bluuebunny/crossref_metadata_2025 huggingface dataset:
Some reactions of carbon monoxide and oxygen with carbodi-imide-modified cytochrome c
Authors: Mathews A J , Brittain T
Published: August 2015

The reactivity of carbodi-imide-modified tuna and horse heart cytochromes c with the ferrous ion ligands CO and O2 has been studied. Both modified cytochromes bind one molecule of CO. Stopped-flow and flash-photolysis experiments indicate the presence of three kinetic processes in the reaction of the cytochromes with CO. The second-order rate constants associated with all three kinetic process are pH-independent being 2.8 x 10(5) M-1.s-1, 3.8 x 10(4) M-1.s-1 and 4 x 10(3) M-1.s-1 under all conditions studied. The concentration-dependence of the contributions made by each of the processes to the overall absorbance change indicates that the fast and slow kinetic phases are associated with two forms of the cytochromes which are in equilibrium, whereas the intermediate phase arises from a separate cytochrome species. The quantum yield for the photodissociation of CO from the ferrous cytochromes is unusually low. Both modified cytochromes are capable of binding and reducing O2. In the presence of excess reductant, the modified cytochromes can catalytically reduce large molar excesses of O2. In the absence of excess reducing agent, the oxy complex initially formed undergoes a pH-dependent intramolecular electron-transfer process with half-life approx. 10 min. EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide]-promoted internal cross-linking is proposed to account for differences between the EDC-modified proteins and carboxymethylated cytochrome c.
Characterization of the human liver vasopressin receptor. Profound differences between human and rat vasopressin-receptor-mediated responses suggest only a minor role for vasopressin in regulating human hepatic function
Authors: Howl J , Ismail T , Strain A J , Kirk C J , Anderson D , Wheatley M
Published: August 2015

The [Arg8]vasopressin (AVP) receptor expressed by human hepatocytes was characterized, and compared with the rat hepatic V1a vasopressin receptor subtype. In addition to determining the pharmacological profile of the human receptor, the cellular responses to AVP were measured in human and rat hepatocytes by assaying glycogen phosphorylase alpha activity and DNA synthesis. Marked differences were observed between human and rat hepatocytes regarding vasopressin receptors and the intracellular consequences of stimulation by AVP. Data presented in this paper demonstrate the following, (i) Vasopressin V1a receptors are present in low abundance on human hepatocytes. (ii) Species differences exist between human and rat V1a receptors with respect to the affinity of some selective antagonists. (iii) AVP-stimulated glycogen phosphorylase a activation in human hepatocytes was approx. 5% of that observed in rat cells. (iv) In contrast with rat hepatocytes, DNA synthesis in human cells in culture was not stimulated by AVP. It is concluded that vasopressin plays only a minor role in the regulation of human hepatic function. Furthermore, conclusions drawn from observations made with AVP and its analogues on rat hepatic function cannot be directly extrapolated to the human situation.
Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer
Authors: Peters C , Rommerskirch W , Modaressi S , von Figura K
Published: August 2015

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI; MPS VI) is a lysosomal storage disease caused by deficiency of the enzyme arylsulphatase B (ASB). A human ASB cDNA has been subcloned into the retroviral vector pXT1 containing the bacterial neomycin-resistance gene and an internal thymidine kinase promoter for transcription of the inserted gene. Replication defective retrovirus was generated by transfecting the construct into the amphotropic packaging cell line PA317. Human MPS VI fibroblasts infected with recombinant retrovirus integrated the provirus into their genome and expressed retrovirus-encoded ASB mRNAs. In infected fibroblasts the level of ASB was up to 36-fold higher than in normal fibroblasts. Biosynthesis and processing of ASB in infected MPS VI fibroblasts was accomplished as in normal fibroblasts, and mature, enzymically active, ASB accumulated in dense lysosomes, indicating that the ASB deficiency in MPS VI fibroblasts was corrected by the retroviral gene transfer.
Rapid inactivation of plant aconitase by hydrogen peroxide
Authors: Verniquet F , Gaillard J , Neuburger M , Douce R
Published: August 2015

Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.
Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP
Authors: Garrett M D , Major G N , Totty N , Hall A
Published: August 2015

Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.
NADH:ubiquinone oxidoreductase from bovine mitochondria. cDNA sequence of a 19 kDa cysteine-rich subunit
Authors: Dupuis A , Skehel J M , Walker J E
Published: August 2015

The sequence of a 19 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria has been determined by a new strategy based on the polymerase chain reaction. The subunits of the enzyme were resolved in a polyacrylamide gel by two-dimensional isoelectric focusing and electrophoresis under denaturing conditions, transferred to a poly(vinylidene difluoride) membrane, and the N-terminal sequence was determined on the stained 19 kDa protein up to residue 27. This information was used to design two mixed oligonucleotide primers and a mixed oligonucleotide probe. With total bovine heart cDNA as template, overlapping cDNAs extending to sequences corresponding to both the 5′ and 3′ extremities of the mRNA coding for the 19 kDa subunit were synthesized in three polymerase chain reactions. These cDNAs were cloned and sequenced and encode a 171-amino-acid mature protein preceded by a methionine residue. The mature protein contains eight cysteine residues spaced at regular intervals through the protein, but the cysteine-rich motifs that are often associated with tetranuclear or binuclear centres in other proteins are not present. However, all eight cysteine residues are strictly conserved in a related protein from Neurospora crassa, suggesting that they have structural and/or functional significance in complex I.
Effect of Mg2+ on the binding of oxytocin to sheep myometrial cells
Authors: Pliška V , Kohlhauf Albertin H
Published: August 2015

Binding isotherms of oxytocin to sheep myometrial cells in a short-term cell culture were investigated in the presence of Mg2+ in the concentration range 0-10 mM. The occurrence of at least three binding sites had been demonstrated earlier. Mg2+ influences individual sites and individual features of the binding isotherm differently. Dissociation constants (Kd) of the high- and medium-affinity sites attain a minimum of 6.8 x 10(-10) and 4.2 x 10(-8) mol/l respectively at 2.75 mM-Mg2+. The two sites display the highest binding capacity (B) at 1 mM-Mg2+ (ratio of high affinity/medium affinity is 1:16). The B/Kd quotients reflecting relative binding (bound-to-free concentration) at the half-saturation of binding sites also have their maxima at 1 mM-Mg2+. The high-affinity site displays a strong positive co-operativity (Hill coefficient at 4 mM-Mg2+ of 2.4), which is amplified in the presence of Mg2+. Positive co-operativity of the medium-affinity site is markedly lower (Hill coefficient at 4 mM-Mg2+ of 1.5) and shows less dramatic Mg(2+)-dependence. Low-affinity sites are not co-operative at any Mg2+ concentration. It is concluded that Mg2+ may display its effect upon the oxytocin-receptor interaction predominantly by influencing positive co-operativity.
Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma. Protective effects of ascorbic acid
Authors: Frei B , Forte T M , Ames B N , Cross C E
Published: August 2015

Cigarette smoke (CS) is known to contain a large number of oxidants. In order to assess the oxidative effects of CS on biological fluids, we exposed human blood plasma to filtered (gas phase) and unfiltered (whole) CS, and determined the rate of utilization of endogenous antioxidants in relation to the appearance of lipid hydroperoxides. Lipid peroxidation was measured with a specific and sensitive assay that can detect lipid hydroperoxides at plasma levels as low as 10 nM. We found that exposure of plasma to the gas phase of CS, but not to whole CS, induces lipid peroxidation once endogenous ascorbic acid has been oxidized completely. In addition, CS exposure caused oxidation of plasma protein thiols and albumin-bound bilirubin, whereas uric acid and alpha-tocopherol were not consumed at significant rates. In plasma exposed to the gas phase of CS, low-density lipoprotein exhibited slightly increased electrophoretic mobility, but there was no apparent degradation of apolipoprotein B. Our results support the concept of an increased vitamin C utilization in smokers, and suggest that lipid peroxidation induced by oxidants present in the gas phase of CS leads to potentially atherogenic changes in lipoproteins.
Expression and site-directed mutagenesis of hepatic glucokinase
Authors: Lange A J , Xu L Z , Van Poelwijk F , Lin K , Granner D K , Pilkis S J
Published: August 2015

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.
Assembly of chick brain MAP2-tubulin microtubule protein. Characterization of the protein and the MAP2-dependent addition of tubulin dimers
Authors: Burns R G
Published: August 2015

The principle proteins present in twice-cycled chick brain microtubule protein were characterized. The protein consists of a stoichiometric mixture of MAP2 and tubulin, together with a number of minor components. Its composition remains unaltered after a third cycle of assembly in a buffer supplemented with 67 mM-NaCl, with the exception of the phosphorylation of MAP2 to a low level (congruent to 1 mol.mol-1). The inclusion of 67 mM-NaCl dissociates the MAP2-tubulin oligomers, and restricts the assembly to the MAP2-dependent addition and loss of tubulin dimers, such that the assembly kinetics approximate to a simple pseudo-first-order reaction. The assembled microtubules exhibit dynamic instability, with no evidence for end-to-end annealing.
Kinetics of GTP hydrolysis during the assembly of chick brain MAP2-tubulin microtubule protein
Authors: Burns R G
Published: August 2015

The kinetics of GTP hydrolysis during microtubule assembly have been examined using chick brain MAP2-tubulin microtubule protein in a NaCl-supplemented buffer. The elongating microtubules terminate in a ‘GTP cap’, since the kinetics of GTP hydrolysis are slower than those of subunit addition. GTP hydrolysis is (a) stoichiometric, (b) occurs as a vectorial wave as the initial rate of hydrolysis is proportional to the molar concentration of microtubule ends and not to the initial rate of subunit addition, and (c) either does not occur, or occurs only at a much lower rate, in the terminal subunits.
Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP)
Authors: Murphy G , Cockett M I , Ward R V , Docherty A J P
Published: August 2015

The abilities of the matrix metalloproteinases 95 kDa and 72 kDa gelatinases (type IV collagenases), stromelysins-1 and -2 and punctuated metalloproteinase (PUMP) to degrade insoluble elastin, type IV collagen films and proteoglycan have been compared. The gelatinases and PUMP were markedly more active in the degradation of elastin than were the stromelysins. PUMP and the stromelysins were more potent proteoglycan-degrading enzymes. All of the enzymes studied degraded soluble native type IV collagen, but the gelatinases were more effective at higher temperatures. These quantitative data allow an analysis of the potential relative roles of these metalloproteinases in the breakdown of the key components of connective tissue matrices.
Complete nucleotide and deduced amino acid sequence of human β2-glycoprotein I
Authors: Steinkasserer A , Estaller C , Weiss E H , Sim R B , Day A J
Published: August 2015

The nucleotide and complete amino acid sequence for the human beta 2-glycoprotein I (beta 2I) was derived by sequencing the cDNA clone pB2I-1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5′ and 3′ untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in beta 2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells.
Biosynthesis and secretion of triacylglycerol in rat liver after partial hepatectomy
Authors: Tijburg L B , Nyathi C B , Meijer G W , Geelen M J H
Published: August 2015

Partially hepatectomized rats were used to investigate the mechanism of fatty-liver development in the regenerating rat liver. After partial hepatectomy the amount of hepatic triacylglycerol increased by almost 4-fold compared with sham-operated rats. The activities of both cytosolic and microsomal phosphatidate phosphohydrolase were enhanced at 12 h after surgery. The activity of diacylglycerol acyltransferase was increased at a later stage of regeneration. Analysis of plasma lipoproteins showed a significant decrease of lipids associated with very-low-density lipoproteins (VLDL). Relative to control, the rate of hepatic triacylglycerol synthesis from [3H]glycerol in vivo was stimulated at 22 h after partial liver resection. However, secretion of glycerol-labelled triacylglycerol in VLDL was the same in control and hepatectomized rats. In cultures of hepatocytes from hepatectomized donor rats, the concentration of triacylglycerol and the biosynthesis of this lipid from [3H]glycerol or from [3H]oleate were enhanced. The secretion of total triacylglycerol into the medium was not affected, resulting in a net accumulation of intracellular triacylglycerol. The rate of secretion of leucine-labelled apolipoproteins B and E associated with VLDL was similar in cell cultures from hepatectomized and sham-operated rats. The results of this study show that the enhancement of the biosynthesis of triacylglycerol in hepatectomized livers is not accompanied by an increase of the secretion of VLDL.
Homodimer and heterodimer subunits of human prostate acid phosphatase
Authors: Lee H , Chu T M , Li S S L , Lee C L
Published: August 2015

Human prostatic acid phosphatase (PAP) isoenzymes, designated PAP-A and PAP-B, were isolated from human seminal plasma by sequential affinity chromatography on concanavalin A and L(+)-tartrate, a classic inhibitor of PAP. Both the major PAP-A and the minor PAP-B isoenzymes exhibited a similar molecular mass (100 and 105 kDa respectively), multiple pI values (5.05-5.35 and 5.05-5.12), and substrate and inhibitor specificity. Immunological characterization revealed that PAP-B possesses distinct antigenic determinants, in addition to the common sites shared with PAP-A. SDS/PAGE indicated that both isoenzymes are composed of two subunits of 50 kDa each. At high salt concentration, PAP-B dissociated completely into single subunits of 50 kDa, whereas PAP-A remained intact at 100 kDa. PAP-B was resolved by reverse-phase h.p.l.c. into three components, designated alpha, beta and gamma, each of 50 kDa, at a molar ratio of approx. 2:1:1. PAP-A contained a single component of molecular mass 50 kDa. The single component of PAP-A and the alpha component of PAP-B possessed identical amino acid compositions and N-terminal sequences, which were different from those of the beta and gamma components. These results indicate that human PAP contains three isoforms, alpha 2, alpha beta and alpha gamma. PAP-A, the major isoenzyme, is a homodimer consisting of two identical subunits (alpha 2), and PAP-B, the minor isoenzyme, is a mixture of two heterodimers, consisting of non-identical subunits (alpha beta and alpha gamma).
Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Purification and properties
Authors: Ruttersmith L D , Daniel R M
Published: August 2015

Exo-1,4-beta-cellobiohydrolase (EC 3.2.1.91) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic eubacterium that grows optimally at 80 degrees C. The enzyme was purified to homogeneity as determined by SDS/PAGE and has an Mr of 36,000. The enzyme is the most thermostable cellulase reported to date, with a half-life at 108 degrees C of 70 min in buffer. In a 40 min assay, maximal activity was recorded at 105 degrees C. Cellobiohydrolase from strain FjSS3-B.1 is active against amorphous cellulose and CM-cellulose but only effects limited hydrolysis of filter paper or Sigmacell 20. The only product identified by h.p.l.c. is the disaccharide cellobiose. The enzyme has a pH optimum around neutral and is stabilized by the presence of 0.8 M-NaCl.
Stoichiometric studies of β-alanine transporters in rabbit proximal tubule
Authors: Jessen H , Sheikh M I
Published: August 2015

The coupling ratio for the transport of beta-alanine and Na+, H+ and Cl- in luminal membrane vesicles isolated from proximal convoluted tubules (pars convoluta) and proximal straight tubules (pars recta) of rabbit kidney was examined. Indirect evidence indicates that 1 H+ and approx. 2 Na+, 1 Cl- (Na(+)-dependent, high-affinity) or 1 Na+ (Na(+)-dependent, low-affinity) are co-transported with beta-alanine in the pars convoluta. In pars recta, the two Na(+)-dependent transporters exhibited the same stoichiometric properties respectively as in pars convoluta.
Estimation of peroxisomal and mitochondrial fatty acid oxidation in rat hepatocytes using tritiated substrates
Authors: Rognstad R
Published: August 2015

The pathways of peroxisomal and mitochondrial fatty acid oxidation were monitored with the use of substrates which produce NAD3H. I used as marker substrates: D-[3-3H]3-hydroxybutyrate for mitochondrial NAD3H production, [2-3H]glycerol for cytosolic NAD3H production, and [2-3H]acetate to measure carbon-bound 3H which was also generated by the metabolism of the commercial 9,10-3H-labelled fatty acids. The assumption that peroxisomal NAD3H can be considered to be equivalent to cytosolic NAD3H was supported using a specific inhibitor of mitochondrial fatty acid oxidation. The approach involves determination of the specific yields, and the relative distribution on carbons 4 and 6, of 3H in glucose from the marker substrates and the labelled fatty acids. In hepatocytes from clofibrate-treated rats, the amount of palmitate or oleate oxidation which starts in the peroxisomes is comparable with that which starts in the mitochondria.
Collagenolytic cysteine proteinases of bone tissue. Cathepsin B, (pro)cathepsin L and a cathepsin L-like 70 kDa proteinase
Authors: Delaissé J M , Ledent P , Vaes G
Published: August 2015

The aim of the work was to identify and characterize the cysteine proteinases of bone tissue, as these enzymes appear necessary for bone resorption. Three cysteine-dependent proteolytic activities were separated from a homogenate of mouse calvaria by a fractionation procedure involving (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The first two are typical cathepsins B and L with respect to (1) their reactivity with anti-(cathepsin B) and anti-(cathepsin L) antibodies respectively, (2) their relative rate constants for inhibition by benzyloxycarbonyl-Phe-Phe-CHN2 and L-3-carboxy-trans-2,3-epoxypropionyl-L-leucylamido-(4-guanid ino)butane and (3) their enzymic properties, such as the higher activities of cathepsin L against collagen and gelatin as compared with cathepsin B, and the fact that benzyloxycarbonyl-Arg-Arg 4-methoxy-2-naphthylamide is hydrolysed only by cathepsin B. Cathepsin L was mainly recovered in its precursor form, as indicated by its apparent 40 kDa molecular mass and its relative stability at pH 7.2. The third enzyme is a cathepsin L-like proteinase with an apparent molecular mass of 70 kDa. It is immunoprecipitated by anti-(cathepsin L) antibodies, and appears as the 25 kDa band of mature cathepsin L in Western blots. It further resembles (pro)cathepsin L with regard to its activities against synthetic substrates and proteins such as collagen, and with regard to its response to various inhibitors. However, unlike (pro)cathepsin L, it is eluted as a 70 kDa protein on gel filtration (even in the presence of 1% Brij or 1 M-NaCl), it is stable at pH values as high as 9, and it exhibits stronger affinity for phenyl-Sepharose. It might thus result from a strong complex between mature cathepsin L and another entity that confers stability at alkaline pH and favours hydrophobic interactions. This 70 kDa activity was also detected in mouse muscle and long bones of Ca(2+)-deficient chicks but not in mouse liver, spleen or kidney.
Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems
Authors: Jimeno P , Garcia-Perez A I , Luque J , Pinilla M
Published: August 2015

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.
Non-enzymic glycation of human extracellular superoxide dismutase
Authors: Adachi T , Ohta H , Hirano K , Hayashi K , Marklund S L
Published: August 2015

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.
Evidence for masking of brown adipose tissue mitochondrial GDP-binding sites in response to fasting in rats made obese by dietary manipulation. Effects of reversion to standard diet
Authors: Puigserver P , Lladó I , Palou A , Gianotti M
Published: August 2015

A specific immunoassay of uncoupling protein (UCP) and measurement of GDP binding were used to study the chronic responses of brown adipose tissue (BAT) mitochondria from rats made obese by dietary means (cafeteria rats) and from obese rats subsequently fed a standard diet (post-cafeteria rats). We studied the response to fasting in order to assess the masking/unmasking responses in these groups. These studies have shown the following. (1) In the obese rats (cafeteria and post-cafeteria) the chronic increase in mitochondrial UCP concentration compared with controls parallels the increase in GDP binding. (2) In 24 h-fasted control rats the decrease in GDP binding is associated with a change in UCP concentration, but in fasting cafeteria and post-cafeteria obese rats the decrease in GDP binding is not associated with any change in UCP concentration. (3) Post-cafeteria obese rats showed increased GDP binding and higher UCP concentrations than the controls, but these values were less than in cafeteria obese rats. (4) Control rats at 8 months old showed greater GDP binding and had a higher UCP concentration than 11-month-old control rats. (5) The responses of GDP binding and UCP concentration to fasting in post-cafeteria obese rats were similar to those in cafeteria obese rats, suggesting that such abbreviations are related to the obese status itself rather than to the composition of the cafeteria diet. The evidence supports the hypothesis that the response of the cafeteria and post-cafeteria obese rats to fasting is associated with a masking of UCP, whereas with chronic manipulation of diet changes in UCP concentration predominate.
Aldehyde reductase is a major protein associated with 3-deoxyglucosone reductase activity in rat, pig and human livers
Authors: Kanazu T , Shinoda M , Nakayama T , Deyashiki Y , Hara A , Sawada H
Published: August 2015

3-Deoxyglucosone reductase activity in the extracts of rat, pig and human livers was potently inhibited by aldehyde reductase inhibitors. The major species of 3-deoxyglucosone reductase purified from human and pig livers were biochemically and immunochemically identical with aldehyde reductase. The two enzymes and rat liver aldehyde reductase exhibited higher catalytic efficiency for 3-deoxyglucosone than for D-glucuronate, a representative substrate of aldehyde reductase.
Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase
Authors: Igarashi P , Karniski L P
Published: August 2015

Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43 kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43 kDa protein. The 43 kDa band-3-like protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330 bp cDNA from rabbit renal cortex. Additional overlapping 272 bp and 1123 bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483 bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45,193 Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosylphosphatidylinositol. In summary, the 43 kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase.
Immunohistochemical distribution of isoenzymes of glutathione transferase in adult rat adrenal gland before and after hypophysectomy
Authors: Mankowitz L , DePierre J W , Mannervik B , Hansson H A
Published: August 2015

The distribution of glutathione transferase subunits 1, 2, 3, 4, 7 and 8 in the different cells of the female and male rat adrenal and the effects of hypophysectomy on these isoenzymes were studied using immunohistochemical methods. All these glutathione transferase subunits, with the exception of subunit 1, were present in the adrenal. Each subunit showed, however, its own characteristic distribution pattern. After hypophysectomy, increased staining for these isoenzymes was generally observed, and this effect was also cell-specific. Staining for subunit 2 increased in intensity in the zona fasciculata and reticularis after hypophysectomy, whereas a decrease was observed in the zona glomerulosa. Staining for subunit 8 was increased in the borderline between the capsule and zona glomerulosa, as well as in medullary chromaffin cells after hypophysectomy. The Mu subunits 3 and 4 increased markedly in fascicular and reticular cells after hypophysectomy and staining for subunit 3 was also increased in the medullary cells. A slight, but more general, increase was observed for subunit 7. We conclude from these experiments that the increases in glutathione transferase subunits observed in the rat adrenal after hypophysectomy are due to increased protein synthesis and/or increased protein stability and not to a selective destruction of cells lacking, or with low levels of, the isoenzymes.
The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells
Authors: Cook S J , Briscoe C P , Wakelam M J O
Published: August 2015

Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.
Restoration of arylsulphatase A activity in human-metachromatic-leucodystrophy fibroblasts via retroviral-vector-mediated gene transfer
Authors: Rommerskirch W , Fluharty A L , Peters C , von Figura K , Gieselmann V
Published: August 2015

Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulphatase A (ASA). A human ASA cDNA was subcloned into the retroviral vector pXT1. Replication-defective retrovirus was generated by transfection of the vector into the amphotropic packaging cell line PA317. Human fibroblasts from a patient suffering from metachromatic leucodystrophy was infected with the recombinant retrovirus. Infected fibroblasts expressed ten times more ASA compared with control fibroblasts from a normal individual. The ASA encoded by the integrated provirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of cerebroside sulphate.
Chitin synthase in encysting Entamoeba invadens
Authors: Das S , Gillin F D
Published: August 2015

Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [3H]GlcNAc ([3H]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.
The short-term regulation of hepatic acetyl-CoA carboxylase during starvation and re-feeding in the rat
Authors: Munday M R , Milic M R , Takhar S , Holness M J , Sugden M C
Published: August 2015

Rapid inhibition of acetyl-CoA carboxylase (ACC) activity in rat liver in response to 6 h starvation and rapid re-activation in response to 2-6 h of re-feeding chow were shown to be due to changes in the expressed activity of existing enzyme. Decreases and increases in ACC concentration occurred at later stages of the transitions, i.e. 6-48 h starvation and 8-24 h re-feeding respectively. The decrease in expressed activity of ACC was due primarily to changes in its phosphorylation state, demonstrated by a significantly decreased Vmax. and significantly increased Ka for citrate of enzyme purified by avidin-Sepharose chromatography from 6 h- or 48 h-starved rats. These effects were totally reversed within 2-4 h of chow re-feeding. Changes in the activity of purified ACC closely correlated with reciprocal changes in the activity of AMP-activated protein kinase (AMP-PK) over the fed to starved to re-fed transition. Increases in the activity ratio of cyclic-AMP-dependent protein kinase in response to starvation lagged behind the increase in AMP-PK and the decrease in ACC activity. Changes in AMP-PK and ACC activities of rat liver closely correlated with changes in plasma insulin concentration in response to time courses of starvation and re-feeding.
Agonist-induced Ca2+ influx into human platelets is secondary to the emptying of intracellular Ca2+ stores
Authors: Alonso M T , Alvarez J , Montero M , Sanchez A , García-Sancho J
Published: August 2015

We have studied the relation between the filling state of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Emptying of the intracellular Ca2+ stores either by incubation in Ca(2+)-free medium or by treatment with low concentrations of the Ca2+ ionophore ionomycin accelerated the influx of Mn2+. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restores low Mn2+ permeability. This Ca(2+)-store-regulated permeability was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Stimulation of platelets with thrombin produced Ca2+ release from the intracellular stores, which was followed, after a temperature-dependent lag (2 s at 37 degrees C; 5 s at 18 degrees C), by an acceleration of Mn2+ influx. Cytochrome P-450 inhibitors prevented the thrombin-induced Mn2+ influx, with little effect on the Ca2+ mobilization from the intracellular stores. Ki values were similar to those estimated for inhibition of the store-regulated permeability in non-stimulated platelets. Similar results were found in platelets stimulated by platelet-activating factor or by ADP. We propose that agonist-induced Ca2+ (Mn2+) influx in platelets is secondary to the emptying of the intracellular Ca2+ stores. The activation of the plasma-membrane Ca2+ (Mn2+) pathway may take place by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & García-Sancho (1991) Biochem. J. 274, 193-197] and neutrophils [Montero, Alvarez & García-Sancho (1991) Biochem. J. 277, 73-79].
Diamines interfere with the transport of l-ornithine in Ehrlich-cell plasma-membrane vesicles
Authors: Medina M A , Urdiales J L , Mates J M , Núñez de Castro I , Sánchez-Jiménez F
Published: August 2015

1. L-Ornithine transport by plasma-membrane vesicles isolated from Ehrlich cells is Na(+)-independent and shows a saturable and a diffusional component. 2. Putrescine, histamine, 5-hydroxytryptamine and 2,3-diaminopropane at 55 microM concentration significantly inhibit 0.5 mM-L-ornithine transport at least for the first 10 min of incubation. 3. There is a trans-stimulatory effect of putrescine on L-ornithine transport.
The effects of the exogenous provision of lactate and the endogenous production of lactate on protein synthesis in the heart
Authors: Fuller S J , Sugden P H
Published: August 2015

We have investigated the effects of exogenous addition of lactate and of the stimulation of endogenous production of lactate on protein synthesis in the anterogradely perfused rat heart. In the absence of exogenous lactate, hearts release lactate into the perfusate. At lactate concentrations of 0.2 mM and greater, the heart takes up lactate. The best fit for lactate uptake plotted against exogenous lactate concentration is a rectangular hyperbola with a maximal rate of 220 mumol/2 h per heart (wet wt. about 1 g). Uptake is half-maximal at about 1.3 mM-lactate. The stimulation of protein synthesis also exhibits a rectangular-hyperbolic dependence on exogenous lactate concentration, with maximal stimulation being about 38%. Half-maximal stimulation occurs at about 0.9 mM-lactate. We stimulated endogenous lactate production by perfusion with 2-cyanocinnamate (an inhibitor of mitochondrial pyruvate transport) at concentrations up to 70 microM. Cardiac outputs, intracellular pH and the concentrations of phosphocreatine and the adenine nucleotides were not altered. Atrial protein-synthesis rates were unchanged, but ventricular rates were decreased. We conclude that endogenous lactate production is unlikely to stimulate protein synthesis and that the stimulation of protein synthesis by exogenous lactate is related to its uptake.
Simultaneous redistribution of mannose 6-phosphate and transferrin receptors by insulin-like growth factors and phorbol ester
Authors: Damke H , von Figura K , Braulke T
Published: August 2015

Insulin-like growth factors I and II (IGF-I and IGF-II) and phorbol ester are known to induce in fibroblasts a rapid redistribution of mannose 6-phosphate (M6P)/IGF II-receptors to the cell surface. We compared the redistribution of the M6P/IGF-II receptor with that of the 46 kDa M6P receptor (MPR46) and of receptors for transferrin, low-density lipoprotein (LDL) and epidermal growth factor (EGF) in human fibroblasts under the influence of these effectors. None of the effectors altered the surface expression of receptors for LDL or EGF, which are predominantly located at the cell surface. IGF-I, IGF-II and phorbol ester increased the surface expression of the M6P/IGF-II receptor and of MPR46. The concentration of the transferrin receptor at the cell surface was increased only by IGF-I and IGF-II, with similar kinetics as for the M6P/IGF-II receptor, suggesting that the same mechanism causes redistribution. The increased surface expression of M6P receptors was accompanied by an increased uptake of receptor ligands. The number of transferrin receptors did not correlate with iron uptake, although neither the rate nor the extent of transferrin internalization was changed. These results indicate that the redistribution of several endocytic receptors induced by IGF-I, IGF-II and phorbol ester shows selectivity, and that the uptake of receptor ligand may become uncoupled from the surface expression of the receptors via distinct mechanisms.
Inositol polyphosphate metabolism and inositol lipids in a green alga, Chlamydomonas eugametos
Authors: Irvine R F , Letcher A J , Stephens L R , Musgrave A
Published: August 2015

Swimming suspensions of Chlamydomonas eugametos were pelleted and homogenized, and the metabolism of inositol polyphosphates by cellular homogenates or supernatants was investigated. Ins(1,4,5)P3 was dephosphorylated under physiological conditions to yield a single InsP2, Ins(1,4]2. In the presence of ATP it was phosphorylated to give Ins(1,3,4,5)P3 as the only InsP4. The Ins(1,4,5)P3 3-kinase activity was predominantly soluble, was not detectably affected by calmodulin or Ca2+, and had a Km for Ins(1,4,5)P3 of 50 microM (two orders of magnitude higher than its mammalian counterpart). Ins(1,3,4,5)P4 was dephosphorylated by the cellular supernatants to Ins(1,3,4)P3 and Ins(1,4,5)P3, and could be phosphorylated to Ins(1,3,4,5,6)P4. No Ins(1,3,4)P3 6-kinase activity could be detected, and experiments with [3H]Ins(1,4,[32P]5)P3 revealed that Ins(1,3,4,5,6)P5 is formed from Ins(1,4,5)P3 with little loss of the 5-phosphate, i.e. the predominant route of synthesis is probably by a direct 6-phosphorylation of Ins(1,3,4,5)P4. Similar experiments with an (NH4)2SO4 fraction of turkey erythrocyte cytosol gave essentially the same result, i.e. direct phosphorylation of Ins(1,3,4,5)P4 in the 6 position is the predominant route of synthesis of InsP5 from that InsP4 in vitro. No InsP6 formation was detected in any of these experiments, but labelling of intact C. eugametos with [3H]inositol revealed that the cells do synthesize InsP6. The lipids of C. eugametos cells contain PtdIns, PtdIns(4)P and PtdIns(4,5)P2 [Irvine, Letcher, Lander, Drøbak, Dawson & Musgrave (1989) Plant Physiol. 64, 888-892]. Further examination of 32P-labelled lipids revealed that about 20% of the PtdInsP was the PtdIns(3)P isomer, and about 1% or less of the PtdInsP2 was the PtdIns(3,4)P2 isomer. The overall inositide metabolism of C. eugametos resembles that of a mammalian cell more closely than it does that of a plant cell or slime mould, and this suggests firstly that the known metabolism of inositol polyphosphates arose at an early time in eukaryotic evolution, and secondly that Chlamydomonas might prove a useful organism for genetic and comparative studies of inositide enzymology.
The pattern of plant annexin gene expression
Authors: Smallwood M F , Gurr S J , McPherson M J , Roberts K , Bowles D J
Published: August 2015

Peptide sequence data derived from a plant annexin, P34 [Smallwood, Keen & Bowles (1990) Biochem. J. 270, 157-161] was used to design amplimers for PCR. A unique fragment of 95 bp, amplified from tomato (Lycopersicon esculertum) genomic DNA, was used in Northern analyses and demonstrated a differential pattern of expression in vegetative tissues of tomato, potato (Solanum tuberosum) and barley (Hordeum vulgare). The tissue-specific abundance of the annexin transcript was found to correlate closely with abundance of annexin protein as revealed by their partial purification and analysis with antisera specific for annexins isolated from tomato suspension-culture cells.