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R B Beechey

Publications and source records attributed to R B Beechey.

At least 55 records · Page 3Linked to original sources
PubMed1987

Identification of the phosphate-translocator from maize mesophyll chloroplasts.

Intact maize mesophyll chloroplasts have been isolated in yields of up to 10 mg of chlorophyll per preparation. The chloroplasts were able to reduce 3-phosphoglycerate at a rate of 2.4 mumol of oxygen/min/mg of chlorophyll. This activity was inhibited by preincubating the intact chloroplasts with pyridoxal 5-phosphate. Chloroplast envelopes have been prepared and the protein profile has been obtained on SDS-polyacrylamide gels. The phosphate-translocator from the chloroplast envelope has been identified as a 30kDa polypeptide.

Carrier Proteins↗
PubMed1987

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation.

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.

Benzimidazoles↗
PubMed1986

The extraction and reconstitution of the alpha-cyanocinnamate-sensitive pyruvate transporter from castor bean mitochondria.

The pyruvate carrier from castor bean mitochondria has been solubilized with Triton X-114 and partially purified using hydroxyapatite column chromatography. SDS-polyacrylamide gel electrophoresis of the hydroxyapatite-eluate showed that there were 6 major protein bands of Mr, 74kDa, 66kDa, 34kDa, 32kDa, 30kDa 12kDa. When the eluate was reconstituted into liposomes it was shown to catalyze a pyruvate exchange reaction which was sensitive to N-ethyl maleimide and a series of analogues of alpha-cyanocinnamate. The characteristics of this pyruvate exchange activity are similar to that found in intact mitochondria, and it is concluded that one or more proteins in the hydroxyapatite-eluate correspond to the pyruvate carrier.

Carrier Proteins↗
PubMed1986

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.

Antimycin A↗
PubMed1986

Evidence for the existence of a channel in the glucose-specific carrier EIIGlc of the Salmonella typhimurium phosphoenolpyruvate-dependent phosphotransferase system.

The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme II (EIIGlc) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl alpha-D-glucopyranoside (alpha-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGlc. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate alpha-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent N-ethylmaleimide was used. Since only one activity-linked dithiol can be detected with sealed inverted membrane vesicles or intact cells and it is accessible to membrane-impermeable sulfhydryl reagents from both sides of the cytoplasmic membrane, we suggest that it is located in a channel constructured by the carrier and that the channel spans the membrane. A second dithiol, not essential for activity, is located near the outer surface of the cytoplasmic membrane.

Binding Sites↗
PubMed1984

Discrimination between the binding sites of modulators of the H+-translocating ATPase activity in rat liver mitochondrial membranes.

The properties of the components of the mitochondrial ATPase which interact with modulators of energy transduction have been examined. The chromatographic behavior and the size of the components which bind trialkyl tins, carbodiimides and uncouplers, have been shown to be different. However, they all appear to be proteolipids with apparent molecular weights around 10,000. On this basis it is proposed that these inhibitors act at different sites in the membrane sector of the ATP synthase of rat liver mitochondria.

Animals↗
PubMed1982

Interactions between the mitochondrial adenosinetriphosphatase and periodate-oxidized adenosine 5'-triphosphate, an affinity label for adenosine 5'-triphosphate binding sites.

Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the alpha and beta subunits and is equally distributed between them. The incorporation of o-ATP into the ATPase is reduced, and the irreversible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number, and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenosine-containing compound and the tripolyphosphate anion in a beta-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the beta-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.

Adenosine Diphosphate↗
PubMed1981

Involvement of the endogenous inhibitor protein in the MgATP-induced inhibition of soluble mitochondrial adenosine triphosphatase activity.

Chloroform-released ATPase from ox heart mitochondria contains significant amounts of inhibitor protein. There is a correlation between processes that affect the interactions between the inhibitor protein and the ATPase molecule and the ability of MgATP to induce an inhibition of ATPase activity. Evidence is presented suggesting that the endogenous inhibitor protein is involved in the process of MgATP-induced inhibition of soluble ATPase activity.

Adenosine Triphosphatases↗
PubMed1981

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.

Adenosine Diphosphate↗
PubMed1981

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

Adenosine Triphosphatases↗
PubMed1981

Potent inhibition of membrane-bound rat intestinal alkaline phosphatase by a new series of phosphate analogues.

The inhibition by phosphonates and phosphate analogues of the alkaline phosphatase activity of rat intestinal brush-border membrane vesicles was studied at pH 7.5 and 30 degrees C. Phenylene-1,3-diphosphonate, 2,6-dinitrophenylphosphonate and phosphonoacetaldehyde were found to be competitive inhibitors, with Ki values in the range 16-80 microM. Adenosine 5'-[beta-thio]diphosphate and adenosine 5'[gamma-thio]triphosphate are also very potent inhibitors, with Ki values of approx. 10 microM. The inhibition produced by these thiophosphates was mainly competitive but with a slight non-competitive element. Adenosine 5'-[beta gamma-imido]triphosphate is also a competitive inhibitor of the alkaline phosphatase, but oxidation of the ribose moiety of this compound with NaIO4 results in an active-site-directed irreversible inhibitor that could be of general use in studies of the mechanism of action of this enzyme.

Adenine Nucleotides↗
PubMed1981

The use of potent inhibitors of alkaline phosphatase to investigate the role of the enzyme in intestinal transport of inorganic phosphate.

In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values less than 5 microM at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogues, was tested, however, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5'-[beta gamma-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity, but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5'-[beta-thio]diphosphate or adenosine 5'-[gamma-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.

Adenine Nucleotides↗
PubMed1980

Dibutylchloromethyltin chloride, a potent inhibitor of electron transport in plant mitochondria.

Dibutylchloromethyl tin chloride (DBCT) inhibits coupled and uncoupled respiration of mitochondria from potato tubers, cauliflower florets and etiolated mung bean hypocotyls with succinate and L-malate but not with external NADH or TMPD/ascorbate as substrates. Using potato and cauliflower mitochondria. DBCT at 200 pmole/mg of protein gives complete inhibition only in KCl-based media and at pH 6.8. DBCT has no effect on the internal pH of mung bean mitochondria, but does cause a decrease in the membrane potential. Electron transport through the alternative oxidase is not inhibited, neither is the ATP-synthase system. DBCT appears to interact with the functionally-distinct pool of ubiquinone associated with the oxidation of succinate and L-malate.

Electron Transport↗