Showing papers in "Biochemical Journal in 1976"

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

Abstract: Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.

Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

Abstract: Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.

Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma

Abstract: The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

Abstract: 1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

Abstract: The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass.

Abstract: The effects of growth-suppressing and muscle-wasting treatments on muscle protein turnover and amino acid concentrations were determined in vivo. All treatments depressed protein synthesis and some treatments depressed protein breakdown. Only prolonged starvation increased protein breakdown. Muscle protein mass is regulated primarily through alterations in protein synthesis in all except emergency conditions. The increased concentrations of the branched-chain amino acids indicate that they are unlikely to be involved in this regulation.

Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus

Abstract: Intervertebral disc is a highly specialized cartilaginous tissue, containing two genetic types of collagen (I and II). Analysis of peptides from a CNBr digest of collagen showed that the proportions of I and II varied gradually and inversely across pig annulus fibrosus, with exclusively type I at the extreme outer edge and exclusively type II in the nucleus pulposus.

Subunit composition and structure of subcomponent C1q of the first component of human complement.

Abstract: 1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.

Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats.

Abstract: Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.

Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver.

Abstract: 1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% alpha-helical form, 18% beta-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing 'high'-and 'low'-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 X 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.

Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick.

Abstract: 1 A method is described for the electrophoretic analysis of intact myosin in polyacrylamide gel in a buffer system containing 002 M-pyrophosphate and 10% (v/v) glycerol, pH 88 2 In this system chicken skeletal-muscle myosins reveal five distinct electrophoretic components, three components from the fast-twitch posterior latissimus dorsi muscle and two slower-migrating components from the slow-twitch anterior latissimus dorsi muscle 3 The Ca2+-activated ATPase (adenosine triphosphatase) activity of myosin components was measured by densitometric scanning of the gel for the Ca3(PO4)2 precipitate formed during the ATPase reaction and subsequently for stained protein Each component from the same muscle appears to have identical ATPase activity, but components from the fast-twitch muscle had an activity 22 times higher than those from the slow-twitch muscle 4 On re-electrophoresis in the same buffer system, individual fractions of fast-twitch myosin did not reproduce the three-band pattern of the original myosin, but migrated at rates consistent with their original mobility 5 Analysis of the mobility of the three fast-twitch myosin components in gels of different concentrations suggests that they are not stable oligomers of each other 6 It is suggested that these components of fast-twitch myosin and slow-twitch myosin are isoenzymes of myosin

Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions.

Abstract: 1. A simple method is described for calculating the free concentrations of all species in a mixture of several ionic components that associate at equilibrium to any extent and with any stoicheiometry. 2. It can readily be adapted to take account of species such as protons for which the free rather than the total concentrations are controlled. 3. It was applied to mixtures of adenine nucleotides, Mg2+ and other ions relevant to the study of glucokinase (EC 2.7.1.2), but the qualitative conclusions are not peculiar to this system. 4. ATP exists in a high and nearly constant proportion (about 80%) as MgATP2- in solutions in which the total MgCl2 concentration exceeds the total ATP concentration by 1-10 mM. 5. By contrast, the proportion of ATP present as MgATP2- varies greatly if the total MgCl2 and total ATP concentrations are varied in constant proportion.

Dipeptidyl peptidase IV, a kidney brush-border serine peptidase.

Abstract: Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.

The primary inhibitor of plasmin in human plasma.

Abstract: A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an α2 protein. By crossed immunoelectrophoresis the α2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The α2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 μmol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and α2 macroglobulin appear.

Reactions involving superoxide and normal and unstable haemoglobins.

Abstract: Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Abstract: Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.

Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation.

Abstract: With dimethyl sulphoxide instead of butanol in the thiobarbituric acid assay for sialic acid, a non-fading chromophore with lambdamax. = 549 nm was produced in a homogeneous solution, allowing dilution of the test mixture in case of high colour yield. This test adapted well to studies on alkaline de-O-acetylation. Bovine and rat submaxillary mucins, and rabbit Tamm-Horsfall urinary sialoproteins contain O-acetyl isomers of neuramine acid that are resistant to the thiobarbituric acid assay. Alkaline de-O-acetylation converted resistant O-acetylneuraminic acid into thiobarbituric acid-reactive sialic acid, and such conversion paralleled de-O-acetylation as measured by the ferric hydroxamate method. The colour increment was similar when the alkaline treatment of bovine submaxillary mucin either preceded or followed the acid hydrolysis. Only alkaline preptreatment was effective with rat submaxillary mucin. By selecting optimal conditions for alkaline de-O-acetylation, O-acetyl isomers can be accurately assessed by the thiobarbituric acid assay.

Myosin light-chain phosphatase.

Abstract: 1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.

L-lactate transport in Ehrlich ascites-tumour cells.

Abstract: Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [ 14 C]lactate or concomitant H + ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH - ions or 1:1 symport with H + ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a V max. as high as 680 nmol/min per mg of protein at pH 6.2 and 37°C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, K i = 6.3 mM), which were themselves transported, (b) the non-transportable analogues α-cyano-4-hydroxycinnamate (K i = 0.5 mM), α-cyano-3-hydroxycinnamate (K i = 2mM) and DL-p-hydroxyphenyl-lactate (K i = 3.6 mM) and (c) the thiol-group reagent mersalyl (K i = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an “inhibitor stop” allowed measurements of the initial rates of net influx and of net efflux of [ 14 C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.

A sparing effect of increased plasma fatty acids on muscle and liver glycogen content in the exercising rat

Abstract: Increasing plasma free fatty acids decreased the degree of glycogen depletion, and increased the citrate concentration, in slow-red (soleus) and fast-red (deep portion of vastus lateralis) muscle during exercise (approx. 50% depletion of glycogen, as against 75% in control animals). There was no effect in fast-white muscle (superficial portion of vastus lateralis). Glycogen concentration in the liver decreased by 83% in controls, but only by 23% in animals with increased free fatty acids during exercise. The decreased glycogen depletion may be partly explained by the findings that (a) plasma-insulin concentration was two- to three-fold higher in animals with increased plasma free fatty acids and (b) the exercise-induced increase in plasma glucagon was lessened by increased free fatty acids. Blood glucose was higher in the animals with increased free fatty acids after the exercise. The rats with increased plasma free fatty acids utilized approx. 50% as much carbohydrate as did the controls during the exercise.

The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit.

Abstract: 1. A series of defined peptides which span the complete sequence were produced from troponin I isolated from white skeletal muscle of the rabbit. 2. Two peptides, CF1 (residues 64-133) and CN4 (residues 96-117) inhibited the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition was potentiated by tropomyosin and the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition, unlike that of troponin I and peptides derived from it, was not potentiated by tropomyosin. 4. The most active inhibitor, peptide CN4, was 45-75% as effective as troponin I when compared on a molar basis. The inhibitory peptide, CN4, and also whole troponin I were shown by affinity chromatography to interact specifically with actin. 5. A strong interaction with troponin C was demonstrated with peptide CF2 (residues 1-47), from the N-terminal region of troponin I. Somewhat weaker interactions were shown with peptides CN5 (residues 1-21) and with the inhibitory peptide CN4. 6. The significance of these interactions for the mechanisms of action of troponin I is discussed.

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

Abstract: Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution

Abstract: An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes Protoporphyrin appears to be highly aggregated under all conditions studied At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization At approx 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions

The regulation of folate and methionine metabolism.

Abstract: 1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the 'methylfolate-trap hypothesis'. This is discussed. 8...

Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation.

Abstract: 1. Melanosomal tyrosinase was isolated from normal C57B1 mice, and a comparison of the tyrosine-hydroxylation and dopa (3,4-dihydroxyphenylalanine)-oxidation activities of this enzyme was made. 2. The results indicate that in the absence of dopa cofactor, this enzyme is capable of tyrosine hydroxylation, but with very little subsequent dopa oxidation and melanin formation. 3. This mechanism of enzyme action may play an important role in the intracellular regulation of melanin formation. 4. Further, dopa appears to act as a positive allosteric effector for tyrosine hydroxylation by tyrosinase, in addition to its known activity as a hydrogen donor for the reaction.

Studies on the catabolism of NG-methylarginine, NG, NG-dimethylarginine and NG, NG-dimethylarginine in the rabbit

Abstract: 1. The routes of elimination of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine were investigated in the rabbit. 2. Analyses showed low plasma concentrations of these amino acids (around 1 nmol/ml) and ratios similar to those found in tissue proteins. The concentrations of these amino acids in extracts of brain, kidney, liver and spleen were similar except that liver had a lower concentration of Ng-methylarginine and Ng, Ng-dimethylarginine. Cerebrospinal fluid contained traces of each amino acid.

Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells.

Abstract: 1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.

Studies on the mechanism of induction of haem oxygenase by cobalt and other metal ions.

Abstract: Cobalt ions (Co2+) are potent inducers of haem oxygenase in liver and inhibit microsomal drug oxidation probably by depleting microsomal haem and cytochrome P-450. Complexing of Co2+ ions with cysteine or glutathione (GSH) blocked ability of the former to induce haem oxygenase. When hepatic GSH content was depleted by treatment of animals with diethyl maleate, the inducing effect of Co2+ on haem oxygenase was significantly augmented. Other metal ions such as Cr2+, Mn2+, Fe2+, Fe3+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ were also capable of inducing haem oxygenase and depleting microsomal haem and cytochrome P-450. None of these metal ions had a stimulatory effect on hepatic haem oxidation activity in vitro. It is suggested that the inducing action of Co2+ and other metal ions on microsomal haem oxygenase involves either the covalent binding of the metal ions to some cellular component concerned directly with regulating haem oxygenase or non-specific complex-formation by the metal ions, which depletes some regulatory system in liver cells of an essential component involved in controlling synthesis or activity of the enzyme.

Ascorbic acid, metal ions and the superoxide radical.

Abstract: 1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.