide (8,21,25). From this result and the observation that purification of the plasma membrane ATPase results in the enrichment of a 100 kD peptide on stained SDS-gels (2,16,23,24), it has been proposed that this 100 kD peptide represents the catalytic subunit of this transport enzyme (22 and references therein).In a previous study from this laboratory (11), radiation inactivation analysis was used to estimate the holoenzyme mol wt of the red beet plasma membrane ATPase when associated with native plasma membrane vesicles. This method involves irradiating protein samples with high energy radiation such as accelerated electrons, x-rays, or '-rays and then analyzing the decrease in enzyme activity with increasing total radiation dose in terms of target theory (4,17 (20).

“…These smaller polypeptides resemble those observed in other plant plasma membrane H+-ATPase preparations (13). The 200-kD phosphopeptide observed in the immunoprecipitates is possibly the dimeric form of the enzyme (6). Because of the presence of <100-kD phosphopeptides in the immunoprecipitates, it is not known if the H+-ATPase is phosphorylated on serine or threonine or both.…”

Section: Discussionmentioning

confidence: 52%

“…Autoradiograms of thinlayer cellulose chromatograms revealed that serine and, to a 5, and 7) or without (lanes 2, 4, and 6) SR and immunoprecipitated with anti-P-1 (lanes 4 and 5), anti-P-2 (lanes 2 and 3), or anti-P-3 (lanes 6 and 7). The immunoprecipitates were hydrolyzed and phosphoamino acids were determined after TLC and autoradiography.…”

Section: Determination Of the Phosphorylated Amino Acidsmentioning

confidence: 97%

Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H⁺-ATPase from Red Beet Storage Tissue

Suzuki

Wang²,

Takemoto³

1992

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H+-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H'-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H -ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca2". Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H+-ATPase and that specific protein kinase(s) are probably associated with the enzyme. SR2 is a virulence factor produced by certain plant pathogenic strains of Pseudomonas syringae pv syringae (30). This 1226-D lipodepsipeptide (9, 28) affects the plasma membranes of plants and yeast (30). SR alters the charge potential and Ca2" and K+ fluxes across these plasma membranes (15,25,31,34). The closure of stomata that it induces in whole leaves of Vicia faba (21) (3,34). It also stimulates the