bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16α-carbonitrile-inducible rat cytochromes P-450
Authors: Barnes T S , Shaw P M , Burke M D , Melvin W T
Published: August 2015

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.
Characterization of rat preadipocytes from normal rat adipose tissue by their effector response
Authors: Swierczewski E , Pello J Y , Arapinis C , Aron Y , Krishnamoorthy R
Published: August 2015

We isolated from normal rat adipose tissue, by a Percoll-density-gradient procedure, two populations of adipocyte precursors. These preadipocytes undergo morphological and biochemical adipose conversion in primary culture. For full adipose conversion, these precursor cells, in addition to the adipogenic factor present in fetal-calf serum, require other effectors differentially. One population completes terminal differentiation in the presence of physiological concentrations of insulin. The second population requires a pre-sensitization with isobutylmethylxanthine at a critical period of the culture in order to respond to insulin. The fact that dibutyryl cyclic AMP could not be substituted for isobutylmethylxanthine suggests that the effect of the latter agent is not through its inhibition of particulate phosphodiesterase activity. These two populations further differ in their response to exogenously added haemin. Thus the existence of at least two developmentally regulated rat adipose-precursor compartments is demonstrated.
Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum
Authors: Robbi M , Beaufay H
Published: August 2015

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.
Heterogeneity of D2 dopamine receptors in different brain regions
Authors: Leonard M N , Macey C A , Strange P G
Published: August 2015

The binding of [3H]spiperone has been examined in membranes derived from different regions of bovine brain. In caudate nucleus, nucleus accumbens, olfactory tubercle and putamen binding is to D2 dopamine and 5HT2 serotonin receptors, whereas in cingulate cortex only serotonin 5HT2 receptor binding can be detected. D2 dopamine receptors were examined in detail in caudate nucleus, olfactory tubercle and putamen using [3H]spiperone binding in the presence of 0.3 microM-mianserin (to block 5HT2 serotonin receptors). No evidence for heterogeneity among D2 dopamine receptors either between brain regions or within a brain region was found from the displacements of [3H]spiperone binding by a range of antagonists, including dibenzazepines and substituted benzamides. Regulation of agonist binding by guanine nucleotides did, however, differ between regions. In caudate nucleus a population of agonist binding sites appeared resistant to guanine nucleotide regulation, whereas this was not the case in olfactory tubercle and putamen.
Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes
Authors: Murthy M S , Pande S V
Published: August 2015

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.
GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability
Authors: Nicchitta C V , Joseph S K , Williamson J R
Published: August 2015

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5′-[gamma-thio]triphosphate and guanosine 5′-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5′-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5′-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.
Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development
Authors: Ganapathi M K , Kwon M , Haney P M , McTiernan C , Javed A A , Pepin R A , Samols D , Patel M S
Published: August 2015

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.
Glycine flanked by hydrophobic bulky amino acid residues as minimal sequence for effective subtilisin catalysis
Authors: Bratovanova E K , Petkov D D
Published: August 2015

The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1′ residue and predilection for bulky hydrophobic P2′ residues. A synergism in the action of S1′ and S2′subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.
A calorimetric study of human CuZn superoxide dismutase
Authors: Biliaderis C G , Weselake R J , Petkau A , Friesen A D
Published: August 2015

Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.
Short- and longer-term regulation of very-low-density lipoprotein secretion by insulin, dexamethasone and lipogenic substrates in cultured hepatocytes. A biphasic effect of insulin
Authors: Bartlett S M , Gibbons G F
Published: August 2015

1. The precise effects of insulin, dexamethasone and lipogenic precursors on the secretion of very-low-density lipoprotein (VLDL) cholesterol and triacylglycerol were dependent on the age of the culture and the duration of treatment. 2. The rates of secretion of triacylglycerol and cholesterol gradually declined with the age of the culture, although there was no detectable decrease within a given 24 h period. 3. Between 4 h and 24 h after cell preparation, insulin inhibited VLDL secretion. Inhibition was maximal between 6 and 12 h after addition of insulin. Longer-term treatment (24-48 h) with insulin resulted in a stimulation of VLDL secretion. This effect was less apparent when dexamethasone was simultaneously present. The secretion of triacylglycerol and cholesteryl ester was more sensitive to insulin than was that of non-esterified cholesterol. 4. Dexamethasone alone stimulated the secretion of VLDL to an extent which increased with the age of the culture. In young cultures (up to 24 h old) dexamethasone protected against inhibition by insulin, but was ineffective in older cultures. 5. In young cultures the stimulatory effect of lipogenic precursors (lactate and pyruvate) on the secretion of triacylglycerol and cholesterol was more pronounced in the presence of dexamethasone. In cultures older than 24 h, the secretion of these components was less sensitive to short-term stimulation by lactate and pyruvate.
Identification by fast atom bombardment mass spectrometry of insulin fragments produced by insulin proteinase
Authors: Savoy L A , Jones R M L , Pochon S , Davies J G , Muir A V , Offord R E , Rose K
Published: August 2015

We describe the isolation by reversed-phase h.p.l.c. of a number of products of the degradation of insulin by insulin proteinase and their direct analysis by fast atom bombardment mass spectrometry (f.a.b.-m.s.). Various semisynthetically labelled insulins were used, including [[2H2]GlyA1]insulin and [18O]LysB29]insulin. The results obtained confirm and extend the results obtained by non-mass-spectrometric methods [Davies, Muir, Rose & Offord (1988) Biochem. J. 249, 209-214, and papers cited therein]. Cleavage sites were identified between positions A13-A14, A14-A15, B9-B10, B13-B14, B24-B25 and B25-B26. The advantages and disadvantages of the application of f.a.b.-m.s. to such studies are discussed.
The switching of electron flux from the cyanide-insensitive oxidase to the cytochrome pathway in mung-bean (Phaseolus aureus L.) mitochondria
Authors: Wilson S B
Published: August 2015

The activities of the mung-bean (Phaseolus aureus L.) mitochondrial cyanide-insensitive oxidase and cytochrome pathways have been measured simultaneously. The results show that electrons can be diverted both from the alternative pathway to the cytochrome pathway and from the cytochrome to the alternative pathway. The competition of the two pathways for the available electron flux is discussed.
Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system
Authors: Link E M , Riley P A
Published: August 2015

1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.
Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats
Authors: Lehman-McKeeman L D , Andrews G K , Klaassen C D
Published: August 2015

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.
Haem synthesis during cytochrome P-450 induction in higher plants. 5-Aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark
Authors: Werck-Reichhart D , Jones O T G , Durst F
Published: August 2015

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.
Myeloperoxidase-oxidase oxidation of cysteamine
Authors: Svensson B E , Lindvall S
Published: August 2015

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.
Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively
Authors: Ballard F J , Ross M , Upton F M , Francis G L
Published: August 2015

1. Competitive binding and receptor cross-linking experiments have been used to examine the receptor-ligand interactions between three bovine insulin-like growth factors (IGF) and monolayer cultures of myoblasts and fibroblasts. 2. Labelled IGF-2 bound predominantly to the type 2 receptor with negligible label cross-linked to the type 1 receptor, notwithstanding the ability of IGF-2 to compete effectively for the binding of IGF-1 to the type 1 receptor. Approx. 100-fold higher concentrations of IGF-1 or the N-terminal truncated (des-Gly-Pro-Glu) IGF-1 (-3N:IGF-1) were required to produce competition equivalent to IGF-2. 3. All IGF peptides, but especially IGF-1, enhanced the binding of labelled IGF-2 to the type 2 receptor of lung fibroblasts. This unusual effect was probably a consequence of the displacement of labelled IGF-2 otherwise bound to a medium protein, a conclusion supported by the demonstration of a 38 kDa membrane protein cross-linked to labelled IGF-2. 4. Both IGF-1 and -3N:IGF-1 bound only to the type 1 IGF receptor in L6 myoblasts, rat vascular smooth-muscle cells and human lung fibroblasts. The peptides competed for labelled IGF-1 binding with potencies in the order -3N:IGF-1 greater than IGF-1 greater than IGF-2 much greater than insulin. Since the IGF peptides were equipotent in skin fibroblasts, it was proposed that the apparently higher affinity of -3N:IGF-1 for receptors in the other cell types was instead a consequence of a low affinity of this peptide for the competing 38 kDa binding protein.
500 MHz 1 H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-β-galactosidase
Authors: Hounsell E F , Feeney J , Scudder P
Published: August 2015

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)
Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents
Authors: Flamigni F , Guarnieri C , Caldarera C M
Published: August 2015

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.
Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells
Authors: Koshio O , Akanuma Y , Kasuga M
Published: August 2015

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.
Effect of recombinant cytokines on glycolysis and fructose 2,6-bisphosphate in rheumatoid synovial cells in vitro
Authors: Taylor D J , Whitehead R J , Evanson J M , Westmacott D , Feldmann M , Bertfield H , Morris M A , Woolley D E
Published: August 2015

Recombinant-derived human interleukin 1 (IL1) alpha and beta and interferon gamma (IFN-gamma) each produced similar increases in rheumatoid synovial cell (RSC) glycolysis, as judged by increased values for glucose uptake, lactate production and cellular fructose 2,6-bisphosphate [Fru(2,6)P2]. Measurement of Fru(2,6)P2 proved to be the most sensitive parameter for an assessment of glycolysis: IL1 alpha, IL1 beta and IFN-gamma all produced a 3-6-fold increase in this metabolite whereas tumour necrosis factor (TNF alpha) was far less effective. Prostaglandin E production was stimulated predominantly by IL1 alpha and IL1 beta rather than by IFN-gamma or TNF alpha. When combinations of cytokines were examined the addition of IFN-gamma with either IL1 alpha, IL1 beta or murine IL1 produced a synergistic increase in cellular Fru(2,6)P2. The three forms of IL1 increased Fru(2,6)P2 via the same pathway, whereas IFN-gamma acted via a different mechanism. The increase in Fru(2,6)P2 in subcultured RSC produced by addition of medium from a primary culture exceeded the maximal effects of any of the single cytokines studied, suggesting the presence of a mixture of cytokines in the primary RSC culture medium.
The effects of surgical stress and short-term fasting on protein synthesis in vivo in diverse tissues of the mature rat
Authors: Preedy V R , Paska L , Sugden P H , Schofield P S , Sugden M C
Published: August 2015

1. We measured fractional rates of protein synthesis, capacities for protein synthesis (i.e. RNA/protein ratio) and efficiencies of protein synthesis (i.e. protein-synthesis rate relative to RNA content) in fasted (24 or 48 h) or fasted/surgically stressed female adult rats. 2. Of the 15 tissues studied, fasting caused decreases in protein content in the liver, gastrointestinal tract, heart, spleen and tibia. There was no detectable decrease in the protein content of the skeletal muscles studied. 3. Fractional rates of synthesis were not uniformly decreased by fasting. Rates in striated muscles, uterus, liver, spleen and tibia were consistently decreased, but decreases in other tissues (lung, gastrointestinal tract, kidney or brain) were inconsistent or not detectable, suggesting that, in many tissues in the mature rat, protein synthesis was not especially sensitive to fasting. 4. In fasting, the decreases in fractional synthesis rate resulted from changes in efficiency (liver and tibia) or from changes in efficiency and capacity (heart, diaphragm, plantaris and gastrocnemius). In the soleus, the main change was a decrease in capacity. 5. Surgical stress increased fractional rates of protein synthesis in diaphragm (where there were increases in both efficiency and capacity) by about 50%, in liver by about 20%, in spleen by about 40%, and possibly also in the heart. In liver and spleen, capacities were increased. In other tissues (including the skeletal muscles), the fractional rates of protein synthesis were unaffected by surgical stress.
The effects of aging on carbonic anhydrase concentrations in rat liver and skeletal muscle
Authors: Jeffery S , Merry B J , Holehan A M , Carter N D
Published: August 2015

The isoenzymes carbonic anhydrase II (CAII) and III (CAIII) have been measured by radioimmunoassay in the livers of male and female rats aged from 21 to 800 days. No sexual dimorphism at 21 days was found, but from 50 to 400 days both isoenzymes show sexual differences. From 600 days onwards, these differences are less apparent. CAIII concentrations in two ‘fast’ fibre muscles and one ‘slow’ fibre muscle have been determined. There is no sexual dimorphism in muscle, but a wide variation between individuals was observed. Fast muscles show maximal CAIII levels at 800 days, whereas in slow muscle the concentration of the isoenzyme is declining at this time.
Evidence for distinct functional molecular sizes of carnitine palmitoyltransferases I and II in rat liver mitochondria
Authors: Zammit V A , Corstorphine C G , Kelliher M G
Published: August 2015

1. Estimates of the functional sizes of the molecular species responsible for the overt (I) and latent (II) activities of carnitine palmitoyltransferase (CPT) in 48 h-starved rat liver mitochondria were obtained from radiation inactivation experiments. 2. The decay in the activity of total CPT and that of CPT II only (after inhibition of CPT I) was measured in mitochondrial samples exposed to different doses of high-energy ionizing radiation. 3. The decay curves obtained by plotting residual activity of total CPT as a logarithm function of irradiation dose suggested the contribution of more than one target towards total CPT activity. 4. By contrast, in mitochondria in which CPT I activity was approximately 95% inhibited, the activity of CPT decayed in a simple mono-exponential manner. Target-size analysis yielded an approximate Mr of 69,700 for this component (CPT II). 5. This information, as well as that on the relative non-irradiated activities of CPT I and CPT II, was used in graphical and statistical methods to obtain the parameters of the decay curve for CPT I. These analyses yielded an approximate Mr of 96,700 for CPT I.
Subcellular localization of transglutaminase. Effect of collagen
Authors: Juprelle-Soret M , Wattiaux-De Coninck S , Wattiaux R
Published: August 2015

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.
Presence of sterol-binding sites in the cytosol of French-bean (Phaseolus vulgaris) roots
Authors: Vega M A , Boland R L
Published: August 2015

Vitamin D3 (cholecalciferol) and stigmasterol have been shown to stimulate Ca2+ uptake and to induce calmodulin synthesis in cultured French-bean (Phaseolus vulgaris) roots. In addition, the appearance of calmodulin in the cultures in response to vitamin D3 could be prevented by RNA-synthesis inhibitors. To investigate the possibility that the sterols affect root DNA transcription through a receptor-mediated mechanism, the existence of sterol-binding sites in P. vulgaris roots was investigated. Specific binding of [3H]vitamin D3 could be demonstrated with intact tissue and the cytosolic fraction obtained therefrom. Equilibrium in the binding reaction with cytosol was attained after 4 h of incubation at 0 degrees C. The [3H]vitamin D3 was reversibly bound, since it could be displaced by an excess of unlabelled sterol. An equilibrium binding constant (KD) of (3.48 +/- 0.09) x 10(-9) M and a maximum binding-site concentration (nmax) of 32 +/- 2.54 (3) pmol/mg of protein could be calculated by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672] analysis. In addition to vitamin D3, stigmasterol and sitosterol were effectively able to compete with [3H]vitamin D3 for binding to root cytosol. Cortisol, oestradiol and progesterone displaced bound labelled vitamin D3 to a lesser extent, whereas 5 beta-dihydrotestosterone, lanosterol and diosgenin were ineffective. The affinity and specificity of the root sterol-binding sites are in agreement with the characteristics of tissue responses to the sterols (Ca2+ uptake and calmodulin synthesis).
Heparan sulphate-degrading endoglycosidase in liver plasma membranes
Authors: Gallagher J T , Walker A , Lyon M , Evans W H
Published: August 2015

An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.
Effects of nutritional status and acute variation in substrate supply on cardiac and skeletal-muscle fructose 2,6-bisphosphate concentrations
Authors: French T J , Holness M J , MacLennan P A , Sugden M C
Published: August 2015

We examined the long-term effects of nutritional status and the acute effects of changes in exogenous carbohydrate- and lipid-substrate supply and utilization on fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations in heart, gastrocnemius and soleus. Starvation decreased Fru-2,6-P2 concentrations in all three muscles. The acute administration of insulin and glucose increased skeletal-muscle Fru-2,6-P2 in the fed, but not in the starved, state, but cardiac Fru-2,6-P2 was unchanged. Cardiac and skeletal-muscle Fru-2,6-P2 concentrations were unaffected by acute increases in fatty acid supply produced by the administration of corn oil plus heparin, or by acute decreases in fatty acid supply produced by inhibition of lipolysis. Differences in cardiac and skeletal-muscle Fru-2,6-P2 concentrations observed in response to starvation were not reversed by administration of glucose or glucose plus insulin, or by inhibition of lipolysis, even though changes in citrate (heart), acylcarnitine (heart) and glycogen (skeletal muscle) were observed. Concentrations remained low for at least 8 h after chow re-feeding, but the fed value was restored by 24 h.
α-amidated peptides derived from pro-opiomelanocortin in normal human pituitary
Authors: Fenger M , Johnsen A H
Published: August 2015

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.
Changes in ornithine decarboxylase and antizyme activities in developing mouse brain
Authors: Onoue H , Matsufuji S , Nishiyama M , Murakami Y , Hayashi S
Published: August 2015

A macromolecular inhibitor to ornithine decarboxylase (ODC) present in mouse brain was identified as ODC antizyme [Fong, Heller & Canellakis (1976) Biochim. Biophys. Acta 428, 456-465; Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1858-1862] on the basis of kinetic properties, Mr and reversal of its inhibition by antizyme inhibitor. The brain antizyme, however, did not cross-react immunochemically with any of seven monoclonal antibodies to rat liver antizyme. ODC activity in mouse brain rapidly decreased after birth, in parallel with putrescine content, and almost disappeared by 3 weeks of age. Free antizyme activity appeared shortly after birth and increased gradually, whereas ODC-antizyme complex already existed at birth and then gradually decreased. Thus total amount of antizyme remained about the same throughout the developmental period in mouse brain. In addition to ODC-antizyme complex, inactive ODC protein was detected by radioimmunoassay in about the same level as the complex at 3 weeks of age. Upon cycloheximide treatment, both free ODC activity and ODC-antizyme complex rapidly disappeared, although free antizyme and the inactive ODC protein were both quite stable.
Cellular content of ubiquitin and formation of ubiquitin conjugates during chicken spermatogenesis
Authors: Agell N , Mezquita C
Published: August 2015

Ubiquitin was purified from chicken testis and its content, biosynthesis and formation of conjugates was determined in germinal cells at successive stages of spermatogenesis. Free ubiquitin increased markedly during spermatogenesis, reaching its maximum level in early spermatids. High levels of ubiquitin were still present in late spermatids but were not detectable in mature spermatozoa. Biosynthesis of ubiquitin occurred in vitro in a fraction containing meiotic and pre-meiotic cells, and during spermiogenesis, in early and late spermatids. The cellular content of free ubiquitin increased after ATP depletion, especially in early spermatids. Lysates of chicken testis cells, particularly those obtained from spermatids, were able to form nuclear (24 and 27 kDa) and extranuclear (55-90 kDa) ubiquitin conjugates in vitro. The presence of increasing levels of ubiquitin and ubiquitin conjugates in chicken spermatids may suggest a possible involvement of this protein in the marked changes of protein turnover, chromatin structure and cell-cell interactions that spermatids undergo during spermiogenesis.
Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase
Authors: Spence J T , Barber M J , Solomonson L P
Published: August 2015

Microcoulometric titrations of NADH:nitrate reductase at 25 degrees C in Mops buffer, pH 7.0, showed that the native enzyme, containing functional FAD, haem and Mo, required addition of five electrons for complete reduction. Reduction of the native enzyme occurred in three waves corresponding to addition of reducing equivalents to the centres in the order: Mo, haem, FAD. Oxidation-reduction midpoint potentials (E'0) for the various redox couples were calculated to be as follows: MoVI/MoV, +16 mV; MoV/MoIV, -27 mV; haemoxidized/haemreduced, -172 mV; FAD/FADH2, -283 mV. The values for the haem and flavin are in excellent agreement with those obtained by visible titrations, namely -164 mV and -288 mV respectively. In contrast, the results for the Mo centre are 28-50 mV more positive than the values previously determined by e.p.r. analysis of frozen enzyme samples poised at defined potentials at 25 degrees C and suggest different pH-dependencies or entropies of reduction for the Mo couples.
Interaction of the trp repressor from Escherichia coli with a constitutive trp operator
Authors: Chandler L R , Lane A N
Published: August 2015

The interaction of the trp repressor from Escherichia coli with a 20 bp fragment of DNA (CGTACTGATT.AATCAGTACG) corresponding to a mutant trp operator was studied by c.d. in the presence and absence of the co-repressor, L-tryptophan, and as a function of the concentration of K+ and Na+ ions. The affinity of the repressor for the mutant operator in the presence of tryptophan is about three orders of magnitude lower than the wild-type sequence. Binding in the absence of tryptophan is about 100-fold weaker than to the wild-type. The dependence of the dissociation constant on the concentration of K+ or Na+ is weak [d(log Ks)/d(log[M+]) = 2.5], and independent of the cation, indicating that electrostatic interactions are not as important for this repressor as for others.