bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of urea-cycle activity by high concentrations of alanine
Authors: Hensgens H E S J , Meijer A J
Published: August 2015

1. Conditions are described in which high intracellular alanine concentrations inhibit urea-cycle flux in isolated hepatocytes. 2. Inhibition of urea-cycle flux by added alanine is DL-cycloserine-insensitive and is accompanied by an increase in intracellular citrulline and a decrease in ornithine. 3. Argininosuccinate synthetase (EC 6.3.4.5) activity in rat liver cytosol is inhibited by alanine in a competitive manner with respect to citrulline. It is concluded that this effects is the primary cause of inhibition of urea-cycle flux by alanine.
The swift increase in alcohol metabolism. Time course for the increase in hepatic oxygen uptake and the involvement of glycolysis
Authors: Yuki T , Thurman R G
Published: August 2015

Gastric intubation of female Sprague-Dawley rats with 5 g of ethanol/kg body wt. nearly doubled oxygen uptake by the isolated perfused rat liver maximally after only 2.5 h of treatment (Swift Increase in Alcohol Metabolism). Inhibition of enhanced oxygen uptake by KCN (2mM) and 4-methylpyrazole (0.8 mM) suggested the involvement of the mitochondrial respiratory chain and alcohol dehydrogenase in this phenomenon. Glycolysis was depressed after ethanol treatment. Diminished ATP generation via glycolysis accounts for a portion (23-50%) of the increased oxygen uptake, assuming that other rates of biosynthesis remain constant. Injection of adrenaline (2 mg/kg) 1 h before perfusion mimicked partially the action of ethanol on hepatic oxygen uptake. The increases produced by ethanol and adrenaline were not additive, suggesting that adrenaline is involved in the action of ethanol. Moreover, the increase in hepatic oxygen uptake produced by 2.5 h of ethanol treatment could be blocked by either alpha-(phenoxybenzamine; 40 mg/kg) or beta-(propranolol; 40 mg/kg) adrenergic blocking agents. Blood glucose increased after ethanol treatment, supporting the involvement of glycogenolytic hormones in this effect. These data indicate that at least part of the stimulated oxygen uptake after treatment with ethanol is a result of lower rates of glycolytic ATP generation resulting from hormone (e.g. adrenaline etc.) action. The ADP not phosphorylated in the cytosol enters the mitochondria, where it stimulates oxygen uptake.
Non-esterified cholesterol-rich adrenal lipid fractions. Preparation, properties and preferential utilization for cholesterol side-chain cleavage by corticotropin-stimulated adrenal mitochondria
Authors: Farese R V , Prudente W J , Chuang L T
Published: August 2015

Rat adrenal 105,000 g supernatant contains two lipid moieties, ‘lipid-I’ and ‘lipid-II’ which contain non-esterified cholesterol and stimulate cholesterol side-chain cleavage in soluble or mitochondrial enzyme systems. Lipid-I contains relatively large low-density heat-stable particles, whereas lipid-II particles are smaller, more dense and heat-labile. Lipid-I and lipid-II can be separated from clear cytosol by ultracentrifugation and gel filtration respectively. Corticotropin plus cycloheximide treatment increases the non-esterified cholesterol concentrations in the lipid fractions, and stimulatory effects of lipids on cholesterol side-chain cleavage appear to correlate with non-esterified cholesterol concentrations therein. On addition of saturating amounts of cholesterol-rich lipid, pregnenolone synthesis and cholesterol binding to cytochrome P-450 are stimulated more in mitochondria from corticotropin-stimulated adrenals than in mitochondria from control or corticotropin-plus cycloheximide-stimulated adrenals. These results support the contention that the corticotropin-induced increase in mitochondrial cholesterol side-chain cleavage involves an increase in cholesterol utilization as well as an increase in cholesterol availability.
Effect of tunicamycin on the metabolism of low-density lipoproteins by control and low-density-lipoprotein-receptor-deficient human skin fibroblasts
Authors: Filipovic I , von Figura K
Published: August 2015

Preincubation of normal human skin fibroblasts with tunicamycin, which inhibits N-glycosylation of glycoproteins, resulted in a dose-dependent and reversible inhibition of binding and internalization of homologous low-density lipoproteins by the cells. The degradation of the internalized lipoproteins was not affected by the drug. Comparative studies with fibroblasts deficient in low-density-lipoprotein receptors indicated that tunicamycin exerts its inhibitory effect only via the receptor-mediated high-affinity binding and uptake of lipoproteins. These results suggest that expression of low-density-lipoprotein receptors on the cell surface of human skin fibroblasts depends on intact N-glycosylation.
Detection of cytochrome b+50 in membranes of Rhodospirillum rubrum isolated from aerobically and phototrophically grown cells
Authors: Niederman R A , Hunter C N , Mallon D E , Jones O T
Published: August 2015

1. Dark equilibrium potentiometric titrations were conducted on membranes purified from Rhodospirillum rubrum in an effort to identify b-type cytochrome components reported in other Rhodospirillaceae. In preparations from aerobically grown cells virtually devoid of bacteriochlorophyll a, three components were observed at 560-540 nm. Their oxidation-reduction midpoint potentials assigned by computer-assisted analysis were +195, +50 and −110 mV at pH 7.0; each of these fitted closely to theoretical single-electron equivalent curves. 2. In chromatophores from phototrophically grown carotenoidless mutant G-9, three components were also observed with E0' +190, +50 and −90mV. 3. The alpha-band of the +50mV component exhibited an absorption maximum near 560nm in difference spectra obtained at fixed oxidation-reduction potentials. 4. This component could be demonstrated most readily in purified membrane preparations and may have been obscured in previous studies by residual cytochrome c'. 5. This is the first definitive report of cytochrome b+50 in membranes from Rs. rubrum and aligns this bacterium with other Rhodospirillaceae in which this component functions in light-driven cyclic electron flow.
Low-level chemiluminescence of bovine heart submitochondrial particles
Authors: Cadenas Enrique , Boveris Alberto , Chance Britton
Published: August 2015

Submitochondrial particles from bovine heart mitochondria showed low-level chemiluminescence when supplemented with organic hydroperoxides. Chemiluminescence seems to measure integratively radical reactions involved in lipid peroxidation and related processes. Maximal light-emission was about 1500 counts/s and was reached 2–10min after addition of hydroperoxides. Ethyl hydroperoxide, cumene hydroperoxide and t-butyl hydroperoxide were effective in that order. Antimycin and rotenone increased chemiluminescence by 50–60%; addition of substrates, NADH and succinate did not produce marked changes in the observed chemiluminescence. Cyanide inhibited chemiluminescence; half-maximal inhibitory effect was obtained with 0.03mm-cyanide and the inhibition was competitive with respect to t-butyl hydroperoxide. Externally added cytochrome c (10–20μm) had a marked stimulatory effect on chemiluminescence, namely a 12-fold increase in light-emission of antimycin-inhibited submitochondrial particles. Stimulation of hydroperoxide-induced chemiluminescence of submitochondrial particles by cytochrome c was matched by a burst of O2 consumption. O2 is believed to participate in the chain radical reactions that lead to lipid peroxidation. Superoxide anion seems to be involved in the chemiluminescence reactions as long as light-emission was 50–60% inhibitible by superoxide dismutase. Singlet-oxygen quenchers, e.g. β-carotene and 1,4-diazabicyclo[2,2,2]-octane, affected light-emission. β-Carotene was effective either when incorporated into the membranes or added to the cuvette. The present paper suggests that singlet molecular oxygen is mainly responsible for the light-emission in the hydroperoxide-supplemented submitochondrial particles.
Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product
Authors: Furneaux H M , Pearson C K
Published: August 2015

Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis–Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a ‘Hofstee’ plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be ‘chased’ into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).
Specific quantification of mouse and human keratin proteins by radioimmunoassay
Authors: Yuspa S H , Poirier M C , Harness J R , Olsom D R , Steinert P M
Published: August 2015

Mouse epidermal keratin proteins were purified and labelled with 125I by chemical techniques. A radioimmunoassay method was established with a rabbit antibody elicited against the mouse keratin. This assay method was utilized to quantify keratin proteins of mouse and human epidermal extracts, both from intact tissues and cells in culture. As little as 30 ng of mouse keratin (0.5 pmol) was quantifiable.
The native forms of the phycobilin chromophores of algal biliproteins. A clarification
Authors: O'Carra P , Murphy R F , Killilea S D
Published: August 2015

Pigments released from phycoerythrins and phycocyanins by treatment with hot methanol are currently regarded as equivalent to the native chromophores phycoerythrobilin and phycocyanobilin. However, evidence presented here confirms the original view of O'Carra & O'hEocha [(1966 Phytochemistry 5, 993-997] that these methanol-released pigments are artefacts differing in their chromophoric conjugated systems from the native protein-bound prosthetic groups. By contrast, the native spectral properties are retained in pigments released by careful acid treatment of the biliproteins and these acid-released phycobilins, rather than the methanol-released pigments, are therefore regarded as the protein-free forms of the native chromophores. The conclusion reached by Chapman, Cole & Siegelman [(1968) J. Am. Chem. Soc. 89, 3643-3645], that all the algal biliproteins contain only phycoerythrobilin and phycocyanobilin, is shown to be incorrect. The identification of a urobilinoid chromophore, phycourobilin, accompanying phycoerythrobilin in B- and R- phycoerythrins is confirmed and supported by more extensive evidence. The cryptomonad phycocyanins are shown to contain a phycobilin chromophore accompanying phycocyanobilin. This further phycobilin has the spectral properties of the class of bilins known as violins and the provisional name “cryptoviolin” is proposed pending elucidation of its structure.
Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin
Authors: Smith G J , Rowlatt C
Published: August 2015

Nucleic prepared from mouse submandibular salivary gland show marked fragility during isolation. Hwever, intact nuclei relatively free from cytoplasmic contamination were obtained by homogenization in buffers containing 0.88 M-sucrose, Ca2+, spermine, spermidine and the proteinase inhibitor aprotinin, followed by centrifugation through 2.2 M-sucrose. The kinetics of digestion by the micrococcal nuclease of chromatin in these nuclei are similar to those of chromatin from mouse liver nuclei. Base-pair size analysis of the solubilized DNA from both organs shows a stable high-molecular weight species of chromatin, which is further digested to mononucleosome and subnucleosome species. With extensive digestion the chromatin becomes insoluble. The mononucleosomes produced from salivary-gland chromatin after the inhibition of endogenous proteinase activity exhibit an s20,w value of 11S and contain histones H1, H2A, H2B, H3 and H4.
Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain
Authors: Lee Lilian M. Y. , Salvatore Antonieta K. , Flanagan Peter R. , Forstner Gordon G.
Published: August 2015

Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J.173, 553–563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3–5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.
Heparin enhances the rate of binding of fibronectin to collagen
Authors: Johansson S , Höök M
Published: August 2015

125I-labelled fibronectin is shown to bind to both native and denatured collagen immobilized on Sephadex beads in reactions that exhibit different kinetics. The rates of both reactions were enhanced by the presence of heparin or highly sulphated dextran sulphate but not by other glycosaminoglycans or dextran sulphates having low sulphate contents.
Crystallographic and molecular-orbital studies on the geometry of antifolate drugs
Authors: Hunt W E , Schwalbe C H , Bird K , Mallinson P D
Published: August 2015

In the common dihydrofolate reductase inhibitors an amino substituent replaces the pteridine carbonyl oxygen atom of folates, with altered hydrogen-bonding properties and size. Flexibility in the amino groups could facilitate enzyme binding. Studies of cycloguanil hydrochloride by neutron diffraction show both in-plane and out-of-plane deformation of amino groups. Molecular-orbital calculations ab initio on 2,4-diamino-5-methylpyrimidinium cation confirm that the 4-amino group is readily deformable. The 2,4-diaminoquinazoline structure is reported. Atomic co-ordinates, thermal parameters, bond distances and bond angles for cycloguanil and 2,4-diaminoquinazoline have been deposited as Supplementary Publication SUP 50108 (13 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.
Proteoglycans in normal and severely osteoarthritic human cartilage
Authors: Vasan N
Published: August 2015

Proteoglycans from osteoarthritic cartilage were compared with those from normal articular cartilage. Normal proteoglycan aggregates are larger in size and more homogeneous than those in osteoarthritis. Proteoglycan monomers from both sources gave two peaks on controlled pore glass-bead chromatography. Although the retarded material from normal cartilage showed an affinity for hyaluronate, the same material from osteoarthritic cartilage did not. The hyaluronate-binding capacity of the material which is partly in the void volume and partly retarded was similar in both types of cartilage. These results suggest that in osteoarthritic cartilage the proteoglycan aggregates are smaller and more heterogeneous and that the chondroitin sulphate side chains are shorter. They also indicate that there are two populations of proteoglycan, one with its hyaluronate-binding-protein region of core protein intact and the other either possessing an inactive binding region or totally lacking it.
Glutathione contents of tissues in the aging mouse
Authors: Hazelton G A , Lang C A
Published: August 2015

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.
Age-associated changes in initiation of ribonucleic acid synthesis in isolated rat liver nuclei
Authors: Miller J K , Bolla R , Denckla W D
Published: August 2015

Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.
The effects of carbon disulphide on rat liver microsomal mixed-function oxidases, in vivo and in vitro
Authors: Obrebska Maria J. , Kentish Peter , Parke Dennis V.
Published: August 2015

An intraperitoneal dose of CS2 (500mg/kg) to male rats resulted in loss of liver microsomal mixed-function-oxidase activity (85% loss of biphenyl 4-hydroxylase), followed by denaturation of liver cytochrome P-450 to cytochrome P-420, and degradative loss of both cytochromes (50% loss). Losses of NADPH–cytochrome c reductase (20%) and cytochrome b5 were considerably less. Intraperitoneal administration of CS2 (100mg/kg) to rats pretreated wtih phenobarbitone or 3-methylcholanthrene resulted in similar losses, but the rate of destruction was greater with cytochrome P-450 than with cytochrome P-448. At 12h after intraperitoneal injection of CS2 to non-pretreated rats, a new cytochrome (P-448) appeared. Rat liver microsomal preparations incubated with CS2 in the presence of NADPH and O2 resulted in loss of cytochrome P-450 and mixed-function-oxidase activity directly related to the concentration of CS2 (10–100μm) and to the period of incubation. Addition of EDTA (1mm) completely inhibited this destruction of cytochrome P-450 by CS2in vitro. Addition of CS2 to liver microsomal preparations resulted in moderate increases in the Ks values for type-I or type-II substrates, but these were insufficient to account for the inhibition of the mixed-function oxidases. We therefore suggest that desulphuration of CS2 leads to binding of the S to cytochrome P-450, denaturation of cytochrome P-450 to cytochrome P-420, and ultimately to destruction of these cytochromes by autoxidation.
Uptake of protoporphyrin IX by isolated rat liver mitochondria
Authors: Koller M E , Romslo I
Published: August 2015

Rat liver mitochondria accumulate protoporphyrin IX from the suspending medium into the inner membrane in parallel with the magnitude of the transmembrane K+ gradient (K+in/K+out). Only protoporphyrin IX taken up in parallel with the transmembrane K+ gradient is available for haem synthesis. Coproporphyrins (isomers I and III) are not taken up by the mitochondria. The results support the suggestion by Elder & Evans [(1978) Biochem. J. 172, 345-347] that the prophyrin to be taken up by the inner mitochondrial membrane belongs to the protoporphyrin(ogen) IX series. Protoporphyrin IX at concentrations above 15 nmol/mg of protein has detrimental effects on the structural and functional integrity of the mitochondria. The relevance of these effects to the hepatic lesion in erythropoietic protoporphyria is discussed.
The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli
Authors: Jones R W
Published: August 2015

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.
Studies on the efflux of metalloporphyrin from rat-liver mitochondria. Effect of albumin, globin, haemin and haemoglobin
Authors: Husby P , Romslo I
Published: August 2015

The mechanism by which metalloporphyrins escape from mitochondria has been studied in isolated rat-liver mitochondria using Co-deuteroporphyrin as the model compound. During the first 10–15 min of incubation the efflux is about 10% of the total amount of Co-deuteroporphyrin synthesized. The efflux then increases to a second steady-state leve of 25–35% after 30–45 min of incubation. The efflux is inversely correlated to the energy state of the mitochondria. Globin at concentrations less than 0.4 mumol/l enhances the efflux of Co-deuteroporphyrin, but has no effect on the degree of energy coupling or on the rate of Co-deuteroporphyrin synthesis. The effect of globin can be competitively inhibited by adding haemin. Haemin (0.5–1.0 mumol/l) when added to the medium in the absence of globin reduces the efflux of Co-deuteroporphyrin by 20–30%, but has no effect on the metal-chelatase activity. Neither albumin nor haemoglobin increases the efflux of Co-deuteroporphyrin from intact mitochondria. The results suggest that the efflux of metalloporphyrin is regulated in part by the energy state of the mitochondria and in part by the presence of metalloporphyrin-binding ligants and unattached haemin in the incubation medium.
N
6-Trimethyl-lysine metabolism. 3-Hydroxy-N6-trimethyl-lysine and carnitine biosynthesis
Authors: Hoppel C L , Cox R A , Novak R F
Published: August 2015

Rats injected with N6-[Me-3H]trimethyl-lysine excrete in the urine five radioactively labelled metabolites. Two of these identified metabolites are carnitine and 4-trimethylammoniobutyrate. A third metabolite, identified as 5-trimethylammoniopentanoate, is not an intermediate in the biosynthesis of carnitine; the fourth and major metabolite, N2-acetyl-N6-trimethyl-lysine, is not a precursor of carnitine. The remaining metabolite (3-hydroxy-N6-trimethyl-lysine) is converted into trimethylammoniobutyrate and carnitine by rat liver slices and into trimethylammoniobutyrate by rat kidney slices. In rat liver and kidney-slice experiments, radioactivity from DL-N6-trimethyl-[1-14C]lysine and DL-N6-trimethyl-[2-14C]lysine was incorporated into N2-acetyl-N6-trimethyl-lysine and 3-hydroxy-N6-trimethyl-lysine, but not into trimethylammoniobutyrate or carnitine. A procedure was devised to purify milligram quantities of 3-hydroxy-N6-trimethyl-lysine from the urine of rats injected chronically with N6-trimethyl-lysine (100 mg/kg body wt. per day). The structure of 3-hydroxy-N6-trimethyl-lysine was confirmed chemically and by nuclear-magnetic-resonance spectrometry [Novak, Swift & Hoppel (1980) Biochem. J. 188, 521–527]. The sequence for carnitine biosynthesis in liver is: N6-trimethyl-lysine leads to 3-hydryxy-N6-trimethyl-lysine leads to leads to 4-trimethylammoniobutyrate leads to carnitine.
Action of calcium ions on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase and other enzymes of the Calvin cycle
Authors: Charles S A , Halliwell B
Published: August 2015

Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase is powerfully inhibited by Ca2+ non-competitively with respect to its substrate, fructose 1,6-bisphosphate. 500 microM-Ca2+ causes virtually complete inhibition and the Ki is 40 microM. Severe inhibition of sedoheptulose bisphosphatase is also caused by Ca2+. A role for Ca2+ in regulation of the Calvin cycle in spinach chloroplasts is proposed.
The determination of reduced nicotinamide-adenine dinucleotide and metabolic intermediates in picomole amounts with bacterial luciferase
Authors: Golden S , Katz J
Published: August 2015

Methods that use bacterial luciferase for the assay of NADH in the range from 1 pmol to 1 nmol are described. Optimal conditions for the assay of glycolytic intermediates, tricarboxylic acid-cycle intermediates and related amino acids from milligram amounts of tissue are presented. The whole spectrum of these intermediates can be determined on about 10 mg of liver tissue. The methods are simple, are suitable for routine use, and the instrumentation is inexpensive. The concentrations of glycolytic intermediates in rat livers were determined by conventional spectrometric methods and with luciferase, and the results found to be in good agreement.
Purine catabolism in isolated rat hepatocytes. Influence of coformycin
Authors: Van Den Berghe Georges , Bontemps Françoise , Hers Henri-Géry
Published: August 2015

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [14C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [14C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.
Preparation of monoclonal antibodies to xanthine oxidase and other proteins of bovine milk-fat-globule membrane
Authors: Mather I H , Nace C S , Johnson V G , Goldsby R A
Published: August 2015

Nine hybridomas secreting monoclonal antibody to proteins of bovine milk-fat-globule membrane were isolated. All nine cell lines continued to secrete monoclonal antibody after serial transfer in culture and after passage as solid tumours in Balb/cJ mice. Four of the cell lines secreted monoclonal antibody specific for xanthine oxidase, one of the major proteins of milk-fat-globule membrane.
The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase
Authors: Ben-Yoseph Yoav , Hungerford Melinda , Nadler Henry L.
Published: August 2015

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.
Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca2+ + Mg2+)-dependent adenosine triphosphatase
Authors: Gietzen K , Tejcka M , Wolf H U
Published: August 2015

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.
Isolation of rat submandibula kallikreins by using immunoadsorption chromatography
Authors: Gautvik K M , Johansen L , Svindahl K , Nustad K , Orstavik T B
Published: August 2015

A one-step immunoadsorption method for the isolation of glandular kallikreins is described using the immunoglobulin fraction from rabbit anti-(rat glandular kallikrein) serum coupled to CNBr-activated Sepharose 4B. The adsorptions of 125I-labelled kallikrein or unlabelled kallifrein from 100 000 g submandibular gland supernatants were more than 97% complete. The elution of kallikrein from the immunoadsorbent using guanidine hydrochloride gave about 20% yield, which could be increased up to 70% by including 0.5% bovine serum albumin in the elution buffer. The electrophoretic mobility of eluted submandibular 125I-labelled kallikrein or submandibular glandular kallikrein was not altered after affinity chromatography, as judged by conventional polyacrylamide disc-gel electrophoresis or by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. In addition, the specific esterase and the kininogenase activities of isolated submandibular kallikreins were more than 90% of those of the reference enzyme. This procedure, which results in the isolation of immunologically and biologically active submandibular kallikrein, may also be used for purificaton of other glandular kallikreins that show immunological homology.
Do photosynthetic bacteria contain cytochrome c1?
Authors: Wood P M
Published: August 2015

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.
Investigations into the effect of acid on the spectral and kinetic properties of purple membrane from Halobacterium halobium
Authors: Edgerton M E , Moore T A , Greenwood C
Published: August 2015

The formation and reversal of the acid species of purple membrane generated below pH 4.00 (22 degrees C) is studied together with the photochemical cycle over the pH range 6.40–3.20. The buffering capacity of the membrane reaches a peak at pH 4.30, indicating the possibility of a conformational change taking place. The generation of the new spectral species can take place in the dark and is unaffected by the addition of reducing agents. Kinetic parameters measured indicate that the group being titrated below pH 4.00 could be the same as that protonated in the formation of intermediate O. The temporal placement of intermediate O after M in the photochemical cycle is shown to be incompatible with the data presented here. Reneutralization of acidified purple membrane shows that the spectral changes in acid are reversible but the phototransient properties are altered.
Carbohydrate composition of the oligosaccharide units of the haemagglutinin from the Hong Kong influenza virus A/Memphis/102/72
Authors: Ward C W , Gleeson P A , Dopheide T A
Published: August 2015

The haemagglutinin from the Hong Kong influenza virus A/Memphis/102/72 contains seven oligosaccharide units attached to asparagine residues 8, 22, 38, 81, 165 and 285 in the heavy chain (HA1) and to residue 154 in the light chain (HA2). The single oligosaccharide unit in HA2 and four of the oligosaccharide units of HA1 (at residues 8, 22, 38 and 81) contain the four monosaccharides N-acetylglucosamine, mannose, galactose and fucose and are of the N-acetyllactosamine (or ‘complex’) type. The two other oligosaccharide units on HA1 are of the oligomannoside (or ‘simple’) type and contain only two residues of N-acetylglucosamine and five or six residues of mannose. The data are discussed in relation to the differences in the carbohydrate compositions of other influenza haemagglutinins.
Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolaevulinate synthase by manganese in the rat brain
Authors: Maines Mahin D.
Published: August 2015

The activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, is differentially distributed in various regions of the rat brain. The cerebellum possessed the highest enzyme activity of the eight regions studied. The cerebral cortex and the midbrain also exhibited high 5-aminolaevulinate synthase activity; the septum, hypothalamus, thalamus, amygdala and the hippocampus possessed much lower enzyme activity. However, the total porphyrin and haem contents of the different brain segments did not vary greatly. Mn2+, when administered subcutaneously to rats, effectively inhibited the activity of 5-aminolaevulinate synthase in the cerebellum, midbrain and cerebral cortex; however, repeated injections of the metal ion neither decreased the haem and porphyrin contents of the brain nor induced haem oxygenase activity. Mn2+ was not an effective inhibitor of 5-aminolaevulinate synthase activity in vitro. On the other hand, studies carried out with the liver in vivo suggested that Mn2+ may alter the turnover rate of cellular haem and haemoproteins. In that event, it is likely that the inhibition of 5-aminolaevulinate synthase by Mn2+ was in part a result of the inhibition of protein synthesis by the metal ion. It is postulated that the haem and porphyrin contents of the brain are maintained at a steady-state level, due in part to the refractoriness to inducers of the regulatory mechanism for haem catabolic enzymes and in part to the ability of the organ to utilize haem precursors derived from extraneuronal sources.
A possible mechanism for the anti-ketogenic action of alanine in the rat
Authors: Nosadini Romano , Datta Harish , Hodson Alan , Alberti K. George M. M.
Published: August 2015

1. The anti-ketogenic effect of alanine has been studied in normal starved and diabetic rats by infusing l-alanine for 90min in the presence of somatostatin (10μg/kg body wt. per h) to suppress endogenous insulin and glucagon secretion. 2. Infusion of alanine at 3mmol/kg body wt. per h caused a 70±11% decrease in [3-hydroxybutyrate] and a 58±9% decrease in [acetoacetate] in 48h-starved rats. [Glucose] and [lactate] increased, but [non-esterified fatty acid], [glycerol] and [3-hydroxybutyrate]/[acetoacetate] were unchanged. 3. Infusion of alanine at 1mmol/kg body wt. per h caused similar decreases in [ketone body] (3-hydroxybutyrate plus acetoacetate) in 24h-starved normal and diabetic rats, but no change in other blood metabolites. 4. Alanine [3mmol/kg body wt. per h] caused a 72±9% decrease in the rate of production of ketone bodies and a 57±8% decrease in disappearance rate as assessed by [3-14C]acetoacetate infusion. Metabolic clearance was unchanged, indicating that the primary effect of alanine was inhibition of hepatic ketogenesis. 5. Aspartate infusion at 6mmol/kg body wt. per h had similar effects on blood ketone-body concentrations in 48h-starved rats. 6. Alanine (3mmol/kg body wt. per h) caused marked increases in hepatic glutamate, aspartate, malate, lactate and citrate, phosphoenolpyruvate, 2-phosphoglycerate and glucose concentrations and highly significant decreases in [3-hydroxybutyrate] and [acetoacetate]. Calculated [oxaloacetate] was increased 75%. 7. Similar changes in hepatic [malate], [aspartate] and [ketone bodies] were found after infusion of 6mmol of aspartate/kg body wt. per h. 8. It is suggested that the anti-ketogenic effect of alanine is secondary to an increase in hepatic oxaloacetate and hence citrate formation with decreased availability of acetyl-CoA for ketogenesis. The reciprocal negative-feedback cycle of alanine and ketone bodies forms an important non-hormonal regulatory system.
The role of Ca2+ in the protoveratrine-induced release of γ-aminobutyrate from rat brain slices
Authors: Minchin M C W
Published: August 2015

1. Protoveratrine A increased the release of gamma-amino[3H]butyrate from small slices of rat cerebral cortex. This effect increased with increasing protoveratrine concentration, reaching a maximum at 100 microM. 2. Removal of Ca2+ from the superfusing medium did not change the increase in release due to 10 microM-protoveratrine; however, the Ca2+ antagonists, compound D-600, La3+, Mn2+, Mg2+ and also high Ca2+ concentration inhibited the effect of the alkaloid, as did procaine. 3. Protoveratrine A increased the uptake of 22Na+ into the slices with a similar dose-response curve to that found for gamma-aminobutyrate release. For the most part, the substances that inhibited protoveratrine-stimulated gamma-aminobutyrate release also inhibited 22Na+ uptake, although the correlation was not perfect. 4. Although extracellular Ca2+ is not required for protoveratrine-induced gamma-aminobutyrate release, an increase in Na+ influx that is susceptible to inhibition by some Ca2+ antagonists does appear to be associated with this phenomenon. However, the possibility remains that changes in the free intracellular Ca2+ concentration may be important for transmitter release induced by depolarizing veratrum alkaloids.
Incorporation of amino acids into soluble and membrane protein fractions of dystrophic hamsters
Authors: Nicholls D M , Creasy R C , Chin-See M W , Carlisle J A , Lange A B , Saleem M
Published: August 2015

The incorporation of labelled leucine was measured in protein fractions of muscle in intact control and dystrophic female hamsters and also in cell-free preparations obtained from these animals. The labelling of the soluble sarcoplasmic protein fraction, the microsomal protein fraction and the sarcolemma protein fraction was increased in the dystrophic hindleg muscle. The specific radioactivities of the sarcolemma protein fraction and other fractions were increased markedly relative to that of free leucine in the dystrophic muscle. In cell-free preparations where ribonuclease effects were avoided, the dystrophic muscle exhibited an increased synthesis of peptide bonds.
Energy conservation by the plant mitochondrial cyanide-insensitive oxidase. Some additional evidence
Authors: Wilson S B
Published: August 2015

Several measures of energy conservation, namely ADP/O ratio, P/O ratio, ATP/O ratio and phosphorylation detected by continuous assay with purified firefly luciferase and luciferin, all show phosphorylation can occur with mung-bean mitochondria at cyanide concentrations sufficient to inhibit the cytochrome oxidase system. Phosphorylation in the presence of cyanide is uncoupler- oligomycin- and salicylhydroxamate-sensitive. The participation of phosphorylation site 1 is excluded, phosphorylation being attributable to a single phosphorylation site associated with the cyanide-insensitive oxidase. The cyanide-insensitive oxidase has also been shown to support a variety of other energy-linked functions, namely, Ca2+ uptake, reversed electron transport and the maintenance of a membrane potential detected by the dye probes 8-anilinonaphthalene-1-sulphonate and safranine. High concentrations of cyanide have uncoupler-like activity, decreasing the ADP/O ratio and the t 1/2 for the decay of a pH pulse through the the mitochondrial membrane. This uncoupler-like effect is most marked with aged mitochondria. The observations of energy conservation attributable to the cyanide-insensitive oxidase are compared with other reports where it is concluded that the alternative oxidase is uncoupled.