Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.     

Human erythrocyte calmodulin. Further chemical characterization and the site of its interaction with the membrane.

Human erythrocyte and bovine brain calmodulins were indistinguishable by tryptic peptide mapping, indicating that the primary sequence of the two proteins is either very similar or identical. Calcium binding determinations of human erythrocyte calmodulin, by equilibrium dialysis and fluorescence titration, were in close agreement with previous studies on other calmodulins. The calcium-activated adenosine triphosphatase which is stimulated by calmodulin was shown to be firmly associated with smooth erythrocyte plasma membranes devoid of spectrin and actin. Kinetic titration demonstrated that there are 4500 calmodulin binding sites per erythrocyte and that the turnover number of this calcium-activated adenosine triphosphatase is 3000 mumol of Pi . (mumol of site)-1 . min-1 which is similar to the turnover numbers of other transport adenosine triphosphatases. Furthermore, calmodulin stimulates calcium-activated adenosine triphosphatase by a simple enzyme-ligand association.     

Proteins of Polyhedral Cytoplasmic Deoxyvirus II. Nucleotide Phosphohydrolase Activity Associated with Frog Virus 3

A nucleotide phosphohydrolase is firmly associated with a purified polyhedral cytoplasmic deoxyvirus, frog virus 3. This adenosine triphosphatase is distinguishable from known mammalian cell adenosine triphosphatases and from adenosine triphosphatase of an unrelated cytoplasmic replicating virus grown in the same host cell. The enzyme activity has a high specificity for adenosine triphosphate; the product of the reaction is adenosine diphosphate. The presence of similar activities in reovirus and poxvirus indicates that adenosine triphosphatase might have a function in the replication of these viruses.     

Molecular mechanism of cardiotoxin action on axonal membranes.

Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.     

Immunological Properties of Micrococcus lysodeikticus Membranes

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.     

A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized-actomyosin

1. The preparation and properties of a myofibrillar protein factor which inhibits the Mg(2+)-activated adenosine triphosphatase of desensitized actomyosin is described. 2. This factor had negligible effect on the Mg(2+)-activated adenosine triphosphatase of natural actomyosin and on the Ca(2+)-activated adenosine triphosphatases of desensitized actomyosin and myosin. 3. The Mg(2+)-activated inosine triphosphatase activity of desensitized actomyosin was not affected by the factor. 4. The inhibitory effect was sensitive to ionic strength. In addition to their ionic effects Mg(2+) and Ca(2+) appeared to have a specific action in reducing the effect of the inhibitor. 5. F-actin reduced the inhibition whereas Bailey-type tropo-myosin had little effect. 6. As far as can be judged from the reported experiments this factor is different from any of the previously described myofibrillar components.     

Localization of Adenosine Triphosphatase Activity on the Chloroplast Envelope in Tendrils of Pisum sativum

When samples of pea tendril tissue were incubated in the Wachstein-Meisel medium for the demonstration of adenosine triphosphatases, deposits of lead reaction product were localized between the membranes of the chloroplast envelope. The presence of Mg²⁺ was necessary for adenosine triphosphatase activity, and Ca²⁺ could not substitute for this requirement. Varying the pH of incubation to 5.5 or 9.4 inhibited enzyme activity, as did the addition of p-chloromercuribenzoic acid or N-ethylmaleimide. The adenosine triphosphatase was apparently inactivated or degraded when the plants were grown in the dark for 24 hours prior to incubation. The enzyme was substrate-specific for adenosine triphosphate; no reaction was obtained with adenosine diphosphate, uridine triphosphate, inosine triphosphate, p-nitrophenyl phosphate, and sodium β-glycerophosphate. Sites of nonspecific depositions of lead are described. The adenosine triphosphatase on the chloroplast envelope may be involved in the light-induced contraction of this organelle.     

Membrane adenosine triphosphatase in synchronous cultures of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis utilizing synchronized cultures of Rhodobacter sphaeroides have revealed that most intracytoplasmic membrane proteins accumulate continuously throughout the cell cycle while new phospholipid appears discontinuously within the intracytoplasmic membrane. The resulting changes in the structure of the membrane lipids was proposed to influence the activities of enzymes associated with the intracytoplasmic membranes (Wraight, C.A., Leuking, D.R., Fraley, R.T. and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). We have extended the study of intracytoplasmic membrane biogenesis in R. sphaeroides to include the membrane adenosine triphosphatase. The membrane bound Mg2+-dependent, oligomycin-sensitive adenosine triphosphatase activity was measured throughout the cell cycle for steady-state synchronized cells of R. sphaeroides and found to accumulate discontinuously. Following treatment with an uncoupling reagent (2,4-dinitrophenol) the intracytoplasmic membrane associated adenosine triphosphatase activity was stimulated uniformly in membranes isolated at different stages of the cell cycle. The adenosine triphosphatase was also measured by quantitative immunoblots utilizing specific antibody to compare the enzyme activity and enzyme protein mass. Immunologic measurement of the adenosine triphosphatase in isolated membranes indicated a constant ratio of enzyme to chromatophore protein exists during the cell cycle in contrast to the discontinuous accumulation of adenosine triphosphatase activity. These results are discussed in light of the cell-cycle specific synthesis of the intracytoplasmic membrane.     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

The mode of inhibition by calcium of cell-membrane adenosine-triphosphatase activity

1. The mechanism of the inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase by calcium was investigated with an enzyme preparation from rabbit kidney cortex and with membranes of human erythrocytes. 2. CaATP, rather than ionic Ca(2+), acts as a competitive inhibitor, competing with MgATP in the Na(+)-plus-K(+)-activated adenosine-triphosphatase reaction. 3. There appears to be no competition between calcium and Na(+) for the activation of adenosine triphosphatase. 4. The inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase of cell membranes by low concentrations of CaATP and the consequent need of intact cells to keep the cytoplasmic concentration of calcium low relative to that of magnesium suggests a raison d'être for the mitochondrial calcium pump.     

The localization of adenosine triphosphatases in morphologically characterized subcellular fractions of guinea-pig brain

1. The distribution of adenosine triphosphatase was studied in morphologically characterized subcellular fractions of guinea-pig brain. The conditions of homogenization were selected so as to favour the survival of nerve endings as organized structures. 2. A fraction consisting mainly of the external membranes of nerve endings was rich in a ouabain-sensitive Na⁺–K⁺-stimulated adenosine triphosphatase which closely resembled that present in the classical microsomal fraction studied by other workers, but which showed a higher specific activity. 3. A dinitrophenol-stimulated adenosine triphosphatase was located in the nerve-ending mitochondria. 4. The synaptic-vesicle fraction contained a small amount of adenosine triphosphatase that differed in its response to several ions and other compounds from the membrane, myelin and mitochondrial fractions, indicating freedom from contamination by these elements.     

The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.     

Epinephrine-binding plasma-membrane antigens in rat liver.

Detergent extracts of isolated rat liver plasma membranes were analysed in two-dimensional immunoelectrophoresis against antiserum to plasma membranes. Enzyme staining of the immunoprecipitates revealed the presence of about ten antigens with nucleoside di- and triphosphatase activity. Most of these were earlier shown also to be NADH-neotetrazolium reductase active. In addition, two of these antigens exhibited L-leucyl-beta-naphthylamidase activity. As judged from autoradiography these plasma membrane antigens earlier characterized as multienzyme complexes bound [14C]epinephrine, and the same antigens were labelled regardless of whether membranes or membrane extracts were incubated with the radioactive hormone. The specificity of this binding was established in displacement experiments with unlabelled hormones or their analogues. Another hormone-binding antigen, also identified in the plasma membrane extract did not exhibit any known enzyme activity while three antigens with different enzyme activities had no epinephrine-binding capacity. [14C]Epinephrine-labelled plasma membrane extracts were chromatographed on Sepharose 4B and the fractions obtained were analysed in two-dimensional immunoelectrophoresis combined with autoradiography. Nucleoside di- and triphosphatases of high molecular weights (5000000) were associated with L-leucyl-beta-naphthylamidase activity, while no such associations were detected in a lower molecular weight region (70000). Further immunological studies on the various fractionated antigens provided evidence that at least two of them occurred in both low and high molecular weight fractions. Hormone-binding membrane components in varying concentrations were found throughout the eluted extract.     

Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.     

Adenosine 5''-triphosphate-linked transhydrogenase in cytoplasmic membranes of colicin-treated and untreated Escherichia coli.

The adenosine 5'-triphosphate (ATP)-linked transhydrogenase reaction, present in the particulate fractions of Escherichia coli, was previously shown to be inhibited in these fractions when the bacteria were treated with colicins K or El. The purpose of this study was to characterized the ATP-linked transhydrogenase reaction and the colicin-caused inhibition of the reaction in purified cytoplasmic membranes. Particulate fractions from bacteria treated or untreated with colicins were separated on sucrose gradients into cell wall membrane and cytoplasmic membrane fractions. The ATP-linked transhydrogenase reaction was found to be exclusively associated with the cytoplasmic membrane fractions. The reaction was inhibited by carbonylcyanide m-chlorophenlhdrazone, dinitrophenol, N,N'-dicyclohexylcarbodiimide, and trypsin. Although the cytoplasmic membrane fractions were purified from the majoriy of the cell wall membrane and its bound colicins, they showed the inhibitory effects of colicins K and El on the ATP-linked transhydrogenase reaction. The inhibition of ATP-linked transhydrogenase reaction induced by the colicin could not be reversed by subjection the isolated membranes to a variety of physical and chemical treatments. Cytoplasmic membranes depleted of energy-transducing adenosine triphosphatase ATPase) complex (coupling factor) lost the ATP-linked transhydrogenase activity. The ATPase complexes isolated from membranes of bacteria treated or untreated with colicins El or K reconstituted high levels of ATP-linded transhydrogenase activity to depleted membranes of untreated bacteria. The same ATPase complexes reconstituted low levels of activity to depleted membranes of the treated bacteria.     

Schistosoma mansoni: fine structural localization of tegumental adenosine triphosphatases

The distribution of Ca2+-dependent adenosine triphosphatase (EC 3.6.1.3.) and nonspecific (Na-K-Mg) adenosine triphosphatase activity in the tegument and subtegumental tissues of Schistosoma mansoni from both mixed and single sex infections was investigated cytochemically. Differences in the distribution of tegumental Ca-adenosine triphosphatase activity in 60- to 70-day-old female worms were found which could be related to the degree of sexual development in the two types of females, with little or no tegumental activity being found in 70-day-old females from single sex infections. In contrast, 28-day-old females from single sex infections showed low levels of tegumental Ca-adenosine triphosphatase activity, suggesting that the lack of tegumental activity in 70-day-old single sex females may be due to a loss or suppression of activity as a consequence of the failure of females in single sex infections to pair and develop to full sexual maturity. No differences in the distribution of nonspecific (Na-K-Mg) adenosine triphosphatase activity between females from mixed and single sex infections were found. The sexual status or age of male worms appeared to have little or no effect on the distribution of tegumental adenosine triphosphatases.