bluuebunny/crossref_metadata_2025 huggingface dataset:
A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath)
Authors: Green J , Dalton H
Published: August 2015

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.
Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase
Authors: Mayr G W
Published: August 2015

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.
X-ray-absorption and electron-paramagnetic-resonance spectroscopic studies of the environment of molybdenum in high-pH and low-pH forms of Escherichia coli nitrate reductase
Authors: George G N , Turner N A , Bray R C , Morpeth F F , Boxer D H , Cramer S P
Published: August 2015

Previous e.p.r. work [George, Bray, Morpeth & Boxer (1985) Biochem. J. 227, 925-931] has provided evidence for a pH- and anion-dependent transition in the structure of the Mo(V) centre of Escherichia coli nitrate reductase, with the low-pH form bearing both an anion and probably a hydroxy-group ligand. Initial e.x.a.f.s. measurements [Cramer, Solomonson, Adams & Mortenson (1984) J. Am. Chem. Soc. 106, 1467-1471] demonstrated the presence of sulphur (or chloride) ligands in the Mo(IV) and Mo(VI) oxidation states, as well as a variable number of terminal oxo (Mo = O) groups. To synthesize the e.p.r. and e.x.a.f.s. results better, we have conducted new e.p.r. experiments and complementary e.x.a.f.s. measurements under redox and buffer conditions designed to give homogeneous molybdenum species. In contrast with results on other molybdoenzymes, attempts to substitute the enzyme with 17O by dissolving in isotopically enriched water revealed only very weak hyperfine coupling to 17O. The significance of this finding is discussed. Experiments with different buffers indicated that buffer ions (e.g. Hepes) could replace the Cl- ligand in the low-pH Mo(V) enzyme form, with only a small change in e.p.r. parameters. E.x.a.f.s. studies of the oxidized and the fully reduced enzyme were consistent with the e.p.r. work in indicating a pH- and anion-dependent change in structure. However, in certain cases non-stoichiometric numbers of Mo = O interactions were determined, complicating the interpretation of the e.x.a.f.s. Uniquely for a molybdenum cofactor enzyme, a substantial proportion of the molecules in a number of enzyme samples appeared to contain no oxo groups. No evidence was found in our samples for the distant ‘heavy’ ligand atom reported in the previous e.x.a.f.s. study. The nature of the high-pH-low-pH transition is briefly discussed.
Effect of choline deficiency on S-adenosylmethionine and methionine concentrations in rat liver
Authors: Zeisel S H , Zola T , daCosta K A , Pomfret E A
Published: August 2015

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.
Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency
Authors: Beckett G J , MacDougall D A , Nicol F , Arthur J R
Published: August 2015

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.
Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes
Authors: Higgins J A , Hitchin B W , Low M G
Published: August 2015

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.
Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase
Authors: Morpurgo L , Agostinelli E , Muccigrosso J , Martini F , Mondovi B , Avigliano L
Published: August 2015

Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.
Genetic regulation of β2-adrenergic receptors in 3T3-L1 fibroblasts
Authors: Nakada M T , Haskell K M , Ecker D J , Stadel J M , Crooke S T
Published: August 2015

The beta 2-adrenergic receptor from mouse 3T3-L1 cells is up-regulated through genetic mechanisms by glucocorticoids and butyrate. To study the genetic regulation of these receptors, we sequenced a 5 kb region of genomic DNA from 3T3-L1 cells, containing the beta-adrenergic receptor gene and approx. 1.5 kb of both 5′ and 3′ flanking sequences. The sequence contained one copy of an 8 bp consensus sequence which can confer phorbol ester-responsiveness to genes. Phorbol esters attenuated the up-regulation of beta 2-adrenergic receptors by glucocorticoids but not by butyrate. This effect was probably due to a phorbol ester-induced decrease in glucocorticoid receptor number. Using methylation-sensitive restriction enzymes, we examined the methylation of a CG-rich region occurring 5′ to the gene and did not detect any changes in methylation of this region upon dexamethasone or butyrate treatment. A total of 16 putative glucocorticoid response elements were found which may mediate the glucocorticoid-induced increase in beta 2-adrenergic receptors. A comparison of the regulatory sequences of the two beta-adrenergic receptor subtypes from human and mouse confirms the observed physiological controls of receptor subtype expression and offers an explanation as to why the subtypes differ in genetic regulation.
Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki
Authors: Bietlot H , Carey P R , Choma C , Kaplan H , Lessard T , Pozsgay M
Published: August 2015

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.
An analysis of the stopped-flow kinetics of gaseous ligand uptake and release by adult mouse erythrocytes
Authors: Brittain T , Simpson R
Published: August 2015

Ligand uptake and release by the haemoglobin contained within adult mouse erythrocytes was studied by using dual-wavelength stopped-flow techniques. The rate of O2 uptake is very much lower than that expected for an equivalent concentration of haemoglobin in free solution. The O2-concentration-dependence found in uptake experiments is greater than first-order. CO uptake shows the same pattern of reactivity as does O2, but the associated rates of uptake are lower and the concentration-dependence of the CO rates is first-order. O2 release from the adult erythrocytes was measured by stopped-flow mixing with Na2S2O4. Under these circumstances the deoxygenation of intracellular haemoglobin shows accelerating time courses. The apparent rate-constant-dependence on dithionite concentration shows a rate limit at high reductant concentrations. Computer simulations of both ligand uptake and release processes were carried out by using a three-dimensional model. The simulations clearly indicate that in rapid-mixing experiments the rather slow experimentally observed O2 uptake rate is due to rate-limiting diffusion through an extracellular stagnant solvent layer. In the case of O2 release, however, the major rate-controlling process is the rate of O2 dissociation from the haemoglobin molecules, which accelerates during the deoxygenation process.
Purification of a unique glycoprotein that enhances phenol oxidase activity in scorpion (Heterometrus bengalensis) haemolymph
Authors: Datta T K , Basu P S , Datta P K , Banerjee A
Published: August 2015

A monomeric glycoprotein (SGP) of Mr 32,000 was isolated to purity from scorpion (Heterometrus bengalensis) haemolymph by (NH4)2SO4 fractionation, chromatofocusing and h.p.l.c. The homogeneity of SGP is confirmed by polyacrylamide-gel electrophoresis. SGP is soluble in 100%-satd. (NH4)2SO4 solution. Needle-shaped crystals of SGP were obtained in an aqueous environment. The glycan part of the molecule contains arabinose, which does not commonly occur in animal glycoproteins. Amino acid analysis demonstrated a preponderance of glycine, tyrosine and glutamic acid. SGP enhances phenol oxidase (EC 1.14.18.1) activity.
Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene
Authors: Lloyd A C , Davies S A , Crossley I , Whitaker M , Houslay M D , Hall A , Marshall C J , Wakelam M J
Published: August 2015

Bombesin stimulation of T15 cells in which the inducible N-ras oncogene is overexpressed caused elevated production of inositol phosphates compared to uninduced cells [Wakelam, Davies, Houslay, McKay, Marshall & Hall (1986) Nature (London) 323, 173-176]. This elevated response is shown here to result from increased generation of inositol 1,4,5-trisphosphate leading to an elevated release of intracellular stored Ca2+. Single-cell analysis of Ca2+ release showed that the elevated response is not a consequence of an increased fraction of responding cells. These amplifications are consistent with p21N-ras acting like a guanine nucleotide coupling protein in this cell line.
Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture
Authors: Newsholme P , Newsholme E A
Published: August 2015

1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response.
Changes in collagen stability and folding in lethal perinatal osteogenesis imperfecta. The effect of α1(I)-chain glycine-to-arginine substitutions
Authors: Baker A T , Ramshaw J A M , Chan D , Cole W G , Bateman J F
Published: August 2015

The effect of glycine-to-arginine mutations in the alpha 1 (I)-chain on collagen triple-helix structure in lethal perinatal osteogenesis imperfecta was studied by determination of the helix denaturation temperature and by computerized molecular modelling. Arginine substitutions at glycine residues 391 and 667 resulted in similar small decreases in helix stability. Molecular modelling suggested that the glycine-to-arginine-391 mutant resulted in only a relatively small localized disruption to the helix structure. Thus the glycine-to-arginine substitutions may lead to only a small structural abnormality of the collagen helix, and it is most likely that the over-modification of lysine, poor secretion, increased degradation and other functional sequelae result from a kinetic defect in collagen helix formation resulting from the mutation.
The biosynthesis of the chromophore of phycocyanin. Pathway of reduction of biliverdin to phycocyanobilin
Authors: Brown S B , Holroyd J A , Vernon D I , Shim Y K , Smith K M
Published: August 2015

The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin.
Postnatal changes in dolichol-pathway enzyme activities in rat liver
Authors: Oda-Tamai S , Kato S , Akamatsu N
Published: August 2015

The activity of hepatic protein N-glycosylation was compared in rats of different ages by incubating UDP-[14C]glucose with liver microsomes. Dolichyl-phosphate [14C]glucose, [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins formed were increased after birth to maximal levels at 2 weeks; thereafter dolichylphosphate [14C]glucose remained constant, while [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins were decreased to constant levels at 4 weeks. The postnatal change in the formation of [14C]glycoproteins was similar to the change in the hexosamine content of N-glycans in liver microsomes and plasma, suggesting that the N-glycosylation of proteins in rat liver increases after birth to a maximum at 2 weeks, and thereafter decreases to a constant level at 4 weeks. The possibility of a regulatory role for dolichyl phosphate in glycoprotein synthesis in rat liver during postnatal development was eliminated by demonstrating the inefficiency of exogenous dolichyl phosphate on the postnatal changes in [14C]glycoprotein formation. The transfer of [14C]glucose from UDP-[14C]glucose to denatured alpha-lactalbumin in liver microsomes increased to a maximum at 2 weeks and then decreased to a constant level, as with transfer to endogenous proteins (i.e. the formation of [14C]glycoproteins). On the other hand, the transfer of oligosaccharide from exogenous [14C]glucosyl-oligosaccharide-lipid to denatured alpha-lactalbumin reached a maximum at 2 weeks and then remained constant. These results strongly suggest that oligosaccharide-lipid available for N-glycosylation is limiting in rat liver after 2 weeks post partum. The activities of dolichyl-phosphate glucose, dolichyl-phosphate mannose and dolichyl-pyrophosphate N-acetylglucosamine synthases increased until 2 weeks post partum. Thereafter, the activity of dolichyl-pyrophosphate N-acetylglucosamine synthase decreased to a constant level at 4 weeks, while the activities of dolichyl-phosphate glucose and dolichyl-phosphate mannose synthases remained constant. These results suggest that N-glycosylation of proteins in rat liver increases until 2 weeks post partum, and that this depends on the activities of dolichol-pathway enzymes as a whole rather than on the activity of specific enzymes. N-Glycosylation then decreases to a constant level at 4 weeks due to decreases in the activities of enzymes responsible for oligosaccharide assembly on lipids, including dolichyl-pyrophosphate N-acetylglucosamine synthase.
Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal β-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver
Authors: Kawashima Y , Uy-Yu N , Kozuka H
Published: August 2015

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2′-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA.
Oxytocin inactivates and phosphorylates rat hepatocyte glycogen synthase
Authors: Ariño J , Bosch F , Gómez-Foix A M , Guinovart J J
Published: August 2015

Incubation of isolated rat hepatocytes with oxytocin produces a time- and dose-dependent inactivation of glycogen synthase. Such inactivation is associated with an increase in the phosphorylation state of the 88 kDa subunit of the enzyme, as observed after electrophoretic analysis of the 32P-labelled enzyme isolated by immunoprecipitation from cells incubated with [32P]phosphate. CNBr cleavage of the immunoprecipitated glycogen synthase showed that multiple sites were phosphorylated after exposure of the cells to the hormone. The effect of oxytocin on hepatocyte glycogen synthase activity was not observed in the absence of extracellular Ca2+. Inactivation of glycogen synthase by oxytocin was partially abolished in the presence of insulin. These results indicate that the effects of oxytocin on glycogen synthase from rat hepatocytes are similar to those observed for other Ca2+-mediated glycogenolytic hormones, such as vasopressin.
Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways
Authors: Ali N , Milligan G , Evans W H
Published: August 2015

1. The distribution of the alpha- and beta-subunits of nucleotide-binding G-proteins among rat liver sinusoidal, lateral and canalicular plasma membranes, endosomes, Golgi membranes and lysosomes was investigated. 2. Pertussis-toxin-catalysed ADP-ribosylation identified a 41 kDa inhibitory alpha-subunit in all liver plasma-membrane functional domains as well as in endosomes. An antibody to a synthetic peptide corresponding to a C-terminal sequence of the inhibitory alpha-subunit also identified the 41 kDa polypeptide in all plasma-membrane domains, in ‘early’ and ‘late’ endosomes and in Golgi membranes; this polypeptide was not detected in lysosomes. The antibody-binding studies showed that bile-canalicular plasma membranes had the highest content of the inhibitory alpha-subunit. 3. Immunofluorescent microscopy confirmed the presence of the inhibitory alpha-subunit in all regions of the hepatocyte's cell surface. 4. An antibody recognizing the beta-subunit showed that a 36 kDa polypeptide was present in all plasma membranes and in ‘early’ and ‘late’ endosomes; it was not detected in lysosomes. The relative distribution among the fractions of this polypeptide was similar to the distribution of the inhibitory alpha-subunit. 5. The presence of high levels of the G-protein inhibitory alpha-subunit in bile-canalicular plasma membranes was confirmed by demonstration of its co-fractionation with marker enzymes in Nycodenz gradients and by free-flow electrophoresis. The significance of this location is discussed.
Ovalbumin and angiotensinogen lack serpin S–R conformational change
Authors: Stein P E , Tewkesbury D A , Carrell R W
Published: August 2015

Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.
Desensitization of α2-adrenergic receptors in NG 108 15 cells by (–)-adrenaline and phorbol 12-myristate 13-acetate
Authors: Convents A , De Backer J P , André C , Vauquelin G
Published: August 2015

alpha 2-Adrenergic receptors on NG 108 15 cell membranes were identified by [3H]rauwolscine binding: Bmax. = 661 +/- 81 fmol/mg of protein, Kd = 6.9 +/- 2.5 nM (mean +/- S.E.M., n = 6). On intact cells, stimulation of these receptors by (-)-adrenaline inhibited the prostaglandin-E1-stimulated adenylate cyclase activity by about 60%. The effect of (-)-adrenaline was pertussis-toxin-sensitive, indicating the involvement of an inhibitory G protein. (-)-Adrenaline/[3H]rauwolscine competition-binding experiments revealed that only 50% of the alpha 2 receptors were coupled to G proteins (i.e. displayed high agonist affinity). Pre-treatment of the cells with 20 microM-(-)-adrenaline provoked homologous desensitization of the alpha 2 receptors. The alpha 2-adrenergic response decreased after a time lag of about 2 h, to reach a minimum after 12 h. The bradykinin and muscarinic responses were not affected. The alpha 2-receptor concentration decreased without time lag. The high-agonist-affinity sites disappeared more rapidly (t1/2 = 42 min) than did the low-affinity uncoupled sites (t1/2 approx. 20 h). In contrast, pertussis-toxin-mediated [32P]ADP-ribosylation of inhibitory G proteins was unaffected by the pre-treatment. Pretreatment of intact NG 108 15 cells with 1 microM-phorbol 12-myristate 13-acetate (PMA) provoked a rapid decrease of the alpha 2-adrenergic response. The effect was nearly complete after 40 min. PMA also decreased the bradykinin response, suggesting a heterologous desensitization process. The alpha 2-receptor concentration, the (-)-adrenaline competition-binding curves and the pertussis- and cholera-toxin-mediated [32P]ADP-ribosylation of their respective G proteins were not affected.
E.p.r.-spectroscopic studies on the molybdenum–iron site of nitrogenase from Clostridium pasteurianum
Authors: George G N , Bare R E , Jin H Y , Stiefel E I , Prince R C
Published: August 2015

The e.p.r. spectroscopy of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum was re-investigated. The sharpness of the delta Ms = +/- 3 g′z peak from the +/- 3/2 Kramer's doublet enables the observation and quantification of incompletely resolved hyperfine splittings from the stable magnetic nuclei 95Mo and 57Fe in samples enriched in these isotopes. No couplings to 1H or 17O could be discerned by examination of spectra from samples exchanged into 2H2O and H2(17)O respectively. Simulation of the spectrum from 95Mo-enriched samples yields a hyperfine coupling of 2.9 MHz, and indicates that the earlier electron-nuclear-double-resonance-derived estimate of 8.1 +/- 0.2 MHz is substantially in error.
Ionic-strength-dependence of the oxidation of native and pyridoxal 5′-phosphate-modified cytochromes c by cytochrome c oxidase
Authors: Kossekova G , Atanasov B , Bolli R , Azzi A
Published: August 2015

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5′-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.
Identification of a novel autophosphorylation site (P4) on the epidermal growth factor receptor
Authors: Hsuan J J , Totty N , Waterfield M D
Published: August 2015

Three major autophosphorylation sites are located near the C-terminus of the epidermal growth factor receptor, but a fourth site is repeatedly detected. We report here the purification and sequencing of a tryptic peptide containing this site, Tyr-1086. Furthermore, we demonstrate that additional phosphopeptides are observed following both partial digestion and overdigestion. Finally, we show that Tyr-1086 can be phosphorylated in intact cells.
Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein
Authors: Taylor M E , Brickell P M , Craig R K , Summerfield J A
Published: August 2015

The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a ‘neck’ region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver.
pH-induced bistable dynamic behaviour in the reaction catalysed by glucose-6-phosphate dehydrogenase and conformational hysteresis of the enzyme
Authors: Aon M A , Cortassa S , Hervagault J F , Thomas D
Published: August 2015

1. Bistable (multiple stationary states) dynamic behaviour in the activity of glucose-6-phosphate dehydrogenase that was subjected to successive pH change was demonstrated in an open continuously stirred tank reactor. Although the enzyme under study did not exhibit an autocatalytic effect and was homogeneously distributed, bistability was shown to occur. 2. The successive pH changes of the enzyme solution corresponded to a pH transition (8.3 in equilibrium 2), i.e. an acidification (forward direction) and an alkalinization (reverse direction). By use of intrinsic protein fluorescence methods, a glucose-6-phosphate dehydrogenase conformational hysteresis was shown to exist concomitant with the pH transition before and after enzyme injection into the reactor. 3. The results obtained suggest that the enzyme behaves, conformationally, as a memory device that stores information about its pH history (i.e. the enzyme records information in its structure about the environment to which it was previously exposed) and transduces it in a non-linear dynamic fashion, producing the bistable behaviour observed in the open reactor.
Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans
Authors: Horner A A
Published: August 2015

Approximately half of all rat skin heparin proteoglycans have polysaccharide chains that have no sites with high binding affinity for antithrombin. The rest have chains with high-affinity antithrombin-binding-site densities ranging from zero to five sites per chain, with a high degree of variation. Proteoglycans vary in size because of diversity in the number of chains per molecule; the relationship between proteoglycan size and high-affinity antithrombin-binding-site density has not been studied previously. Polydisperse heparin proteoglycans from rat skin, labelled biosynthetically with 35S, were fractionated by gel filtration on Bio-Gel A-150m and arbitrarily divided into five fractions of decreasing average molecular size. Fractionation of these products on antithrombin-agarose showed that the proportion of proteoglycans with high affinity for antithrombin decreased from 39% to 25% as molecular size decreased. However, as the molecular size of high-affinity proteoglycans decreased, the proportion of their chains that had high affinity increased from 29% to 59%. Therefore molecular size is a significant factor in determining the proportion of high-affinity chains in heparin proteoglycans. A model of heparin biosynthesis is proposed in which areas of specific enzyme activity that control the synthesis of the antithrombin-binding-site sequence are sparsely and nonrandomly distributed on mast-cell Golgi membranes. It is postulated that the likelihood of a developing proteoglycan encountering one of these hypothetical areas is molecular-size-dependent.
Characterization of hepatic lactogen receptor. Subcellular distribution and characterization of N-linked carbohydrate chains
Authors: Haldosén L A , Andersson G , Gustafsson J A
Published: August 2015

The types of carbohydrate chains present in a rat liver lactogenic hormone-binding receptor species with an Mr of 82,000, and in its hormone-binding subunits with Mr values of 40,000 and 35,000, were characterized using carbohydrate-chain-cleaving enzymes and affinity cross-linking. The subcellular distribution of lactogenic hormone-binding species was studied in organelle-enriched fractions. The monomeric Mr-40,000 and Mr-35,000 species contain N-linked tri- or tetra-antennary complex and high-mannose chains respectively. The Mr-82,000 species exists in two forms, where the Mr-40,000 and Mr-35,000 subunits are each combined with unglycosylated and, with the technique used, unlabelled subunit(s). Studies with organelle-enriched fractions revealed that the Mr-35,000 species was found in an endoplasmic reticulum-enriched fraction. The Mr-40,000 species was the predominant monomeric binding species in Golgi/endosome- and plasma membrane-enriched fractions. It is suggested that the Mr-35,000 species is a precursor to the Mr-40,000 species. In lysosome/endosome- or lysosome-enriched fractions, a broad distribution in Mr (35,000-40,000) was characteristic of the hormone-binding species. The Mr-82,000 species was only found in a Golgi/endosome-enriched fraction. Labelling of endosome lactogen receptor by injection of 125I-labelled ovine prolactin in vivo and cross-linking yielded only the Mr-40,000 species. Thus, the Mr-40,000 and Mr-35,000 lactogenic hormone-binding species each appear to be combined with the unglycosylated receptor subunit(s) in the Golgi complex to form Mr-82,000 heterodimeric complexes.
Primary structure of a mouse mastocytoma proteoglycan core protein
Authors: Kjellén L , Pettersson I , Lillhager P , Steen M L , Pettersson U , Lehtonen P , Karlsson T , Ruoslahti E , Hellman L
Published: August 2015

The complete nucleotide sequence of a mouse mastocytoma proteoglycan core protein mRNA was determined. The mRNA, estimated to contain 1.1 kb, encodes a protein with an Mr of 16715. A 21-amino acid-residue region of the protein is composed of alternating serine and glycine residues. Southern-blot analysis of mouse genomic DNA with cDNA containing sequences corresponding to the Ser-Gly repeat region revealed more than 15 gene fragments. Hybridization with a probe corresponding to the N-terminal portion of the core protein identified two fragments, and cDNA covering the C-terminal part of the core protein and the 3′ untranslated part of the mRNA hybridized to a single fragment. Antibodies against the core protein, obtained after immunization of rabbits with a fusion protein, reacted with both chondroitin sulphate proteoglycans and heparin proteoglycans produced by the tumour. In immunoblotting of a microsomal fraction from the mastocytoma, the antiserum recognized a single protein (Mr 17,000), which probably represents the core protein before glycosylation.
Brain chemiluminescence and oxidative stress in hyperthyroid rats
Authors: Adamo A M , Llesuy S F , Pasquini J M , Boveris A
Published: August 2015

Newborn Wistar rats were made hyperthyroid by injection of tri-iodothyronine and assayed for survival, brain oxygen uptake, brain chemiluminescence and activity of antioxidant enzymes. Brain chemiluminescence was measured (1) by removing the parietal bones or (2) through the translucid parietal bones. Control animals showed a brain chemiluminescence of 130 +/- 12 c.p.s./cm2 and 99 +/- 10 c.p.s./cm2 for procedures (1) and (2) respectively. Hyperthyroid rats showed increases in the spontaneous brain photoemission of 46 and 70% compared with controls, measured by procedures 1 and 2 respectively. The hyperthyroid state did not modify the oxygen-dependent chemiluminescence of brain homogenates. The hyperthyroid animals showed a 30% increase in the oxygen uptake of brain slices and a dramatic shortening of life-span to about 16 weeks. Superoxide dismutase (the Cu-Zn enzyme), catalase and Se-dependent glutathione peroxidase activities of brain homogenates were increased by 18, 36 and 30% respectively in the hyperthyroid animals. Isolated brain mitochondria produced 0.18-0.20 nmol of H2O2/min per mg of protein in state 4 in the presence of succinate as substrate. No difference was observed between control and hyperthyroid animals. It is concluded that hyperthyroidism leads to hypermetabolism and oxidative stress in the brain. The increased levels of oxygen and peroxyl radicals may contribute to premature ageing in these animals.
Evidence for tight metabolic control of the receptor-activated polyphosphoinositide cycle in human platelets
Authors: Steen V M , Tysnes O B , Holmsen H
Published: August 2015

The [32P]PIP2/[32P]PA and the [32P]PIP/[32P]PA relationships were demonstrated to be remarkably similar after stimulation of [32P]Pi-prelabelled platelets for 90 s with various combinations and concentrations of agonists and inhibitors. Thus the activity of the PI and PIP kinases with the corresponding phosphomonoesterases may be tightly controlled during receptor-mediated platelet stimulation involving phospholipase C activation.
Kinetic analysis of protein modification reactions at equilibrium
Authors: Rakitzis E T
Published: August 2015

A kinetic analysis is presented of reactions of protein modification, and/or of modification-induced enzyme inactivation, which can formally be described by a single exponential function, or by a summation of two exponential functions, of reaction time plus a constant term. The reaction schemes compatible with the kinetic formalism of these cases are given, and a simple kinetic criterion is described whereby the identification of one of these cases, strong negative protein modification co-operativity, may be carried out. The treatment outlined in this paper is applied to a case from the literature, the inactivation of glyceraldehyde-3-phosphate dehydrogenase by butane-2,3-dione [Asriyants, Benkevich & Nagradova (1983) Biokhimiya (Engl. Transl.) 48, 164-171].
Pathways of glycogen synthesis from glucose during the glycogenic response to insulin in cultured foetal hepatocytes
Authors: Bismut H , Plas C
Published: August 2015

The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.