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PubMed1979

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.

Ascorbic Acid↗
PubMed1982

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell.

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.

Animals↗
PubMed1982

Stimulation of Na+ -dependent amino acid uptake by activation of the Ca2+ -dependent K+ channel in the Ehrlich ascites tumor cell.

The activation of Ca2+ -dependent K+ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na+ -dependent transport of alpha-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K+ conductance. The same treatment does not modify the Na+ -independent uptake of the norbornane amino acid.

Amino Acids↗
PubMed1984

Effects of electron donors on Ca2+-dependent K+ transport in one-step inside-out vesicles from the human erythrocyte membrane.

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.

Ascorbic Acid↗
PubMed1986

Analysis of the all or nothing behaviour of Ca-dependent K channels in one-step inside-out vesicles from human red cell membranes.

The all or nothing behaviour of Ca2+-dependent K+ channels has been analyzed in one-step inside-out vesicles. There is a threshold for Ca2+ below which the K+ channels remain silent, and which ranges between the 10(-6) and 10(-8) M for different vesicles under the experimental conditions tested, in the absence of Mg2+. The increase of Ca2+ concentration within this range recruits a larger fraction of the vesicles to the active (permeable to 86Rb+) state. The apparent rate of 86Rb+ transport through each individual channel was found to increase, however, with Ca2+ concentration. This finding is not an artefact due to size heterogeneity of the vesicle population, and it is consistent with the variations of the mean open time of the channels with Ca2+ concentration reported previously in patch-clamp experiments. The electron donor system ascorbate + phenazine-methosulphate increases the rate of 86Rb+ transport through the channels whereas oxidized cytochrome c has the opposite effect.

Biological Transport↗
PubMed1987

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain.

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.

Alcaligenes↗
PubMed1989

Involvement of thiol groups in the function of the dipeptide/proton cotransport system in rabbit renal brush-border membrane vesicles.

The role of thiol groups in H+-gradient-dependent dipeptide transport in rabbit renal brush-border membrane vesicles was investigated using glycylsarcosine as the substrate. Treatment of the membrane vesicles with a thiol-group-reducing agent, dimercaptopropanol, stimulated Gly-Sar transport. On the other hand, treatment with thiol group oxidants such as 5,5'-dithiobis(2-nitrobenzoic acid), plumbagin and phenazine methosulfate inhibited Gly-Sar transport. These effects were irreversible, because washing the membranes after treatment failed to reverse the effects. Incubation of the membrane vesicles with phenylarsine oxide, a reagent which interacts specifically with vicinal dithiols, significantly inhibited Gly-Sar transport. In all cases, the stimulation or the inhibition of the dipeptide transport was primarily due to changes in the maximal velocity of the transport system, the apparent affinity constant remaining unaltered. These results demonstrate the involvement of one or more vicinal dithiol groups in the function of the renal dipeptide transport system and that these thiol groups must exist in reduced form to maintain maximal transport activity. In addition, these data indirectly suggest that a dithiol-disulfide interchange may play a role in the function of the renal dipeptide transport system.

Animals↗
PubMed1976

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.

Alcaligenes↗
PubMed1976

Trimethylamine dehydrogenase from a methylotrophic bacterium. I. Isolation and steady-state kinetics.

1. The isolation of trimethylamine dehydrogenase (EC 1.5.99.7) from a restricted facultative methylotroph to electrophoretic homogeneity is described. 2. The molecular weight and subunit molecular weights were found to be 146800 for the enzyme by sedimentation equilibrium ultracentrifugation and 70000-80000 for the two non-identical subunits by sodium dodecyl sulphate gel electrophoresis. 3. Initial velocity studies indicate that the enzymatic reaction proceeds by a Ping-Pong mechanism. 4. Further kinetic evidence was obtained by analysis of product inhibition patterns using the alternate substrate diethylamine and the products acetaldehyde and ethylamine as product inhibitors, for the release of ethylamine before the addition of phenazine methosulphate and for the existence of an enzyme-two-carbon unit complex as a stable form of the enzyme. 5. Some properties of the unusual prosthetic group of trimethylamine dehydrogenase and its photodegradation product are described in preliminary form.

Acetaldehyde↗
PubMed1977

Fluorimetric assay of tobacco leaf dehydrogenases with resazurin.

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.

Dihydrolipoamide Dehydrogenase↗
PubMed1978

Methylamine dehydrogenase of Pseudomonas sp. J. Purification and properties.

Methylamine dehydrogenase was purified in a homogeneous form from methylamine-grown Pseudomonas sp. J. The specific activity of the purified enzyme was 5.19 at 19 degrees C. The molecular weight was estimated to be 105 000, and the enzyme was composed of two kinds of subunit with molecular weights of 40 000 and 13 000, respectively. The enzyme contained little phosphorus, iron and copper. The enzyme had absorption maxima at 278, 330, 430 and 460 nm (shoulder). On addition of methylamine, the peaks at 430 and 460 nm decreased, while that at 330 nm increased. Primary amines served as substrates, but secondary and tertiary amines did not. Phenazine methosulfate was the most effective electron acceptor and oxygen was ineffective. The enzyme was inhibited by carbonyl reagents, cuprizone and HgCl2 but not by other chelators or sulfhydryl reagents. Some of other physical and biochemical properties of the enzyme were studied. These results show that the enzyme purified from Pseudomonas sp. J is essentially similar to the enzyme obtained from Pseudomonas AM1, although it differs slightly in some properties.

Amino Acids↗
PubMed1979

Properties of Pseudomonas AM1 primary-amine dehydrogenase immobilized on agarose.

1. The primary-amine dehydrogenase of Pseudomonas AM1 (primary amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.-) was purified by an improved method and covalently attached to cyanogen bromide-activated Sepharose 4B. The immobilized enzyme showed very little change in its sensitivity to heat and to inhibition by semicarbazide as compared with the soluble enzyme, but had enhanced stability at 0 degrees C. The pH optimum of the immobilized enzyme remained unchanged at pH 7.4. 2. A new type of spectrophotometric assay is described in which sedimentation of the immobilized enzyme in the cuvette is prevented by increasing the viscosity by the presence of 10% (w/w) polyethylene glycol (M1 20 000). Detailed kinetic analysis using this assay showed only insignificant differences in the Km values for n-butylamine and phenazine methosulphate between the soluble and Agarose-bound enzymes. The results are compared with those for other oxidoreductase enzymes immobilized on Sepharose.

Enzymes, Immobilized↗
PubMed1981

Purification and some properties of the soluble hydrogenase from Chromatium vinosum.

A routine procedure for the growth and harvesting of large (600 1) batches of Chromatium vinosum and the isolation of hydrogenase (hydrogen: (acceptor) oxidoreductase, EC 1.12.-.-) are described. The enzyme is pure according to polyacrylamide gel electrophoresis, has a molecular weight of 61,000-63,000 and consists of a single polypeptide chain. The enzyme is stable in air but not active. Activity is obtained only after complete removal of oxygen. EPR spectroscopy at 9 GHz shows a signal indicative for a [4Fe-4S]3+(3+,2+) cluster and in addition a rather complex signal of unknown origin. This additional signal completely disappears upon removal of oxygen, by incubation with 2-mercaptoethanol or by reduction with ferrocytochrome c. No EPR signals are detected in the enzyme reduced with H2 or dithionite. The intensity of the EPR signal of the [4Fe-4S] cluster corresponds to one-quarter of the enzyme concentration, both in the untreated as well as in the He- or N2-activated enzyme. If the enzyme is activated under He and then brought in contact with air the signal increases 4-fold and represents about one free spin/enzyme molecule. When measured at 35 GHz the line shape and peak positions of the additional signal change, indicating that the signal is not originating from a simple S = 1/2 system. None of the inhibitors of the hydrogenase activity has any effect on the shape or intensity o the EPR signal fo the Fe-S cluster, 2H2O also has no effect. All EPR signals disappear after reduction with NADH or ascorbate in the presence of phenazine methosulphate. It is suggested that the Fe-S cluster is not the primary site of interaction of H2 with the enzyme.

Chromatium↗
PubMed1981

The function of ascorbate with respect to prolyl 4-hydroxylase activity.

1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.

Animals↗
PubMed1989

Alkylthioacetic acid (3-thia fatty acids)--a new group of non-beta-oxidizable, peroxisome-inducing fatty acid analogues. I. A study on the structural requirements for proliferation of peroxisomes and mitochondria in rat liver.

The induction of peroxisome proliferation was examined in rat liver after administration of equal concentrations (1 mmol/kg body weight) of 1,10-bis(carboxymethylthiodecane) (BCMTD), 1-mono(carboxymethylthiotetradecane) (CMTTD), 1-mono(carboxymethylthiooctane) (CMTO), 1-mono(carboxyethylthiotetradecane) (CETTD), palmitic acid and hexadecanedioic acid (HDDA). BCMTD, a non-beta-oxidizable and non-omega-oxidizable sulphur-substituted fatty acid analogue was considerably more potent than CMTTD (only non-beta-oxidizable) in inducing enlargement of the liver and increasing peroxisomal activities (monitored by peroxisomal beta-oxidation, palmitoyl-CoA hydrolase and catalase activities). Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number and size of peroxisomes and the relative volume fraction of the peroxisomes. All these cellular responses were more marked with BCMTD than compared with CMTTD. CMTO, a non-beta-oxidizable fatty acid analogue containing a lower hydrophobic alkyl-end than CMTTD and CETTD (a beta-oxidizable fatty acid analogue), showed a slight increase (1.4-1.8-fold) of peroxisomal beta-oxidation and caused marginally morphological changes of peroxisomes compared with CMTTD and BCMTD. The most striking effect of the alkylthiopropionic acid (CETTD) was an enhancement of the hepatic triacylglycerol level. Palmitic acid and hexadecanedioic acid only marginally affected the peroxisomal activities, but no morphological changes of peroxisomes and fat droplets were observed. The presented data strongly suggest that a minimal structural requirement for a peroxisome proliferator may be (1) a carboxylic acid group linked to (2) a hydrophobic backbone which (3) cannot be beta-oxidized i.e., the fatty acid analogues have a sulphur atom in the beta-position. It is also conceivable that blockage for omega-oxidation may potentiate the peroxisome-proliferating activities in as much as BCMTD was more potent than CMTTD. Two mitochondrial marker enzymes, carnitine palmitoyltransferase and succinate phenazine methosulphate oxidoreductase were differently affected after administration of the investigated compounds. Furthermore, BCMTD and CMTTD as well as HDDA treatments increased the number of mitochondria, but the mitochondria tended to be smaller. The overall results presented here indicate that the structural requirements for proliferation of mitochondria are not identical to those for proliferation of peroxisomes.

Animals↗
PubMed1987

Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein.

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2-. species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.

Alcaligenes↗
PubMed1982

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats. Effects of 3-methylcholanthrene and beta-naphthoflavone.

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b5, NADH- and NADPH- dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-488) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and beta-naphthoflavone, increased the cytochrome content 3- to 10-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 micrometers alpha-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.

2-Acetylaminofluorene↗
PubMed1984

Pentose pathway of glucose metabolism in isolated granular pneumocytes. Metabolic regulation and stimulation by paraquat.

Activity of the pentose phosphate pathway of glucose metabolism was measured in isolated granular pneumocytes under a variety of metabolic conditions known to alter this pathway in intact lungs. Granular pneumocytes were isolated by trypsinization of rat lungs and maintained in primary culture for 24 hr before use. Cells were incubated for 1 hr at 37 degrees with 5.5 mM glucose specifically labeled as 1-14C, 6-14C, U-14C, or 5-3H for determination of glucose utilization, pentose cycle activity, and partition of CO2 production between mitochondrial and pentose pathways. With control cells, total glucose utilization was 111 +/- 4.8 nmoles X hr-1 X (10(6) cells)-1 (mean +/- S.E., N = 19), and 2.2% was metabolized by the pentose cycle. Pentose cycle CO2 production was 7.3 nmoles X hr-1 X (10(6) cells)-1 representing 34% of total CO2 production. Dinitrophenol (50 microM) stimulated mitochondrial CO2 production 5-fold but had no effect on the pentose cycle activity. Phenazine methosulfate (5 microM) had no effect on mitochondrial activity but stimulated pentose cycle activity 15-fold. Antimycin A (0.4 micrograms/ml) markedly inhibited both pathways. After a 30-min preincubation with paraquat (3 mM), the pentose cycle CO2 production increased to 107 nmoles X hr-1 X (10(6) cells)-1 accounting for 39.6% of glucose utilization and 88.4% of CO2 production. Mitochondrial CO2 production was unchanged with paraquat. These studies demonstrate that the pentose cycle in resting granular pneumocytes accounts for a major fraction of the CO2 production from glucose and that activity of this pathway is regulated by the utilization of cytoplasmic reducing equivalents. Paraquat produces marked stimulation of pentose cycle activity in granular pneumocytes, resulting in maximal utilization of cytoplasmic NADPH.

Animals↗