bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes
Authors: Jamal Z , Saggerson E D
Published: August 2015

Diabetic rats were used as a source of brown-adipose-tissue mitochondria 2 days after a single subcutaneous injection of streptozotocin (100 mg/kg). Diabetes caused an 80% decrease in carnitine-dependent oxidation of palmitoyl-CoA and a 50-60% decrease in overt carnitine palmitoyltransferase activity. An additional lesion in brown-adipose-tissue mitochondrial oxidative capacity was also indicated, since diabetes increased by 30-50% the rate of oxidation under uncoupled conditions of several respiratory substrates (i.e. malate + palmitoylcarnitine, malate + pyruvate, succinate, NNN′N′-tetramethyl-p-phenylenediamine + ascorbate). This decrease in mitochondrial function was accompanied by an approx. 30% decrease in the abundance of cytochromes (a + a3) and total cytochromes b.
Expression of a gene encoding a novel ferredoxin in the cyanobacterium Synechococcus 6301
Authors: Cozens A L , Walker J E
Published: August 2015

A gene was discovered in the cyanobacterium Synechococcus 6301 that encodes a protein highly related to members of the [2Fe-2S] ferredoxin family found in chloroplasts and cyanobacteria. It follows a cluster of seven genes encoding subunits of the cyanobacterial ATP synthase complex. It is transcribed as a monocistronic mRNA of 408 nucleotide residues. Transcription starts at a site 55 bp upstream of the initiator methionine codon. Transcriptional initiation and termination signals with sequences similar to those found in Escherichia coli are not present. Comparison of the predicted sequence of the ferredoxin protein with those of other cyanobacterial and plant ferredoxins shows an average sequences identity of about 40%. Twelve amino acid residues are invariant, including the four cysteine residues that provide ligands for the [2Fe-2S] cluster. The deduced Synechococcus ferredoxin protein sequence has a C-terminal extension of eight amino acid residues relative to most other 2Fe-2S ferredoxins except for those from halobacteria, which also have a C-terminal extension. The sequence of the Synechococcus protein is most closely related to ferredoxins from the two complex cyanobacteria Chlorogloeopsis fritschii and Mastigocladus laminosus. The deduced protein sequence is not that of the major soluble ferredoxin that has been isolated from Synechococcus 6301 and is reported in the accompanying paper [Wada, Masui, Matsubara & Rogers (1988) Biochem. J. 252, 571-575]. So it appears to be a novel [2Fe-2S] ferredoxin and Synechococcus 6301 contains at least two [2Fe-2S] ferredoxins, which may have different roles in vivo.
Release of copper from yeast copper-thionein after S-alkylation of copper-thiolate clusters
Authors: Felix K , Weser U
Published: August 2015

Our knowledge on the release of copper from Cu-thionein in biological systems is limited. Other than oxidative cleavage or direct transfer, the possibility of an alkylation mechanism seemed attractive. Iodoacetamide and methyl methanesulphonate were successfully employed to alkylate the Cu-thiolate sulphur atom of homogeneous Cu(I)-thionein from yeast. The alkylation caused a weakening of the Cu-S bonding, which led to the release of copper. After equilibrium dialysis a proportion of the released copper was found in the dialysis buffer. When iodoacetamide was used carboxymethylcysteine was detected in the protein hydrolysate. A 10-fold molar excess over cysteine was sufficient for complete alkylation, which could be conveniently monitored by c.d. at 328 and 359 nm. The reaction proceeded under both aerobic and anaerobic conditions. E.p.r. measurements of Cu2+ revealed unequivocally the complete cleavage of the Cu-thiolate bonding in less than 5 h. It is possible that this mode of copper release might be of relevance to the molecular transport of this biochemically important transition metal.
Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping
Authors: Tavaré J M , Denton R M
Published: August 2015

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.
Recruitment of actin to the cytoskeletons of human monocyte-like cells activated by complement fragment C5a. Is protein kinase C involved?
Authors: Banks P , Barker M D , Burton D R
Published: August 2015

U-937 cells differentiated by exposure to dibutyryl cyclic AMP respond to complement fragment C5a with a marked increase in cytoskeletal F-actin, which can be detected by fluorescence-activated cell sorting (f.a.c.s.) analysis of their rhodamine phalloidin-stained cytoskeletons. The C5a-induced increase in F-actin content can be prevented by prior exposure of the cells to cytochalasin B and pertussis toxin. It is insensitive to removal of extra cellular Ca2+, to cholera toxin or to neomycin. Phorbol myristate acetate (PMA), an activator of protein kinase C, does not induce actin polymerization in the differentiated cells. Both C5a and PMA stimulate superoxide production. The action of C5a on superoxide formation is also inhibited by neomycin, a phospholipase inhibitor. These results suggest that the cytoskeletal response to C5a requires activation of a G protein, but probably does not involve phospholipase C and protein kinase C, and is not highly dependent on the availability of Ca2+. Phospholipase C and kinase C may, however, be components of the pathway leading from C5a binding to superoxide production.
Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers
Authors: Béliveau R , Demeule M , Ibnoul-Khatib H , Bergeron M , Beauregard G , Potier M
Published: August 2015

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5′-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.
5′-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities
Authors: Truong V L , Collinson A R , Lowenstein J M
Published: August 2015

Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5′-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.
Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C
Authors: Thams P , Capito K , Hedeskov C J
Published: August 2015

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.
pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea)
Authors: Cadet F , Meunier J C
Published: August 2015

The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 × 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 × 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
Methylation of the α2(I) collagen gene in chemically transformed rat liver epithelial cells
Authors: Smith B D , Marsilio E
Published: August 2015

W8 cells, 2-N-(acetoxyacetyl)aminofluorene-transformed rat liver epithelial-like cells, secrete no alpha 2(I) collagen chains. This paper reports the first demonstration of DNA methylation in the promoter-5′ region of an alpha 2(I) collagen gene which occurs in W8 cells. Since inhibition of methylation by azacytidine induces transcription of the alpha 2(I) gene, DNA methylation of W8 alpha 2(I) promoter-5′ region could contribute to altered collagen production in these cells.
Imipenem as substrate and inhibitor of β-lactamases
Authors: Monks J , Waley S G
Published: August 2015

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.
A xylosyltransferase involved in the synthesis of a protein-associated xyloglucan in suspension-cultured dwarf-French-bean (Phaseolus vulgaris) cells and its interaction with a glucosyltransferase
Authors: Campbell R E , Brett C T , Hillman J R
Published: August 2015

A particulate enzyme preparation made from suspension-cultured dwarf-French-bean (Phaseolus vulgaris) cv. Canadian Wonder cells was shown to incorporate xylose from UDP-D-[14C]xylose into polysaccharide. The reaction was dependent upon the presence of UDP-D-glucose and was stimulated, and apparently protected, by GDP-D-glucose and GDP-D-mannose, though neither was able to replace UDP-D-glucose as a glycosyl donor. The product of the reaction was identified as xyloglucan by analysis of products of enzyme breakdown and acid hydrolysis. Mr determination after proteinase K digestion indicated that the nascent xyloglucan is closely associated with protein. Preincubation of the enzyme with UDP-D-glucose stimulated incorporation from UDP-D-[14C]xylose, suggesting an ‘imprecise’ mechanism of biosynthesis, as defined by Waldron & Brett [(1985) in Biochemistry of Plant Cell Walls (Brett, C. T. & Hillman, J. R., eds.) (SEB Semin. Ser. 28), pp. 79-97, Cambridge University Press, Cambridge].
Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands
Authors: Takasaki C , Kimura S , Kokubun Y , Tamiya N
Published: August 2015

A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.
Expression of the renin gene in extra-renal tissues of the rat
Authors: Samani N J , Swales J D , Brammar W J
Published: August 2015

Expression of the renin gene in several rat organs is demonstrated by the detection of renin mRNA using a ribonuclease-protection technique. In two of these sites, the brain and the liver, renin mRNA levels are unaffected by changes in dietary salt which markedly affect renal renin mRNA levels. The findings provide the basis for an important ubiquitous local regulatory role for the renin-angiotensin system extending beyond the circulation.
NADP-linked malic enzyme. Purification from maize leaves, Mr and subunit composition
Authors: Thorniley M S , Dalziel K
Published: August 2015

1. The isolation of NADP-linked malic enzyme (EC 1.1.1.40) from maize leaves is described, together with studies of its Mr and subunit composition. 2. The enzyme was purified to apparent homogeneity by affinity chromatography on N6-aminohexyl-2′,5′-bisphosphoadenosine-agarose, gel filtration with Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-50. A purification of 140-fold with a 30% yield was obtained. 3. A detailed study of the Mr by several methods revealed the existence of different Mr forms in solution. 4. In the presence of dithiothreitol the enzyme appears to be present in triethanolamine buffer, pH 7.5, as a tetramer with a subunit Mr of 60,000 and an S20,w of 10.75 S. 5. In phosphate buffer, pH 7.0, it seems to be a dimer of Mr 120,000 with an S20,w of 7.95 S. 6. In the absence of dithiothreitol, lower-Mr forms were detected by sedimentation-equilibrium and sedimentation-velocity studies in triethanolamine buffer. 7. Results from gel filtration gave Mr values of about 340,000 in both buffers.
Use of 3-aminopropanol as an ethanolamine analogue in the study of phospholipid metabolism in Tetrahymena
Authors: Smith J D , Barrows L J
Published: August 2015

About 50% of the ethanolamine in phosphatidylethanolamine in Tetrahymena is replaced by 3-aminopropan-1-ol when the compound is added to the growth medium. The phosphatidylpropanolamine which is formed is not converted into the corresponding phosphatidylcholine analogue by methylation. There is an increase in phosphatidylcholine formed by the phosphotransferase pathway from free [3H]choline and a decrease in the phosphatidylcholine formed by the methylation pathway from [14C]methionine. The nature of the observed phospholipid alterations suggests that the regulation of phosphatidylcholine biosynthesis in Tetrahymena may be different from that found in higher eukaryotes.
Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187
Authors: Davidson A M , Halestrap A P
Published: August 2015

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.
Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline
Authors: Jones P M , Salmon D M W , Howell S L
Published: August 2015

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.
Isolation of an active form of the ATP + Mg2+-dependent protein phosphatase stimulated by the deinhibitor protein and by p-nitrophenyl phosphate
Authors: Goris J , Merlevede W
Published: August 2015

A 75 kDa + 37 kDa-subunit high-Mr form of the ATP + Mg2+-dependent phosphatase, containing no modulator or inhibitor-1 and specific towards the beta-subunit of phosphorylase kinase, was isolated from dog liver. The phosphorylase phosphatase activity is stimulated by the deinhibitor protein, by p-nitrophenyl phosphate or by trypsin treatment. The sensitivity to modulator or inhibitor-1 can be increased dramatically by partial proteolysis. The enzyme inactivation by modulator after trypsin treatment can be reversed by protein kinase FA. The effects of p-nitrophenyl phosphate and the deinhibitor protein are synergistic, and p-nitrophenyl phosphate does not influence the sensitivity of the phosphatase to the heat-stable inhibitor proteins. These observations confirm that p-nitrophenyl phosphate stimulation is a suitable criterion for the identification of the active enzyme forms of the ATP + Mg2+-dependent phosphatase.
Mechanism of cellular effect of phorbol esters on action of arginine vasopressin and angiotensin II on rat vascular smooth muscle cells in culture
Authors: Caramelo C , Tsai P , Schrier R W
Published: August 2015

The inhibitory effect of phorbol-12-myristate-13-acetate (PMA) on the Ca2+-mobilization mechanisms by arginine vasopressin (AVP) and angiotensin II (AII) was analysed in rat vascular smooth muscle cells (VSMC) in culture. PMA inhibited the Ca2+-mobilizing effect of both AVP and AII in a dose-dependent manner, including the rise in cytosolic free Ca2+ ([Ca2+]i) and Ca2+ efflux. In addition, inositol trisphosphate (IP3) production induced by AVP or AII was more than 50% reduced by PMA. The involvement of protein kinase C was implicated by the diminution of the PMA effect by the specific protein kinase C inhibitor isoquinoline-sulphonyl-O-2-methylpiperazine (H7) and the lack of effect of an inactive phorbol. Thus, these results suggest that there is a blocking site that is common or similar for both AVP and AII signal transduction, and that it is a substrate for protein kinase C. This blocking action of protein kinase C occurred at least in part by inhibition of IP3 production and, subsequently, a reduction in cytosolic Ca2+ release. In the presence of ionomycin, which produces an increase in [Ca2+]i that is not altered by PMA, 45Ca2+ efflux was increased instead of inhibited by PMA, thus suggesting that protein kinase C activation also stimulates a Ca2+-extrusion mechanism in VSMC.
Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus
Authors: Tan K H , Meyer D J , Gillies N , Ketterer B
Published: August 2015

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.
Skeletal-muscle glycogen synthesis during the starved-to-fed transition in the rat
Authors: Holness M J , Schuster-Bruce M J L , Sugden M C
Published: August 2015

The pattern of glycogen deposition in skeletal muscles of varying fibre composition was examined in rats during the starved-to-fed transition. In all the muscles studied, glycogen concentrations steadily increased during the first 8 h after chow re-feeding, and the fed value was exceeded. Rates of glycogen deposition varied, not with muscle fibre composition, but with the extent of glycogen depletion during starvation. There was no evidence for skeletal-muscle glycogen breakdown during the period of hepatic glycogenesis, making it unlikely that recycling of carbon from muscle glycogen to lactate is quantitatively important for the provision of glycogenic precursors to the liver, but moderate glycogen loss was observed from 8 to 24 h after re-feeding, when the liver is in the lipogenic mode. The factors influencing glucose disposal by skeletal muscle after re-feeding are discussed.
Chelation of transferrin iron by desferrioxamine in K562 cells. The partition of iron between ferrioxamine and ferritin
Authors: Roberts S , Bomford A
Published: August 2015

In this study we have determined whether desferrioxamine can chelate iron delivered to human leukaemic cells by the transferrin endocytic cycle. The cellular uptake of desferrioxamine was investigated by an indirect method in which the conversion of repeated pulses of [59Fe]transferrin to [59Fe]ferrioxamine was determined at two concentrations of the drug. Maximum generation of [59Fe]ferrioxamine occurred in cells exposed to either 100 microM- or 500 microM-desferrioxamine after 40-60 min. Thereafter (up to 180 min) [59Fe]ferrioxamine levels remained steady with 20% of a 59Fe pulse partitioning to chelator at 100 microM and 50% at 500 microM. Of the cellular [59Fe]ferrioxamine loss 50% occurred within 90-120 min. In cells preloaded with desferrioxamine for 1 or 4 h the partitioning of iron during a 3 h incubation with [59Fe]transferrin was dependent upon the extracellular concentration of the chelator. Above 1 mM more than 80% of entering iron was converted to ferrioxamine and less than 5% partitioned to ferritin. Below this concentration (50-500 microM) a proportion of the iron became ferritin associated (7-41%). There was a linear increase in the total amount of intracellular [59Fe]ferrioxamine in accordance with cellular iron uptake showing that transferrin continued to cycle in the presence of high concentrations of desferrioxamine. The uptake of iron and generation of ferrioxamine were markedly reduced by 5 mM-methylamine, which prevented endosome acidification and uncoupling of iron from endocytosed transferrin.
The structure of the human glutathione S-transferase π gene
Authors: Cowell I G , Dixon K H , Pemble S E , Ketterer B , Taylor J B
Published: August 2015

The human glutathione S-transferase pi gene has been isolated from a cosmid library. The gene spans approximately 3 kb, is interrupted by six introns and the region around its 5′ end has the high G + C and CpG content typical of an HTF (HpaII tiny fragment) island. In addition to a TATA box at position -28 relative to the transcription initiation site and two G + C boxes (GGGCGG), the promoter contains a sequence motif matching the phorbol ester- and ras-responsive element from the polyoma virus enhancer.
1d-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface
Authors: Rubiera C , Velasco G , Michell R H , Lazo P S , Shears S B
Published: August 2015

We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types.
Carnitine metabolism in the vitamin B-12-deficient rat
Authors: Brass E P , Stabler S P
Published: August 2015

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.
dl-α-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of dl-α-difluoromethylarginine to dl-α-difluoromethylornithine
Authors: Slocum R D , Bitonti A J , McCann P P , Feirer R P
Published: August 2015

DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [(3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.
Second-messenger generation in PC12 cells. Interactions between cyclic AMP and Ca2+ signals
Authors: Gatti G , Madeddu L , Pandiella A , Pozzan T , Meldolesi J
Published: August 2015

Changes in cyclic AMP concentrations were studied in intact PC12 pheochromocytoma cells exposed to a variety of treatments. A marked increase was triggered by N-(L-2-phenylisopropyl)adenosine, the activator of an adenosine receptor, whereas a decrease (observed even after phosphodiesterase blockade) was induced by carbachol, working through a muscarinic receptor inhibited by the selective muscarinic blocker pirenzepine, only at high concentration (Ki 450 nM). A decrease in cyclic AMP was also induced by clonidine, an alpha 2-adrenergic-receptor agonist. Both the alpha 2-adrenergic and the muscarinic inhibitions were prevented by pretreatment of the cells with pertussis toxin, and were unaffected by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. The latter drug caused a decrease in the resting cyclic AMP concentrations, and a potentiation of the increase induced by adenosine-receptor activation. Except for clonidine, all these treatments were found to be effective in both growing PC12 cells and, although to a smaller degree, in cells that had stopped growing and had acquired a neuron-like phenotype after prolonged treatment with nerve growth factor (NGF). Neither forskolin (a direct activator of adenylate cyclase) nor the activation of adenosine and alpha-adrenergic receptors was able to modify the resting cytosolic Ca2+ concentration [Ca2+]i in PC12 cells. Likewise, the K+-induced [Ca2+]i transients were unchanged after these treatments, whereas the transients induced by carbachol through the activation of a muscarinic receptor highly sensitive to pirenzepine were moderately potentiated by forskolin (and, to a lesser degree, by the adenosine analogue) and attenuated by clonidine. These results characterize in further detail the spectrum and the mutual interrelationships of the intracellular signals induced by receptor activation in PC12 cells, also as a function of the NGF-induced differentiation.
Characterization of the autophosphorylation of chicken gizzard caldesmon
Authors: Scott-Woo G C , Walsh M P
Published: August 2015

Caldesmon, an actin- and calmodulin-binding protein of smooth muscle, is a protein serine/threonine kinase capable of Ca2+/calmodulin-dependent autophosphorylation [Scott-Woo & Walsh (1988) Biochem. J. 252, 463-472]. Phosphorylation nullifies the inhibitory effect of caldesmon on the actin-activated Mg2+-ATPase activity of smooth-muscle myosin [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have characterized the kinase activity of caldesmon of chicken gizzard smooth muscle. Autophosphorylation requires Ca2+/calmodulin, but is unaffected by other second messengers (Ca2+/phospholipid/diacylglycerol, cyclic AMP or cyclic GMP), and is inhibited by the calmodulin antagonists chlorpromazine and compound 48/80, with 50% inhibition at 39.8 microM and 12.0 ng/ml respectively. Half-maximal activation of autophosphorylation occurs at 60-80 nM-Ca2+ and 0.14 microM-calmodulin, and maximal activity at 0.14-0.18 microM-Ca2+ and 1 microM-calmodulin; activation is gradually lost at higher Ca2+ and calmodulin concentrations. Autophosphorylation is pH-dependent, with maximal activity over the range pH 7-9, and requires free Mg2+ in addition to the MgATP2- substrate. The Km for ATP is 15.6 +/- 4.1 microM (mean +/- S.D., n = 4), and kinase activity is inhibited by increasing ionic strength [half-maximal inhibition at I = 0.094 +/- 0.009 M (mean +/- S.D., n = 4)]. Autophosphorylation does not affect the rate of hydrolysis of caldesmon (free or bound to calmodulin) by alpha-chymotrypsin. However, a slight difference in peptides generated from phospho- and dephospho-forms of caldesmon is observed. The binding of phospho- or dephospho-caldesmon to F-actin protects the protein against chymotryptic digestion, but does not alter the pattern of peptide generation. Characterization of proteolytic fragments of caldesmon generated by alpha-chymotrypsin and Staphylococcus aureus V8 protease enables localization of the phosphorylation sites and the kinase active site within the caldesmon molecule.
Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes
Authors: Isaac R E
Published: August 2015

Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.
Structural interpretation of the binding of 9-azidoacridine to d-amino acid oxidase
Authors: Nicholson B H , Batra S P
Published: August 2015

D-Amino acid oxidase (EC 1.4.3.3) forms an inhibited complex with the nucleotide- and aromatic-binding-site affinity reagent 9-azido[3H]acridine. Tryptic digestion of the photolysed complex yielded two radioactive peptides, 222-265 (T23) and 298-328 (T29), which core and secondary structure analysis revealed to be exposed, but which also comprised the propargylglycine-binding residues. This suggests that at least parts of the peptides containing these residues are in the active centre and that they are spatially close to the flavin-binding site.
Activation of rat liver plasma-membrane diacylglycerol kinase by vasopressin and phenylephrine
Authors: Rider M H , Baquet A
Published: August 2015

Plasma-membrane fractions were prepared from the livers of rats injected with 0.15 M-NaCl (controls) or vasopressin (1 nmol/kg body wt.). When assayed in the presence of deoxycholate, vasopressin increased the Vmax. of plasma-membrane diacylglycerol kinase 2-4-fold, and the apparent Km of the enzyme for 1,2-dioleoyl-sn-glycerol was doubled. The effect of vasopressin on the Vmax. of plasma-membrane diacylglycerol kinase was twice as great between pH 7 and 8.5 than at pH 6 or 6.5. Vasopressin doubled the activity of diacylglycerol kinase in the plasma-membrane fraction when the enzyme was assayed with phosphatidylserine rather than deoxycholate as stimulator, and when either 1-stearoyl-2-arachidonoyl-sn-glycerol or 1,2-dioleoyl-sn-glycerol was the substrate. In perfused livers vasopressin (10 nM) increased the Vmax. of plasma-membrane diacylglycerol kinase 2-fold, and phenylephrine (3 microM) gave a similar effect. Vasopressin doubled diacylglycerol kinase activity in hepatocytes that had been preincubated for 55 min, but not in cells that had only been preincubated for 15 min.
Effect of serum proteins on haem uptake and metabolism in primary cultures of liver cells
Authors: Sinclair P R , Bement W J , Gorman N , Liem H H , Wolkoff A W , Muller-Eberhard U
Published: August 2015

A role of haemopexin in transporting haem to hepatocytes for degradation has been inferred from the high affinity of haemopexin for haem. We have examined this question in primary cultures of chick-embryo and adult rat liver cells. We present here the results of four sets of experiments which indicate that haemopexin retarded haem uptake by hepatocytes in culture. (1) Haem bound to bovine serum albumin is known to repress the activity of delta-aminolaevulinate synthase in chick cultures as indicated by decreased porphyrin accumulation. When haem-albumin was added in the presence of excess purified or freshly secreted chicken haemopexin, no haem-mediated repression of porphyrin production was observed. The haem-mediated repression of porphyrin accumulation was partially prevented when human, but not chicken, albumin was added to cultures. This finding reflected the higher affinity of human albumin for haem compared with that of chicken albumin. (2) Haemopexin inhibited the ability of haem to be incorporated into cytochrome P-450 induced in the chick cultures in the presence of the iron chelator desferrioxamine. (3) The rate of association of [55Fe]haem with cultured rat hepatocytes when [55Fe]haem-haemopexin was added was one-eighth of the rate observed when [55Fe]haem-bovine serum albumin was used as the haem donor. (4) The presence of haemopexin also diminished the catabolism of haem by both rat and chick-embryo liver cell cultures. It is concluded that the uptake and subsequent metabolic effects of haem are inhibited in cultured hepatocytes by proteins such as haemopexin which have a high affinity for haem.
A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles
Authors: Tiruppathi C , Balkovetz D F , Ganapathy V , Miyamoto Y , Leibach F H
Published: August 2015

An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.