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PubMed1990

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits.

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.

Adenosine Triphosphatases↗

PubMed1983

Subunit interaction in the mitochondrial H+-translocating ATPase. The role of oligomycin sensitivity conferral protein and coupling factor 6 in ATPase binding and Pi-ATP exchange in mitochondrial membranes.

Oligomycin sensitivity conferral protein, in the absence of coupling factor 6 (F6), is able to bind the ATPase to mitochondrial membranes with an apparent association constant of 10(6) M-1. The F6-dependent ATPase binding has an apparent association constant 1 to 2 orders of magnitude lower than that obtained with oligomycin sensitivity conferral protein. The oligomycin sensitivity conferral protein-dependent, membrane-bound ATPase activity is sensitive to rutamycin while the F6-dependent, membrane-bound ATPase activity is insensitive to rutamycin. F1-ATPase and Type II ATPase require F6 in addition to oligomycin sensitivity conferral protein and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. This F6 requirement for the 32Pi-ATP exchange is not related to the F6 effect on the ATPase binding. The Type I ATPase and therefore the 26,500-dalton subunit associated with it requires F6 and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. Oligomycin sensitivity conferral protein can be interchanged with the 26,500-dalton ATPase binding protein in the binding of the ATPase and the 32Pi-ATP exchange.

Adenosine Triphosphatases↗

PubMed1980

Activities of Ca2+-ATPase and Na+ + K+-ATPase in human placenta.

Although there have been a few reports on Ca2+-ATPase and Na+ + K+-ATPase in human placenta, they are still insufficient for getting more detailed information about human placenta. In this study we investigated some characteristics of the Ca2+-ATPase and measured the activity of Ca2+-ATPase and Na+ + K+-ATPase at various gestational periods. Maximum activity of Ca2+-ATPase was seen at 3 mM and pH 9.0. The enzyme was distinguished from ALP (i.e., Alkaline phosphatase) by such tests as adding L-phenylalanine and heat treatment. Our measurement results of Ca2+-ATPase activity (mumoles Pi/mg protein/30 min) in human placental chorion were 21.9 +/- 2.4 (Mean +/- S.D.) at 10-15 gestational weeks, 19.1 +/- 1.4 at 16-27 gestational weeks, 31.4 +/- 7.2 at 28-35 gestational weeks, and at 36-42 gestational weeks, the AFD group showed 25.8 +/- 4.6, the LFD group, 27.9 +/- 2.1, and the SFD group, 21.2 +/- 1.9. Those of Na+ + K+-ATPase activity (mumoles Pi/mg protein/30 min) were 0.98 +/- 0.05 at 10-15 gestational weeks, 0.82 +/- 0.02 at 16-27 gestational weeks, 1.85 +/- 0.42 at 28-35 gestational weeks and at 36-42 gestational weeks, the AFD group showed 1.58 +/- 0.45, the LFD group, 1.49 +/- 0.37, and the SFD group, 1.68 +/- 0.36. As a result, Ca2+-ATPase activity tended to be higher at 28-35 gestational weeks than for other periods, while it showed lower for the SFD group than for the AFD group. As regards to Na+ + K+-ATPase activity, there were not seen any significant differences between the AFD placenta and the LFD placenta as compared with the AFD placenta.

Alkaline Phosphatase↗

PubMed1993

Purification and characterization of lysosomal H(+)-ATPase. An anion-sensitive v-type H(+)-ATPase from rat liver lysosomes.

Lysosomal H(+)-ATPase was purified to homogeneity from rat liver lysosomes. It is a bafilomycin A1-sensitive Mg(2+)-ATPase, which reacts with antibodies against the 16- and 70-kDa subunits of vacuolar H(+)-ATPase (Nezu, J., Motojima, K., Tamura, H., and Ohkuma, S. (1992) J. Biochem. (Tokyo) 112, 212-219), and has been separated from both the N-ethylmaleimide (NEM)-sensitive/bafilomycin A1-insensitive Mg(2+)-ATPase (ATPase I) and the NEM-insensitive Mg2+/Ca(2+)-ATPase (ATPase II) (Hayashi, H., Arai, K., Sato, O., Shimaya, A., Sai, Y., and Ohkuma, S. (1992) Chem. Pharm. Bull. 40, 2783-2786). The purified enzyme had the subunit structure of vacuolar H(+)-ATPase, consisting of 110-, 70-, 56-, 42-, 39-, 34-, (32-,) and 16-kDa proteins. It had optimal activity at a pH of 7.0-8.0, with an apparently single Km value for ATP of 95 microM. It hydrolyzed ATP > or = dATP >> GTP, ITP >> UTP, but not CTP, and was inhibited by ADP. It demonstrated divalent cation specificity in the order of Mg2+, Mn2+ > Fe2+, Co2+ > Ca2+. Among various anions, Cl-, Br-, and F- activated ATPase activity, whereas NO3- inhibited activity. It was inhibited not only by bafilomycin A1 but also by NEM, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and dicyclohexylcarbodi-imide. The purified enzyme was incorporated into proteoliposomes where its proton pump activity was reconstituted. This suggested that the isolated enzyme maintains its H+ translocation activity. These findings suggest that the isolated enzyme is an anion-sensitive vacuolar type H(+)-ATPase that functions as a lysosomal proton pump.

Animals↗

PubMed1993

Molecular cloning of a P-type ATPase gene from the cyanobacterium Synechocystis sp. PCC 6803. Homology to eukaryotic Ca(2+)-ATPases.

With oligonucleotide primers derived from P-type ATPase genes of different sources, a part of Synechocystis sp. PCC 6803 genomic DNA was amplified and used as hybridization probe for the Synechocystis gene. A 4.7 kb HindIII fragment was cloned and sequenced; it contains the open reading frame of the E1E2-ATPase. The Synechocystis ATPase (named PMA1) consists of 915 amino acids with a M(r) of 98,902; it has ten putative transmembrane domains and contains the conserved regions a to j common to all P-type ATPases. Its amino acid sequence shows less than 20% identity to prokaryotic ATPases but about 30% identity to eukaryotic Ca(2+)-ATPases. An alignment to rat kidney and yeast Ca(2+)-ATPase protein sequences shows homology in stalk regions and transmembrane domains domains which are thought to be involved in calcium binding and transport; these three ATPases reveal very similar hydropathy plots and form a separate group in the phylogenetic tree of P-type ATPases. The results strongly support the assumption that PMA1 of Synechocystis is a calcium translocating ATPase, possibly involved in regulatory processes with calcium as second messenger.

Adenosine Triphosphatases↗

PubMed1985

What family of ATPases does the vacuolar H+-ATPase belong to?

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [14C]DCCD and [14C]NEM bound to a 9 kDa polypeptide, while [14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [gamma-32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H+-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H+-ATPase of yeast.

Adenosine Triphosphatases↗

PubMedDate not supplied

Characterization of the F1F0-ATPase and the tightly-bound ATPase activities in submitochondrial particles from human term placenta.

In a previous study we demonstrated the existence of a tightly-bound ATPase in the human placental mitochondria (Martínez et al., 1993). The current study characterizes the ATP hydrolysis produced by the F1F0-ATPase and the tightly-bound ATPase in submitochondrial particles from the human term placenta. Both enzymes were not differentiated by pH. Inhibitors were necessary to distinguish the activity of each enzyme. The kinetic of the total ATP hydrolysis fitted into a model of two enzymes. During the characterization, it was observed that the tightly-bound ATPase activity was partially inhibited by vanadate and Mg2+, whereas the F1F0-ATPase was totally inhibited by Mg2+. Different nucleotides were hydrolyzed by the tightly-bound ATPase; the F1F0-ATPase hydrolyzed exclusively ATP. Glucose-6-phosphate, p-nitrophenylphosphate, or pyrophosphate were not hydrolyzed by the F1F0-ATPase, although some hydrolysis was observed with the tightly-bound ATPase. It is concluded that the tightly-bound ATPase activity corresponded to a 5'-nucelotidase, and that the human placental mitochondria could participate in the metabolism of nucleotides.

Adenosine Triphosphate↗

PubMed1999

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.

Adenosine Triphosphatases↗

PubMed1987

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor.

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.

Binding, Competitive↗

PubMed1990

Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria.

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.

Adenosine Triphosphatases↗

PubMed1995

Functional consequences of substitution of the disulfide-bonded segment, Cys127-Cys150, located in the extracellular domain of the Na,K-ATPase beta subunit: Arg148 is essential for the functional expression of Na,K-ATPase.

The Cys127-Cys150 disulfide-bonded loop (L1) of the Torpedo californica Na,K-ATPase beta 1 subunit was substituted with the corresponding loop of the rat beta 1, mouse beta 2, or pig H,K-ATPase beta subunit. All the substituted mutant beta subunits assembled with the Na,K-ATPase alpha subunit in a trypsin-resistant manner. The mutants with L1 from the Na,K-ATPase beta subunit isoforms (rat beta 1 and mouse beta 2) each formed a functional complex with the Na,K-ATPase alpha subunit. On the other hand, the complex of the alpha subunit with the mutant beta subunit that was substituted with the pig H,K-ATPase beta subunit L1 was inactive as to ATP hydrolysis. Ser131 and Phe148 located within L1 of the pig H,K-ATPase beta subunit-substituted mutant were back-mutated to Pro131 and Arg148, respectively. The Phe148 to Arg mutation restored the ability of the mutant beta subunit substituted with the H,K-ATPase beta subunit L1 to form a functional complex with the alpha subunit. These results suggested that the Cys127-Cys150 loop of the Na,K-ATPase beta 1 subunit, especially Arg148, plays a critical role in the functional expression of Na,K-ATPase.

Amino Acid Sequence↗

PubMed1993

Cloning of a Ca(2+)-ATPase gene of Plasmodium falciparum and comparison with vertebrate Ca(2+)-ATPases.

A Ca(2+)-ATPase gene was cloned from the genomic libraries of Plasmodium falciparum. From the deduced amino acid sequence of the gene, a 139 kDa protein with a total of 1228 amino acids was predicted. Sequence of a partial cDNA clone of the gene identified two introns near the 3'-end at the regions identical to the regions assumed for the Ca(2+)-ATPase gene of P. yoelii, a rodent malaria species. As compared with a variety of Ca(2+)-ATPases, the P. falciparum Ca(2+)-ATPase had the highest amino acid sequence homology (78%) to the P. yoelii Ca(2+)-ATPase, moderate homology (45-50%) to vertebrate sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), and lowest homology (20%) to a plasma membrane Ca(2+)-ATPase. The P. falciparum protein conserved sequences and residues that are important for the function and/or structure of the organellar type Ca(2+)-ATPase, such as high affinity Ca(2+)-binding sites, fluorescein isothiocyanate (FITC)-binding regions, and the phosphorylation site, but the protein did not contain calmodulin-binding regions that occur in the plasma membrane type Ca(2+)-ATPase. Thus we concluded the cloned gene was the organellar type Ca(2+)-ATPase of P. falciparum. In a region between the phosphorylation site and FITC-binding region, the P. falciparum protein was about 200 residues longer than the rabbit SERCA and lacked a sequence that binds to phospholamban, a protein that regulates the activity of the rabbit SERCA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase.

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.

Animals↗

PubMed1993

Cloning and sequencing of the genes coding for the A and B subunits of vacuolar-type Na(+)-ATPase from Enterococcus hirae. Coexistence of vacuolar- and F0F1-type ATPases in one bacterial cell.

The eubacterium Enterococcus hirae ATCC 9790 possesses a H(+)-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F0F1-type ATPase (Shibata, C., Ehara, T., Tomura, K., Igarashi, K., and Kobayashi, H. (1992) J. Bacteriol. 174, 6117-6124). We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na(+)-translocating ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60% identical) to the A (large or alpha) and the B (small or beta) subunits, respectively, of vacuolar-type H(+)-ATPases which have been found in eukaryotic endomembrane systems (Neurospora crassa, Saccharomyces cerevisiae, Arabidopsis thaliana, and carrot) and archaebacterial cell membranes (Sulfolobus acidocaldarius and Methanosarcina barkeri). The A and B subunits of Na(+)-ATPase showed about 23-28% identities with the beta and alpha subunits of E. hirae F1-ATPase and of Escherichia coli F1-ATPase, respectively. These results indicate that E. hirae Na(+)-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.

Adenosine Triphosphatases↗

PubMed1999

Na,K-ATPase and V-ATPase in ovarian follicles of Drosophila melanogaster.

Uncovering the cause and meaning of bioelectric phenomena in developing systems requires investigations of the distribution and activity of ion-transport mechanisms. In order to identify and localize ion pumps in ovarian follicles of Drosophila, we used immunofluorescence microscopy, immunoelectron microscopy, subcellular fractionation, immunoblots, and acridine-orange staining. We applied various antibodies directed against the Na,K-pump (Na,K-ATPase) and against vacuolar-type proton pumps (V-ATPase). During all phases of oogenesis, Na,K-ATPase were found in apical and lateral follicle-cell membranes and, during rapid follicle growth (beginning with stage 10), also in nurse-cell membranes and in the oolemma. V-ATPase were detected in various cytoplasmic vesicles and in yolk spheres and, beginning with stage 10, also in apical follicle-cell membranes and in the oolemma. Given these and earlier results, we propose that: 1) V-ATPase coupled to secondary active antiporters represent the ouabain-intensitive potassium pumps described previously; 2) both Na,K-ATPase and V-ATPase are involved in bioelectric phenomena as well as in osmoregulation and follicle growth, especially during stages 10-12; 3) organelle-associated V-ATPase play a role in vesicle acidification and in yolk processing; and 4) the channel-forming protein ductin is a component of both V-ATPase and gap junctions in ovarian follicles of Drosophila.

Animals↗

PubMed1991

[HPLC method for measuring (Na(+)-K(+)) ATPase and (Ca(++)-Mg(++)) ATPase in erythrocytes from different species of mammals].

(Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase are enzymes located in erythrocyte plasma membranes, driving back ions against the electrochemical gradient; (Na(+)-K+)ATPase transports 3 Na+ ions out of the cell, and 2 K+ ions into it for each hydrolyzed ATP molecule, whereas the Ca(2+)-pump transports Ca2+ ions out of the cells, by utilizing still the ATP hydrolysis. The method used to test the activity of the above mentioned enzymes is based on the measuring of the ADP quantity released during the reaction by HPLC, that is High Performance Liquid Chromatography; the chromatographic type is a Ion-Pair Reversed-Phase. This method presents the following important advantages for the assay of the enzymes we analysed: 1) It is reproducible through time; 2) It is perfectly linear; 3) It is extremely sensitive. This method allowed us to carry out a comparative study of (Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of several species of mammalia: man, horse, rabbit, lamb, rat. We recovered different values in ATPase activity; (Ca(++)-Mg++)ATPase shows a higher activity than Na(+)-K+)ATPase; moreover, some differences exist in the various Mammalia considered, with relation to each pump: the lamb shows the lowest activity for both pumps, whereas the rabbit shows the highest one. At present, the different values obtained are being interpreted and analysed. This method is also very versatile, since it allowed us to assess the Km value for Ca++ of the (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of rabbit. The value resulted to be 100 microMs, thus 10 times higher than the human Km value for the Ca++.

Adenosine Diphosphate↗

PubMed1992

THE INSECT V-ATPase, A PLASMA MEMBRANE PROTON PUMP ENERGIZING SECONDARY ACTIVE TRANSPORT: IMMUNOLOGICAL EVIDENCE FOR THE OCCURRENCE OF A V-ATPase IN INSECT ION-TRANSPORTING EPITHELIA.

Active electrogenic K+ transport in insects serves as the energy source for secretion or absorption in gastrointestinal epithelia or for the receptor current in sensory epithelia. In the larval midgut of the tobacco hornworm Manduca sexta, a vacuolar-type proton pump (V-ATPase) and a K+/nH+ antiport represent the functional elements of the potassium pump. Several immunological findings support the hypothesis that active K+ transport in other insect epithelia may also be energized by a V-ATPase. In immunoblots, crude homogenates of sensilla-rich antennae and Malpighian tubules of M. sexta cross reacted with an immune serum directed to the purified plasma membrane V-ATPase from the midgut; the M. sexta midgut V-ATPase cross reacted with polyclonal antibodies to endomembrane V-ATPases from xenic origin. In immunocytochemical investigations of larvae of M. sexta and adults of Antheraea pernyi, monoclonal antibodies to defined subunits of the purified midgut V-ATPase or polyclonal antibodies to xenic endomembrane V-ATPase labelled the sites of active K+ transport: the goblet cell apical membrane in the midgut, the brush border of Malpighian tubules and the apical projections of the auxiliary cells in antennal sensilla. The functional mechanism of a primary H+-pumping V-ATPase and a secondary H+-dependent K+ transport postulated for K+-transporting insect epithelia may be further applicable to active Na+ or Cl- transport and would provide a unifying concept for all ouabain-insensitive electrogenic ion transport in insects. The findings from the midgut investigations, however, are the first instance in which a V-ATPase provides an alternative to the Na+/K+-ATPase in energizing secondary active transport in animal plasma membranes.

Journal Article↗

PubMed1984

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+-ATPase.

Periodical changes in Ca2+-ATPase and Mg2+-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca2+-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt , when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg2+-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca2+-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg2+-ATPase activity. There results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.

Adenosine Triphosphatases↗