Subunit composition of vacuolar membrane H+-ATPase from mung bean (original) (raw)
Related papers
Journal of Biological Chemistry
A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 ~mol/mg*min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67-and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase.
The Journal of biological chemistry, 1988
Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-c...
Journal of Biological Chemistry
Vacuolar ATPases constitute a novel class of Nethylmaleimide-and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble FoF1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the -70-kDa subunit. Using antibodies to the -70-kDa subunit of corn to screen a carrot root Xgtll cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative FoF1-ATPase Bchains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to &chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the FoF1-type ATPases. A major functional difference between the 69-kDa and &subunits is the location of 3 critical cysteine residues: two in the putative catalytic region and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the aminoterminal end of the subunit.
Subunit composition and Ca2+-ATPase activity of the vacuolar ATPase from barley roots
Archives of Biochemistry and Biophysics, 1992
The vacuolar ATPase was purified from a tonoplastenriched membrane fraction from barley (Hordeurn vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KN09 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 116,68,63,46,42,34,32, 17,13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purifled holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca"-ATPase were obtained from the Sephacryl S-400 column. A peak of Caa+-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO; and DCCI. It is proposed that this Ca2'-ATPase is a partial reaction of the plant vacuolar ATPase. The second Caa+-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO; or DCCI. The second Ca2+-ATPase is a major component of ATP hydrolysis by the native membranes. o 1ee2~~~,demio Plws, Inc. The vacuolar ATPases, or V-type ATPases (l), are proton pumping ATPases that acidify endomembrane compartments of eukaryote cells. V-type ATPases have been purified from plants (l-3), fungi (4), and animals (5-9). The V-type ATPase provides the driving force for accumulation of ions, sugars, and amino acids in the plant vacuole. It may play a role in salt tolerance since it provides the energy for sequestering Na+ and Cl-in the vac-1 To whom correspondence should be addressed.
Journal of Biological Chemistry, 2000
Immunoblot analyses and partial amino acid sequencings revealed that both the 40-(E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35-(D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V max and K m of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H ؉ pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.
Journal of Biological Chemistry, 1986
Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72-and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro['4C]benzo-2-oxa-1,3-diazole (['4C]NBD-Cl) and N,N'-['4C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-C1 preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit. Plant vacuoles are acidic organelles in which ions, sugars, organic acids, and hydrolytic enzymes are stored (1). Studies with isolated vacuoles and tonoplast (plant vacuolar membrane) vesicles have indicated that a proton-translocating ATPase present on the tonoplast generates an electrochemical gradient, which may be responsible for the observed accumulation of ions and solutes (reviewed in Ref. 2). A similar ATP-dependent proton pump is also present on the Golgi of maize coleoptiles (3). Several recent reports have described the partial purification of a novel ATPase from plant and ~~
The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H1ATPase
Vacuolar ATPases constitute a novel class of Nethylmaleimide-and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble FoF1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the -70-kDa subunit. Using antibodies to the -70-kDa subunit of corn to screen a carrot root Xgtll cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative FoF1-ATPase Bchains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to &chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the FoF1-type ATPases. A major functional difference between the 69-kDa and &subunits is the location of 3 critical cysteine residues: two in the putative catalytic region and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the aminoterminal end of the subunit.
European Journal of Biochemistry, 1998
The plasma membrane H ϩ -ATPase was purified from tobacco cells (line BY-2). After solubilization by lysophosphatidylcholine followed by separation on a glycerol gradient, a fraction with a high specific activity of 9 µmol ATP · min Ϫ1 · mg protein Ϫ1 was obtained, in which the H ϩ -ATPase polypeptide represented at least 80% of the protein. The incubation of this fraction in the presence of alkaline phosphatase increased H ϩ -ATPase activity by 40%, in a manner consistent with dephosphorylation of the enzyme itself. The hydrolytic activity of the solubilized enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline phosphatase treatment was also accompanied by a 92% decrease in the H ϩ -ATPase phosphothreonine content, whereas the phosphoserine residues were almost unaffected. The dephosphorylation induced a slight decrease of the affinity of the enzyme towards ATP. The purified enzyme was not activated by lysophosphatidylcholine addition nor by trypsin-mediated proteolysis, two treatments reported to release the inhibitory control by the C-terminal domain of the H ϩ -ATPase and to increase the affinity of the enzyme towards ATP. Based on these results, the regulatory phosphorylation evoked by alkaline phosphatase most likely differs from the autoinhibitory control of the H ϩ -ATPase by its C-terminal domain.
Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase
Plant Cell Reports, 2015
Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H ?-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H ?-ATPase activity.