Showing papers in "Biochemical Journal in 1980"
TL;DR: An O2- -dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.
Abstract: Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2- -dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.
1,611 citations
TL;DR: A rapid procedure for measuring the specific radioactivity of phenylalanine in tissues was developed, which facilitates the accurate determination of rates of protein synthesis in a wide range of tissues by injection of 150 mumol of L-[4-(3)H]phenylAlanine/100 g body wt.
Abstract: A rapid procedure for measuring the specific radioactivity of phenylalanine in tissues was developed. This facilitates the accurate determination of rates of protein synthesis in a wide range of tissues by injection of 150 mumol of L-[4-(3)H]phenylalanine/100 g body wt. The large dose of amino acid results in a rapid rise in specific radioactivity of free phenylalanine in tissues to values close to that in plasma, followed by a slow but linear fall. This enables the rate of protein synthesis to be calculated from measurements of the specific radioactivity of free and protein-bound phenylalanine in tissues during a 10 min period after injection of radioisotope.
903 citations
TL;DR: Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate,osphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4 degrees C.
Abstract: Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C Liposomal stability, ie the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum Whole blood and to some extent plasma were less detrimental to stability than was serum (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro
602 citations
TL;DR: The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.
Abstract: The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.
530 citations
TL;DR: In a search for new inhibitors of the nuclear enzyme poly(ADP-ribose) synthetase, it was found that various benzamides substituted in the 3-position were the most inhibitory compounds found to date.
Abstract: In a search for new inhibitors of the nuclear enzyme poly(ADP-ribose) synthetase, it was found that various benzamides substituted in the 3-position were the most inhibitory compounds found to date. Two of the benzamides, 3-aminobenzamide and 3-methoxybenzamide, were found to be competitive inhibitors, with Ki values or less than 2 microM.
498 citations
TL;DR: An improved perfusion system for the isolated rat heart is described and insulin was not required for maximal rates of glucose consumption at near-physiological workloads, in contrast with subphysiological, workloads when glucose was the sole added substrate.
Abstract: 1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.
446 citations
TL;DR: Benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide was found to be an excellent substrate for the fluorimetric assay of cathepsin B, and arginine 4,methyl- 7-cOUmarylamides for cathepsypsin H.
Abstract: Benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide was found to be an excellent substrate for the fluorimetric assay of cathepsin B, and arginine 4-methyl-7-coumarylamide for cathepsin H. Procedures were developed that are very convenient, and avoid the hazards associated with the use of naphthylamides.
383 citations
TL;DR: The internal mitochondria must be considered in any study of synaptosomal transport, as evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase.
Abstract: A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.
377 citations
TL;DR: Evidence is presented to suggest that swainsonine is a reversible active site-directed inhibitor of lysosomal alpha-mannosidase.
Abstract: An indolizidine alkaloid (swainsonine) was isolated from the plant Swainsona canescens. Swainsonine is a specific and potent inhibitor of alpha-mannosidase (EC 3.2.1.24) and when administered to animals produces a phenocopy of the genetically based lysosomal storage disease, mannosidosis. Evidence is presented to suggest that swainsonine is a reversible active site-directed inhibitor of lysosomal alpha-mannosidase.
346 citations
TL;DR: Inhibition and substrate-specificity studies suggest that pig aortic endothelial and smooth-muscle cells in culture possess three distinct ectonucleotidase systems that could be of importance in the regulation of neurotransmission, blood platelet function and vasodilation.
Abstract: 1. Pig aortic endothelial and smooth-muscle cells in culture rapidly catabolize exogenous ATP, ADP or AMP. 2. In both cell types catabolism is due to Mg2+-stimulated ectoenzymes. 3. Inhibition and substrate-specificity studies suggest that both cell types possess three distinct ectonucleotidases, namely nucleoside triphosphatase (EC 3.6.1.15), nucleoside diphosphatase (EC 3.6.1.6) and 5′-nucleotidase (EC 3.1.3.5), as well as nucleoside diphosphate kinase (EC 2.7.4.6). 4. These ectonucleotidase systems could be of importance in the regulation of neurotransmission, blood platelet function and vasodilation.
321 citations
TL;DR: Several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which the authors have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.
Abstract: 1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese ( fa/fa ) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O 2 consumption, and glucose oxidation to CO 2 . The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.
TL;DR: The low-molecular-weight stimulator of phosphofructokinase has been purified from rat liver and is tentatively identified as fructose 2,6-bisphosphate.
Abstract: The low-molecular-weight stimulator of phosphofructokinase [Van Schaftingen, Hue & Hers (1980) Biochem. J. 192, 887-895] has been purified from rat liver. It was completely destroyed upon incubation with 0.01 M-HCl for 10 min at 20 degrees C and fructose 6-phosphate and a reducing power equivalent in amount to the acid-labile organic phosphate were formed. It was therefore tentatively identified as fructose 2,6-bisphosphate.
TL;DR: The results indicate that the preponderance of glycosaminoglycans in the proteoglycan molecule is a main reason for both polydispersity and hydrophilicity of the proteglycan preparation, and suggest that the enzymic procedures could prove useful as a method to obtain new information about the structure and properties of proteogly can core molecules.
Abstract: Digestion of chick-embryo cartilage proteoglycan (type H) with chondroitin AC II lyase or keratanase, in the presence of EDTA, N-ethylmaleimide, phenylmethanesulphonyl fluoride and pepstatin, resulted in the removal of the bulk of the chondroitin sulphate or keratan sulphate chains respectively, without altering the protein portion of the macromolecule. An exhaustive treatment of the proteoglycan with chondroitin AC II lyase followed by digestion with keratanase yielded a core fraction having the enzymically modified linkage oligosaccharides. Zonal sedimentation of this core preparation on a sucrose gradient in 0.5% SDS resulted in a single narrow band with a sedimentation coefficient of 6S. In 4 M-guanidinium chloride, the core preparation showed a tendency to aggregate to multiple-molecular-weight forms which could dissociate in the presence of Triton X-100. The results indicate that the preponderance of glycosaminoglycans in the proteoglycan molecule is a main reason for both polydispersity and hydrophilicity of the proteoglycan preparation, and further suggest that the enzymic procedures could prove useful as a method to obtain new information about the structure and properties of proteoglycan core molecules.
TL;DR: A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O(2) (-) production, is proposed as the molecular mechanism for the enhancement of H( 2)O(2%) formation rates observed on addition of protophores, ionophores and Ca(2+).
Abstract: Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3-1.0nmol of H(2)O(2)/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca(2+) also markedly stimulated H(2)O(2) production by rat heart mitochondria. The enhancement of H(2)O(2) generation in antimycin-supplemented mitochondria and the increased O(2) uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca(2+) concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate-ubiquinone reductase activity, also decreased mitochondrial H(2)O(2) production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O(2) (-), the precursor of mitochondrial H(2)O(2). This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c(1) (3+) to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O(2) (-). The effect of protophores, ionophores and Ca(2+) is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c(1) [Wikstrom & Berden (1972) Biochim. Biophys. Acta283, 403-420; Mitchell (1976) J. Theor. Biol.62, 337-367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O(2) (-) production, is proposed as the molecular mechanism for the enhancement of H(2)O(2) formation rates observed on addition of protophores, ionophores and Ca(2+).
TL;DR: Results extend the previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations, consistent with the hypothesis that the reducing potential of tissues decreases in senescence.
Abstract: 1. Previous results from this laboratory demonstrated that the erythrocyte content of reduced glutathione (GSH) decreased as a function of both increasing cell age and mouse age [Abraham, Taylor & Lang (1978) Biochem. J. 174, 819-825]. In the present investigation glutathione concentrations were determined in other tissues of the C57BL/6J mouse of different ages (6--31 months) throughout the life-span. 2. At all ages the total glutathione and the GSH concentrations in liver were 3 times that in kidney and 10 times that in heart. In the old (31 months) mouse the GSH contents were lower by 30% in the liver, 34% in the kidney and 20% in the heart than in the mature (17--23 months) animals. 3. The oxidized glutathione (GSSG) concentrations of the tissues did not vary with age and constituted less than 3% of the total glutathione. 4. The decreased in GSH concentrations were not due to changes in organ weights, which were constant from 10 to 36 months of age. 5. These findings extend our previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations. Further, this is consistent with our hypothesis that the reducing potential of tissues decreases in senescence.
TL;DR: In this paper, a cationic phthalocyanin-like dye, based on cinchomeronic acid, was used to stain foetal and adult rat tail tendon and adult rabbit Achilles tendon.
Abstract: Proteoglycan in foetal- and adult-rat tail tendon and adult-rabbit achilles tendon was stained for electron microscopy with a cationic phthalocyanin-like dye, based on cinchomeronic acid, in a ‘critical electrolyte concentration’ method [Scott (1973) Biochem. Soc. Trans. 1, 787-806). Provided that the tissue was fixed with glutaraldehyde or formaldehyde, regular orthogonal perifibrillar arrays of filamentous material (proteoglycan) were observed, but no intra-fibrillar proteoglycan was seen. Specific proteoglycan-collagen interactions are inferred, and a model is proposed. Without fixation, the filamentous arrays disaggregated in the MgCl2 solutions (0.3 M) used during staining. End-to-end proteoglycan aggregation is implied. Tendon and cartilage are compared. Problems of electron-histochemical localization of extended space-filling polyanions by the use of cationic electron-dense precipitants are discussed, particularly polyanion-domain collapse, specificity of staining and fixation. A two-stage staining procedure that markedly enhances contrast is described, based on the multivalent nature of the dye, and the consequent anion-exchange properties of the dye-polyanion complex.
TL;DR: The present studies indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intram itochondrial enzymes by Ca2- is of likely physiological significance.
Abstract: 1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.
TL;DR: It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor.
Abstract: The concentration-dependence on exogenous ATP of activation and inhibition of mast-cell histamine secretion, phosphatidylinositol labelling and leakage of metabolites shows that all these functions are regulated by the free acid ATP4-. Maximal histamine secretion and phosphatidylinositol labelling occur with ATP4- at approx. 2 microM, but higher concentrations, which cause inhibition of secretion and phosphatidylinositol labelling, are required to maximize leakage of 32P-labelled metabolites. Both enhancement and inhibition of phosphatidylinositol labelling (due to low and high concentrations of ATP4- respectively) are rapid in onset; histamine secretion is characterized by a delay, especially at low concentrations of ATP4- (approx. 1 microM). Phosphatidylinositol labelling and histamine secretion are dependent on extracellular Ca2+. Metabolite leakage due to the presence of exogenous ATP4- is slow and does not require Ca2+. Of 18 analogues of ATP that were tested, only four were agonists for secretion, and only these four permitted leakage of 32P-labelled metabolites. It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor. For mast cells stimulated with ATP4- enhancement of phosphatidylinositol metabolism is not sufficient by itself to cause Ca2+-dependent secretion.
TL;DR: The idea that zinc-thionein can function in zinc homeostasis as a reservoir of zinc, releasing the metal to zinc-requiring metalloenzymes according to need is supported.
Abstract: The ability of rat liver zinc-thionein to donate its metal to the apo-enzymes of the zinc enzymes horse liver alcohol dehydrogenase, yeast aldolase, thermolysin, Escherichia coli alkaline phosphatase and bovine erythrocyte carbonic anhydrase was investigated. Zinc-thionein was as good as, or better than, ZnSO4, Zn(CH3CO2)2 or Zn(NO3)2 in donating its zinc to these apo-enzymes. Apo-(alcohol dehydrogenase) could not be reactivated by zinc salts or by zinc-thionein. Incubation of the other apo-enzymes with near-saturating amounts of zinc as ZnSO4, Zn(CH3CO2)2, Zn(NO3)2, or zinc-thionein resulted in reactivation of the apo-enzymes. With apo-aldolase zinc-thionein gave 100% reactivation within 30min. Reactivation by ZnSO4 and Zn(CH3CO2)2 was complete and instantaneous. Zinc-thionein was somewhat better than Zn(NO3)2 in completely reactivating apo-thermolysin. With apo-(alkaline phosphatase) 43% reactivation was obtained with Zn(CH3CO2)2 and 18% with zinc-thionein. With apo-(carbonic anhydrase) zinc-thionein was better than ZnSO4, Zn(CH3CO2)2 or Zn(NO3)2, with a maximal reactivation of 54%. That zinc was really being transferred from zinc-thionein to apo-(carbonic anhydrase) was shown by the fact that 2,6-pyridine dicarboxylic acid and 1,10-phenanthroline had minimal effects on the reactivation of apo-(carbonic anhydrase) when added after the incubation {[apo-(carbonic anhydrase)+zinc thionein]+chelator}, but inhibited reactivation when added before the incubation {apo-(carbonic anhydrase)+[zinc-thionein+chelator]}. These observations support the idea that zinc-thionein can function in zinc homeostasis as a reservoir of zinc, releasing the metal to zinc-requiring metalloenzymes according to need.
TL;DR: Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure and changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo.
Abstract: Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.
TL;DR: An enzyme was isolated from chlorobenzoate-grown cells, which converted the 4-carboxymethylenebut-2-en-4-olides into maleoylacetic acid, and values from an ordinary muconate cycloisomerase II were obtained.
Abstract: 1. An enzyme for the cycloisomerization of 2- and 3-chloro-cis,cis-muconic acid was isolated from 3-chlorobenzoate-grown cells of Pseudomonas sp. B13. It was named muconate cycloisomerase II, because it could it clearly be differentiated by its Km and Vmax. values from an ordinary muconate cycloisomerase, which functioned in benzoate catabolism and exhibited low activity with the chlorinated substrates. 2-Chloro-cis,cis-muconic acid was converted into trans- and 3-chloro-cis,cis--muconic acid into cis-4-carboxymethylenebut-2-en-4-olide together with dehalogenation. 2. An enzyme was isolated from chlorobenzoate-grown cells, which converted the 4-carboxymethylenebut-2-en-4-olides into maleoylacetic acid.
TL;DR: Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described, and there are significant differences between the catalytic properties of G SH S- transferase omega and the cationic GSHS-transferases.
Abstract: Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.
TL;DR: Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-t RNAs) and has no effect on the transfer of [14C]isoleucine from the enzyme .
Abstract: Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.
TL;DR: The activity of phosphofructokinase present in a liver Sephadex filtrate was increased by a low-molecular-weight effector, which could be isolated from a liver extract by ultrafiltration, gel filtration or heat treatment, but was rapidly destroyed in trichloroacetic acid, even in the cold.
Abstract: 1 Recycling of metabolites between fructose 6-phosphate and triose phosphates has been investigated in isolated hepatocytes by the randomization of carbon between C((1)) and C((6)) of glucose formed from [1-(14)C]galactose 2 Randomization of carbon atoms was regularly observed with hepatocytes isolated from fed rats and was then little influenced by the concentration of glucose in the incubation medium It was decreased by about 50% in the presence of glucagon 3 Randomization of carbon atoms by hepatocytes isolated from starved rats was barely detectable at physiological concentrations of glucose in the incubation medium, but was greatly increased with increasing glucose concentrations It was nearly completely suppressed by glucagon These large changes can be attributed to parallel variations in the activity of phosphofructokinase 4 The main factors that appear to control the activity of phosphofructokinase under these experimental conditions are the concentration of fructose 6-phosphate, the concentration of fructose 1,6-bisphosphate and also the affinity of the enzyme for fructose 6-phosphate 5 The affinity of phosphofructokinase for fructose 6-phosphate was diminished by incubation of the cells in the presence of glucagon and also by filtration of an extract of hepatocytes through Sephadex G-25 and by purification of the enzyme When assayed at 025 or 05mm-fructose 6-phosphate, the activity of phosphofructokinase present in a liver Sephadex filtrate was increased by a low-molecular-weight effector, which could be isolated from a liver extract by ultrafiltration, gel filtration or heat treatment, but was rapidly destroyed in trichloroacetic acid, even in the cold This effector appears to be a highly acid-labile phosphoric ester Its concentration was greatly increased in hepatocytes incubated in the presence of glucose and was decreased in the presence of glucagon
TL;DR: The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydration complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
Abstract: A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
TL;DR: The feeding of high-fat diets gave a 1.4-2.4% increase in total liver peroxisomal beta-oxidation and a similar increase in specific activity, and a 2.5-fold increase was seen in the specific activity of purified peroxISomal preparations.
Abstract: Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.
TL;DR: Observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.
Abstract: Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.
TL;DR: In this article, the authors investigated the role of peroxidase in the defense against H2O2-related free radicals in epimastigotes, and found that the enzyme was not sufficient to defend against free radicals.
Abstract: The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.
TL;DR: Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium.
Abstract: Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1′-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous “priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.
TL;DR: The sequence of three alcohol dehydrogenase alleloenzymes from the fruitfly Drosophila melanogaster has been determined by the sequencing of peptides produced by trypsin, chymotrypsin), thermolysin, pepsin and Staphylococcus aureus-V8-proteinase digestion.
Abstract: The sequence of three alcohol dehydrogenase alleloenzymes from the fruitfly Drosophila melanogaster has been determined by the sequencing of peptides produced by trypsin, chymotrypsin, thermolysin, pepsin and Staphylococcus aureus-V8-proteinase digestion. The amino acid sequence shows no obvious homology with the published sequences of the horse liver and yeast enzymes, and secondary structure prediction suggests that the nucleotide-binding domain is located in the N-terminal half of the molecule. The amino acid substitutions between AdhN-11 (a point mutation of AdhF), AdhS and AdhUF alleloenzymes were identified. AdhN-11 alcohol dehydrogenase differed from the other two by a glycine-14-(AdhS and AdhUF)-to-aspartic acid substitution, the AdhS enzyme from AdhN-11 and AdhUF enzymes by a threonine-192-(AdhN-11 and AdhUF)-to-lysine (AdhS) substitution and the AdhUF enzyme was found to differ by an alanine-45-(AdhS and AdhN-11)-to-aspartic acid (AdhUF) charge substitution and a 'silent' asparagine-8-(AdhS and AdhN-11)-to-alanine (AdhUF) substitution. Detailed sequence evidence has been deposited as Supplementary Publication SUP 50107 (36 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.