bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism of palmitoyl-CoA inhibition of Ca2+ uptake in liver and heart mitochondria
Authors: Beatrice M C , Pfeiffer D R
Published: August 2015

The mechanism by which palmitoyl-CoA inhibits Ca2+ uptake in liver and heart mitochondria was examined. At a given concentration of palmitoyl-CoA, the extent of inhibition is inversely related to the concentration of the respiratory substrate succinate. Palmitoyl-CoA inhibition of uncoupler-stimulated respiration and respiration stimulated by ionophore-A23187-induced Ca2+ cycling is also relieved by high succinate concentrations. These effects of palmitoyl-CoA and succinate concentration are distinct from the increase in inner-membrane permeability, which can be produced by palmitoyl-CoA and Ca2+ [Beatrice, Palmer & Pfeiffer (1980) J. Biol. Chem. 255, 8663-8671]. The apparent K0.5 of the mitochondrial Ca2+ pump is not altered by palmitoyl-CoA. No or negligible effects of palmitoyl-CoA on the Ca2+-uptake rate are observed when ascorbate replaces succinate as an energy source. These findings, together with the known activity of palmitoyl-CoA as a competitive inhibitor of the dicarboxylate carrier [Morel, Lauquin, Lunardi, Duszynski & Vignais (1974) FEBS Lett. 39, 133-138], indicate that palmitoyl-CoA inhibits energy-linked Ca2+ transport by limiting the rate of electron transport through limitation of succinate entry into the mitochondria rather than by directly inhibiting the Ca2+ carrier.
α-Lactalbumin and lactose concentrations in rat milk during lactation
Authors: Nicholas K R , Hartmann P E , McDonald B L
Published: August 2015

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.
A comparison of ornithine decarboxylases from normal and SV40-transformed 3T3 mouse fibroblasts
Authors: Weiss J M , Lembach K J , Boucek R J
Published: August 2015

Ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) has been purified from 3T3- and SV40-transformed 3T3 mouse fibroblasts by affinity chromatography, and the physicochemical properties of the two enzymes compared. Measured properties include molecular weight of the active species, subunit molecular weight and specific activity of the purified enzymes, kinetic parameters, thermostability, degradation rate in vivo and immunological cross-reactivity. Although crude extracts of the transformant possess more ornithine decarboxylase activity per mg of protein than the parent strain, there is no evidence for the appearance of an altered form of the enzyme in these cells. The results reported in the present paper indicate that the increased ornithine decarboxylase activity in the transformed cells is the result of higher enzyme biosynthesis de novo.
2,3-Dihydroxybenzoate pathway in Pseudomonas putida. 1H n.m.r. study on the ring-cleavage site
Authors: Andreoni V , Canonica L , Galli E , Gennari C , Treccani V
Published: August 2015

1. Ring cleavage of 2,3-dihydroxybenzoate by cell-free extracts of Pseudomonas putida leads to 2-hydroxy-6-oxo-(2Z,4E)-hexa-2,4-dienoic acid and CO2. 2. The 1H n.m.r. spectrum of the ring-fission product obtained in a 2H2O solution suggests that the extra-diol cleavage occurs between C-3 and C-4.
Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study
Authors: Burton G , Jordan P M , MacKenzie N E , Fagerness P E , Scott A I
Published: August 2015

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.
Reactivity of photoreduced cytochrome aa3 complexes with molecular oxygen
Authors: Nicholls P , Chanady G A
Published: August 2015

Cytochrome c oxidase (ox heart cytochrome aa3) is reduced on illumination in the presence of a photocatalyst system containing deazaflavin and EDTA. The photo-reduced enzyme reacts with oxygen at neutral pH to give a form of ferric enzyme, whereas a corresponding sample partially reduced by light in the absence of any photocatalyst reacts with oxygen to give an oxyferri species (‘oxygenated’ enzyme). Reduction by the photocatalyst system at an alkaline pH value (9.0) also gives rise to fully reduced oxidase (both haem groups ferrous). At these pH values the immediate product after oxygen addition is a species with a 605-606 nm absorption band, not identical with ferrous cytochrome a, but capable of oxidizing added cytochrome c. This intermediate, which is unstable at neutral pH, may be analogous to the ‘compound B’ obtained by Chance and co-workers [Chance, Saronio & Leigh (1975) J. Biol. Chem. 250, 9226-9237; Chance, Saronio & Leigh (1979) Biochem. J. 177, 931-941] at low temperatures.
Prostaglandin biosynthesis and lipolysis in subcellular fractions from rabbit kidney medulla
Authors: Erman A , Raz A
Published: August 2015

Three separate prostaglandin-generating activities are associated with plasma membranes, mitochondria and microsomal fractions from rabbit kidney medulla. In the plasma membranes and mitochondria, but not in microsomal fractions, Ca2+ ions stimulate the activity of phospholipase A2, yielding selective release of arachidonic acid and linoleic acid and concomitant increase in prostaglandin E2 formation.
Vitamin K-dependent carboxylation and vitamin K epoxidation. Evidence that the warfarin-sensitive microsomal NAD(P)H dehydrogenase reduces vitamin K1 in these reactions
Authors: Wallin R , Suttie J W
Published: August 2015

Passage of a Triton X-100-solubilized microsomal systems in vitro that are used to study these reactions is the warfarin-sensitive NAD(P)H dehydrogenase.
Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes
Authors: Janski A M , Cornell N W
Published: August 2015

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.
Chemical, immunological and catalytic properties of 2′:3′-cyclic nucleotide 3′-phosphodiesterase purified from brain white matter
Authors: Kurihara T , Nishizawa Y , Takahashi Y , Odani S
Published: August 2015

The amino acid composition, isoelectric point, specificity of the antibody raised and various catalytic properties were determined for 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) purified from bovine brain white matter by a procedure involving solubilization with elastase (EC 3.4.21.11).
Investigations of rat liver uroporphyrinogen decarboxylase. Comparisons of porphyrinogens I and III as substrates and the inhibition by porphyrins
Authors: Smith A G , Francis J E
Published: August 2015

1. The decarboxylations of uroporphyrinogens, hepta-, hexa- and penta-carboxyporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in rat liver supernatant have been compared as functions of substrate concentrations. Although Km and Vmax. (for total porphyrinogens formed) were estimated, prophyrinogens and CO2 produced at 1 microM were considered to be a better indication of real relative rates, owing to substrate/product inhibitions. Uroporphyrinogen III was the best substrate by the criteria of Km/Vmax. and decarboxylation at 1 microM and was converted into coproporphyrinogen more quickly than its series-I isomer. 2. The difference between uroporphyrinogens I and III as substrates was confirmed by using a mixture of [14C8]uroporphyrinogens, the discrimination occurring principally in the first decarboxylation. 3. Porphyrins, especially oxidation products of the substrates, inhibited the enzyme. Heptacarboxyporphyrin III was the most effective inhibitor of both uroporphyrinogen III and heptacarboxyporphyrinogen III conversion into coproporphyrinogen. 4. Rapid analysis of the livers from rats made porphyric with hexachlorobenzene demonstrated that substantial quantities of the tetrapyrroles were present in vivo as the porphyrinogens (21-42%). 5. Enzymic decarboxylation of uroporphyrinogen III in 2H2O-containing buffer gave [2H4]coproporphyrinogen. 6. Rats treated with cycloheximide for 10h showed no decrease in uroporphyrinogen decarboxylase activity/mg of protein, suggesting a relatively slow turnover of the enzyme.
Characterization of human muscle myosins with respect to the light chains
Authors: Volpe P , Biral D , Damiani E , Margreth A
Published: August 2015

Isolated myosins from human predominantly fast and slow muscles, human neonatal and foetal muscle were examined for light chain composition by one- and two-dimensional electrophoresis. The LC1F, LC2F and LC3F light chains were identical with their counterparts from rabbit fast myosin. Human LC1S was identified by correlative criteria as a single component having a molecular weight slightly lower than, but an electric charge similar to, that of rabbit LC1Sb. Consequently, human LC1S appears to be much less heterogeneous relative to LC1F than is the case with other mammalian species. A high immunological cross-reactivity was likewise observed, with antibody specific to rabbit LC1F, between the isolated myosins from several human mixed muscles and rabbit fast myosin, though reactivity was highest with foetal myosin (having a pure-fast-light-chain pattern).
Transglycosylation reactions catalysed by two β-mannanases
Authors: Coulombel C , Clermont S , Foglietti M J , Percheron F
Published: August 2015

By using [3H]mannobiose as a labelled acceptor, it was possible to demonstrate transfer reactions catalysed by two beta-mannanases, with mannotetraose and mannopentaose as substrates. The enzyme from Streptomyces transfers one mannose unit from the oligosaccharides, whereas the enzyme from fenugreek (Trigonella foenum-graecum) seeds is able to transfer oligomannose residues.
Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver
Authors: Ramakrishna S , Benjamin W B
Published: August 2015

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.
Identification of the AraE transport protein of Escherichia coli
Authors: MacPherson A J , Jones-Mortimer M C , Henderson P J
Published: August 2015

1. Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000. 2. An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000. 3. An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000. 4. N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity. 5. N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent. 6. These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene. 7. The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.
The effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells
Authors: Guenther H L , Guenther H E , Fleisch H
Published: August 2015

Investigations were performed to assess the effects of dichloromethanediphosphonate on the synthesis of collagen by (1) isolated rabbit articular chondrocytes, (2) isolated rat calvaria bone cells and (3) bone explants from rats treated with the diphosphonates. The studies showed that dichloromethanediphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, causes articular chondrocytes to increase net collagen biosynthesis, both when measured as 3H-labelled or as non-radioactive material, in a dose-related fashion. The increment in collagen synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture; however, it declined with cells in tertiary culture and was absent after the fourth subculture. The type of collagen was not affected by the diphosphonate. The synthesis of collagen by bone cells was likewise increased with dichloromethanediphosphonate. No effects were detected with 1-hydroxyethane-1,1-diphosphonate was tested. Finally, when calvaria and tibiae from diphosphonate-treated rats were cultured in vitro, the positive effect of dichloromethanediphosphonate on collagen synthesis was also evident. 1-Hydroxyethane-1,1-diphosphonate, on the other hand, decreased the incorporation of [3H]proline into the collagen of calvaria and osseous tibial shafts and showed no effect on the collagen synthesis of the cartilaginous tibial heads.
Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration
Authors: McCarthy J E , Ferguson S J , Kell D B
Published: August 2015

1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.
Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils
Authors: Segal A W , Garcia R , Goldstone H , Cross A R , Jones O T
Published: August 2015

A cytochrome b with a midpoint oxidation-reduction potential of −245mV (cytochrome b-245) that is a major component of the microbicidal oxidase system of human neutrophil leucocytes has been identified in human eosinophils, monocytes and macrophages at concentrations similar to that found in human neutrophils. It was absent from a variety of other cells. This cytochrome is present in phagocytic leucocytes and probably plays an important part in the specialized activities of these cells.
Destruction of liver haem by norethindrone. Conversion into green pigments
Authors: White Ian N. H.
Published: August 2015

1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with 14C by using 5-amino[4-14C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major 14C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with 59Fe by using 59FeCl3, norethindrone administration resulted in the detection of 59Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H2SO4. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.
Action of low-density lipoprotein and compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, on the synthesis of dolichol-linked oligosaccharides and low-density-lipoprotein receptor in human skin fibroblasts
Authors: Filipovic I , Menzel B
Published: August 2015

To explore whether there is an inter-relationship between the rate of low-density (LD) lipoprotein binding to its receptor and the formation of dolichol-linked oligosaccharides, experiments were performed with human fibroblasts where the synthesis of lipoprotein receptor and dolichyl saccharides was under control of LD lipoprotein and compactin. Pretreatment of the cells with nonlabelled LD lipoprotein resulted in a suppression of both the binding of 125I-labelled LD lipoprotein to the receptor and the synthesis of dolichyl saccharides from [14C]acetate and [3H]mannose, but not from [3H]mevalonolactone. Compactin, in contrast, inhibited only the formation of dolichol-linked oligosaccharides. Mevalonolactone (1 microM) abolished the inhibitory effect of LD lipoprotein on dolichyl saccharide formation, but was not able to restore the receptor-binding capacity, thus suggesting that the synthesis of lipoprotein receptor is not coupled to the formation of dolichyl saccharides.
Aspects of adipose-tissue metabolism in foetal lambs
Authors: Vernon R G , Robertson J P , Clegg R A , Flint D J
Published: August 2015

1. The mean volume of adipocytes, the rates of fatty acid and acylglycerol glycerol synthesis from various precursors (in vitro), the rates of oxidation of acetate and glucose (in vitro) and the activities of lipoprotein lipase and various lipogenic enzymes were determined for perirenal adipose tissue from foetal lambs during the last month of gestation. 2. The fall in the rate of growth of perirenal adipose tissue during the last month of gestation is associated with a diminished capacity for fatty acid synthesis and lipoprotein lipase activity, but no change in the rate of acylglycerol glycerol synthesis was observed. There was no fall in the activities of cytosolic acetyl-CoA synthetase or the NADP-linked dehydrogenases, suggesting that the decrease in the rate of fatty acid synthesis was due to an impairment at the level of acetyl-CoA carboxylase or fatty acid synthetase. 3. The rate of fatty acid synthesis from acetate was greater than that from glucose. The rate of fatty acid synthesis from glucose per adipocyte of foetal lambs was similar to that of young sheep. The characteristic metabolism of adipose tissue of the adult sheep is thus present in the foetus, despite the relatively large amounts of glucose in the foetal ‘diet’.
Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes
Authors: Ghiselli G , Sirtori C R , Nicosia S
Published: August 2015

Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km.
Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation
Authors: Teng C T , Teng C S
Published: August 2015

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.
The polyphosphoinositide phosphodiesterase of erythrocyte membranes
Authors: Downes C. Peter , Michell Robert H.
Published: August 2015

1. A new assay procedure has been devised for measurement of the Ca2+-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [32P]Pi. They are incubated under appropriate conditions for activation of the phosphodiesterase and the released32P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca2+. For both esters, optimum rates of release were seen at pH6.8–6.9. Mg2+did not provoke release of either ester. 3. Ca2+provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca2+. 4. In the presence of Mg2+, the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca2+and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg2+-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.
Inhibition of plant fatty acid synthesis by nitroimidazoles
Authors: Jones A V , Harwood J L , Stratford M R , Stumpf P K
Published: August 2015

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.
Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats
Authors: Peavy D E , Taylor J M , Jefferson L S
Published: August 2015

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.
Ontogeny of the glucocorticoid receptor and its relationship to tyrosine aminotransferase induction in cultured foetal hepatocytes
Authors: Cake M H , Yeoh G C T , Oliver I T
Published: August 2015

The glucocorticoid receptor activity that can be detected in the liver from 15-day foetal rats would appear to be associated with the haemopoietic cells. In hepatocytes, purified by culture for 1-2 days from 15-day foetal rats, the glucocorticoid receptor activity is low and dexamethasone does not induce the enzyme tyrosine aminotransferase. If culture is continued both receptor activity and steroid responsiveness are acquired. Cultured hepatocytes from 19-day foetal liver contain receptor from the first day of culture and, furthermore, the subsequent level of response to glucocorticoids is directly correlated with the actual receptor concentration. It would appear that the glucocorticoid receptor is not acquired by hepatocytes until after 18 days of gestation. Nevertheless, the fact that bromodeoxyuridine has no effect on the rate of accumulation of receptor in hepatocytes suggests that the differentiative event leading to the subsequent appearance of the receptor has already occurred before day 15 of gestation. However, the acquisition of the receptor would appear to be dependent on mitosis as cytosine arabinoside can inhibit the process.
Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells
Authors: Bakker Ger H. , Hoogerbrugge Jos W. , Rommerts Focko F. G. , Van Der Molen Henk J.
Published: August 2015

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ‘LH’). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9±7.4ng/60min per 106 cells; stimulated production was 156.1±39.5ng/60min per 106 cells (means±s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [32P]Pi incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3±6.1% (mean±s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6±28.6ng/30min per mg of protein; lutropinstimulated production: 498.8±91.5ng/30min per mg of protein; means±s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.
Lipogenesis in vivo in maternal and foetal tissues during late gestation in the rat
Authors: Lorenzo M , Caldés T , Benito M , Medina J M
Published: August 2015

The rate of 3H2O incorporation into lipid in vivo progressively decreased in liver but increased in parametrial adipose tissue during the last 3 days of gestation. These changes seem to be related to those occurring in plasma insulin and progesterone concentrations during the same period. Foetal liver showed a high rate of lipogenesis, which sharply decreased before parturition. foetal lung lipogenesis increased during days 20 and 21 of gestation.
Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin
Authors: Assimacopoulos-Jeannet F , Denton R M , Jeanrenaud B
Published: August 2015

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.
Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes
Authors: Mayor F , Marvizón J G , Aragón M C , Gimenez C , Valdivieso F
Published: August 2015

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.
Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures
Authors: Wilson K J , Honegger A , Hughes G J
Published: August 2015

The chromatographic properties of peptides ranging in length from 2 to 65 residues have been compared on reverse-phase packings in three buffer systems at low pH. Of the buffers examined, two are widely used in connection with u.v. detection [(i) phosphate/acetonitrile or (ii) phosphate/propan-2-ol] and the third for fluorescence detection [(iii) pyridine/formate-pyridine/acetate/propan-1-ol]. The addition of a chaotropic salt, NaClO4, to the phosphate buffers, as first described by Meek (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1632-1636, is shown to significantly improve the chromatographic behaviour of more hydrophobic peptides. The two most commonly used detection systems, u.v. and fluorescence, are compared in terms of ease of use and sensitivity.
Chemical modification of peptides by hydrazine
Authors: Honegger A , Hughes G J , Wilson K J
Published: August 2015

High pressure (‘performance’) liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.
Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase
Authors: McCarthy A D , Johnson P , Tipton K F
Published: August 2015

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.
A convenient zymogram stain for cellulases
Authors: McHale Anthony , Coughlan Michael P.
Published: August 2015

The release of dye from phosphoric acid-swollen cellulose azure by endocellulases and exocellulases is the basis of a technique for locating these enzymes on gels.
The hydrophobicities of cholera toxin, tetanus toxin and their components
Authors: Ward W H J , Britton P , van Heyningen S
Published: August 2015

1. Charge-shift electrophoresis showed that cholera toxin and its subunits have no hydrophobic surfaces. 2. Amino-acid composition and sequence data suggested that the proteins have no masked hydrophobic regions. 3. The A subunit of cholera toxin may interact with polar molecules in the membrane to exert its effect inside the cell. 4. The only hydrophobic part of tetanus toxin was the H-chain.
The effects of anions on fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli
Authors: Robinson J J , Weiner J H
Published: August 2015

A broad range of anions was shown to stimulate the maximal velocity of purified fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli, while leaving the Km for fumarate unaffected. Reducing agents potentiate the effects of anions on the activity, but have no effect by themselves. Thermal stability, conformation as monitored by circular dichroism and susceptibility to the thiol reagent 5,5′-dithiobis-(2-nitrobenzoic acid) are also altered by anions. The apparent Km for succinate in the reverse reaction (succinate dehydrogenase activity) varies as a function of anion concentration, but the maximal velocity is not affected. The membrane-bound activity is not stimulated by anions and its properties closely resemble those of the purified enzyme in the presence of anions. Thus it appears that anions alter the physical and chemical properties of fumarate reductase, so that it more closely resembles the membrane-bound form.
Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification
Authors: Sugiyama Yuichi , Yamada Tadataka , Kaplowitz Neil
Published: August 2015

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; Mr 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to Mr 52000 (PLAT Y1) and Mr 45000 (PLAT Y2) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y2 had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y1- and PLAT Y2-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y2 accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y1 and PLAT Y2 fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y1 and PLAT Y2 for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y1 and PLAT Y2. The major glutathione S-transferase from the PLAT Y2 fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y2-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.
The conversion of alanine into glutamine in guinea-pig renal cortex. Essential role of pyruvate carboxylase
Authors: Forissier M , Baverel G
Published: August 2015

1. The metabolism of L-alanine was studied in isolated guinea-pig kidney-cortex tubules. 2. In contrast with previous conclusions of Krebs [(1935) Biochem. J. 29, 1951-1969], glutamine was found to be the main carbon and nitrogenous product of the metabolism of alanine (at 1 and 5 mM). Glutamate and ammonia were only minor products. 3. At neither concentration of alanine was there accumulation of glucose, glycogen, pyruvate, lactate, aspartate or tricarboxylic acid-cycle intermediates. 4. Carbon-balance calculations and the release of 14CO2 from [U-14C]alanine indicate that oxidation of the alanine carbon skeleton occurred at both substrate concentrations. 5. A pathway involving alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, pyruvate carboxylase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of alanine into glutamine. 6. Strong evidence for this pathway was obtained by: (i) suppressing alanine removal by amino-oxyacetate, and inhibitor of transaminases, (ii) measuring the release of 14CO2 from [1-14C]alanine, (iii) the use of L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from alanine, and (iv) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from alanine. 7. In this pathway, the central role of pyruvate carboxylase, which explains the discrepancy between our results and those of Krebs (1935), was also demonstrated.
Stimulation of heparan sulphate synthesis in cultured rat hepatocytes by (+)-catechin
Authors: Sinn W , Sudhakaran P R , Von Figura K
Published: August 2015

The secretion of heparan sulphate by cultured rat hepatocytes was increased in the presence of (+)-catechin. The increase was due to a new species of heparan sulphate that lacked the carbohydrate-protein linkage between xylose and serine in normal heparan sulphate proteoglycan. The mean molecular weight of this heparan sulphate varied between 6300 and 9500, was not affected by treatment with alkali or Pronase and was 2-3-fold lower than that of chains released from heparan sulphate proteoglycan. After digestion with Pronase, only a minor fraction of chains contained serine, and after treatment with alkali and NaB3H4 reduction less than 5% of the chains exposed [3H]xylitol at the reducing terminals. These results suggested that (+)-catechin or metabolites of it acted as acceptors of heparan sulphate synthesis. In cultures treated wih cycloheximide, synthesis of heparan sulphate decreased to less than 5%. (+)-Catechin could restore the heparan sulphate synthesis to almost normal values. The (+)-catechin-induced heparan sulphate was secreted. Only a small fraction was incorporated into the plasma membrane or other cellular compartments. This may indicate that the protein core is essential for association of heparan sulphate with cellular compartments.