bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Ca2+-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins
Authors: Blackwood R A , Ernst J D
Published: August 2015

The annexins are a family of structurally similar, Ca2(+)-dependent, phospholipid-binding proteins. We compared six members of this family (calpactin I heavy chain, lipocortins I and III, endonexin II, p68 and protein II) to determine their phospholipid-binding specificities, as well as their ability to promote aggregation and fusion of phospholipid vesicles. The Ca2+ requirement for all of the proteins was lowest for binding to vesicles composed of phosphatidic acid, followed by phosphatidylserine and then phosphatidylinositol. Only protein II, p68, lipocortin III and endonexin II bound to vesicles composed of phosphatidylethanolamine, and none bound to phosphatidylcholine. Both calpactin I heavy chain and lipocortin I promoted aggregation of phosphatidylserine- or phosphatidylinositol-containing vesicles in the presence of less than 10 microM-Ca2+. Lipocortin I promoted fusion of liposome membranes by lowering threshold Ca2+ concentrations. Although calpactin I heavy chain did not affect threshold Ca2+ concentrations, it did increase the rate and extent of spontaneous fusion. In contrast, p68 inhibited fusion at threshold Ca2+ concentrations. Whereas previous reports have emphasized properties that the annexins have in common, these findings reveal quantitative and qualitative differences among the annexins which may relate to distinct intracellular functions.
Dephosphorylation of insulin-receptor autophosphorylation sites by particulate and soluble phosphotyrosyl-protein phosphatases
Authors: King M J , Sale G J
Published: August 2015

Insulin stimulates autophosphorylation of the insulin receptor on multiple tyrosines in three domains: tyrosines 1316 and 1322 in the C-terminal tail, 1146, 1150 and 1151 in the tyrosine-1150 domain, and possibly 953, 960 or 972 in the juxtamembrane domain. In the present work the sequence of dephosphorylation of the various autophosphorylation sites by particulate and cytosolic preparations of phosphotyrosyl-protein phosphatase from rat liver was studied with autophosphorylated human placental insulin receptor as substrate. Both phosphatase preparations elicited a broadly similar pattern of dephosphorylation. The tyrosine-1150 domain in triphosphorylated form was found to be exquisitely sensitive to dephosphorylation, and was dephosphorylated 3-10-fold faster than the di- and monophosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains. The major route for dephosphorylation of the triphosphorylated tyrosine-1150 domain involved dephosphorylation of one of the phosphotyrosyl pair, 1150/1151, followed by phosphotyrosyl 1146 to generate a species monophosphorylated mainly (greater than 80%) at tyrosine 1150 or 1151. Insulin receptors monophosphorylated in the tyrosine-1150 domain disappeared slowly, and overall the other domains were completely dephosphorylated faster than the tyrosine-1150 domain. Dephosphorylation of the diphosphorylated C-terminal domain yielded insulin receptor in which the domain was singly phosphorylated at tyrosine 1322. Triphosphorylation of the insulin receptor in the tyrosine-1150 domain appears important in activating the receptor tyrosine kinase to phosphorylate other proteins. The extreme sensitivity of the triphosphorylated form of the tyrosine-1150 domain to dephosphorylation may thus be important in terminating or regulating insulin-receptor tyrosine kinase action and insulin signalling.
The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [l-(+)-lactate dehydrogenase]
Authors: Genet R , Lederer F
Published: August 2015

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed.
Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes
Authors: Das A M , Harris D A
Published: August 2015

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.
Study of the mode of action and site-specificity of the endo-(1→4)-β-d-glucanases of the fungus Penicillium pinophilum with normal, 1-3H-labelled, reduced and chromogenic cello-oligosaccharides
Authors: Bhat K M , Hay A J , Claeyssens M , Wood T M
Published: August 2015

The modes of action of the five major endo-(1→4)-beta-D-glucanases (I, II, III, IV and V) purified from Penicillium pinophilum cellulase were compared by h.p.l.c. analysis, with normal, 1-3H-labelled and reduced cello-oligosaccharides and 4-methylumbelliferyl glycosides as substrates. Significant differences were observed in the preferred site of cleavage even when substrates with the same number of glycosidic bonds were compared. Thus, although endoglucanase I was unable to attack normal cello-oligosaccharides shorter than degree of polymerization 6, it hydrolysed reduced cellopentaose to yield cellotriose and cellobi-itol, and it produced cellotriose and 4-methylumbelliferyl glucoside from 4-methylumbelliferyl cellotetraoside. Endoglucanase IV hydrolysed [1-3H]cellotriose but did not attack either cellotri-itol or 4-methylumbelliferyl cellobioside. These and other anomalous results indicated clearly that modification of the reducing glycosyl residue on the cello-oligosaccharides induces in an apparent change in the mode of action of the endoglucanases. It is suggested that, although cello-oligosaccharide derivatives are useful for differentiating and classifying endoglucanases, conclusions on the mechanism of cellulase action resulting from these measurements should be treated cautiously. Unequivocal information on the mode of endoglucanase action on cello-oligosaccharides was obtained with radiolabelled cello-oligosaccharides of degree of polymerization 3 to 5. Indications that transglycosylation was a property of the endoglucanases were particularly evident with the 4-methylumbelliferyl cello-oligosaccharides. Turnover numbers for hydrolysis of the umbelliferyl cello-oligosaccharides were calculated, and these, along with the other analytical data collected on the products of hydrolysis of the normal, reduced and radiolabelled cello-oligosaccharides, suggested that the various endoglucanases had different roles to play in the overall hydrolysis of cellulose to sugars small enough to be transported through the cell membrane.
P2-purinoceptor regulation of surfactant phosphatidylcholine secretion. Relative roles of calcium and protein kinase C
Authors: Rice W R , Dorn C C , Singleton F M
Published: August 2015

Ca2+ and protein kinase C have both been proposed as intracellular signals for subsequent phosphatidylcholine secretion by alveolar Type II cells. We have determined the relative roles of Ca2+ and protein kinase C in regulating surfactant phosphatidylcholine secretion by utilizing exogenous ATP and the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) as secretagogues, along with MAPTAM to chelate intracellular Ca2+ and sphingosine to inhibit endogenous protein kinase C. Exposure of Type II cells to the P2-purinoceptor agonist, ATP, results in a dose-dependent increase in surfactant phosphatidylcholine secretion from isolated alveolar Type II cells with an EC50 (concn. producing 50% of maximal response) of 2 microM. Administration of exogenous ATP to Type II cells also results in a dose-dependent increase in inositol trisphosphate production, Ca2+ mobilization and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding as a measure of protein kinase C translocation. The EC50 in each case is 1-5 microM, indicating association of these events with surfactant phosphatidylcholine secretion. Loading Type II cells with non-hydrolysable GTP analogue (GTP[S]) inhibited ATP-induced Ca2+ mobilization, supporting the hypothesis that Type II cell P2-purinoceptors are coupled to phospholipase C via a GTP-binding protein. The ATP-induced elevation of cytosolic Ca2+ was also inhibited by MAPTAM (a cell-permeant EGTA analogue) by 90%, but MAPTAM was without effect on surfactant phosphatidylcholine secretion induced by ATP. Sphingosine inhibited both ATP- and TPA-induced surfactant phosphatidylcholine secretion as well as [3H]PDBu binding with a similar IC50 (concn. producing 50% of maximal inhibition) (10 microM). Sphingosine did not affect surfactant phosphatidylcholine secretion induced by terbutaline and did not have a significant effect on Ca2+ mobilization induced by exogenous ATP. These results are consistent with a prominent role for protein kinase C in regulation of P2-purinoceptor-induced surfactant phosphatidylcholine secretion, and indicate that Ca2+ mobilization is not a necessary step for ATP-induced surfactant phosphatidylcholine secretion.
Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids
Authors: Preston G G , Wall J D , Emerich D W
Published: August 2015

Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.
Photoaffinity labelling of atrial natriuretic factor (ANF)-R1 receptor by underivatized 125I-ANF. Involvement of lipid peroxidation
Authors: Larose L , McNicoll N , Rondeau J J , Escher E , De Lean A
Published: August 2015

In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.
δ-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2
Authors: McKenzie F R , Milligan G
Published: August 2015

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.
The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues
Authors: McCool D J , Marcon M A , Forstner J F , Forstner G G
Published: August 2015

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.
1H-n.m.r. investigation of naturally occurring and chemically oversulphated dermatan sulphates. Identification of minor monosaccharide residues
Authors: Bossennec V , Petitou M , Perly B
Published: August 2015

The 1H-n.m.r. spectra of various dermatan sulphate preparations present, besides the major signals of the basic disaccharide unit, several other minor signals. We have assigned most of them by n.m.r., using two-dimensional proton-proton double-quantum-correlation and nuclear-Overhauser-effect spectroscopy experiments. This allowed us to identify 2-O-sulphated L-iduronic acid and D-glucuronic acid residues as well as 6-sulphated N-acetylgalactosamine (presumably 4-O-sulphated as well). 2-O-Sulphated iduronic acid was present to similar extents (6-10% of total uronic acids) in pig skin dermatan sulphate and pig intestine dermatan sulphate, whereas glucuronic acid represented 17% of the uronic acid of pig skin dermatan sulphate and was virtually absent (1%) from the other preparation. 6-O-Sulphated N-acetylgalactosamine was present in minor amounts in pig intestine dermatan sulphate only. The influence of sulphation of iduronic acid units on their conformation was assessed by using chemically oversulphated pig intestine dermatan sulphate. Introduction of sulphate groups in this unit in dermatan sulphate tends to shift the conformational equilibrium towards the 1C4 conformer.
Protein degradation in cultured fetal hepatocytes. Absence of an inhibitory effect of insulin
Authors: Peavy D E , DeSante D C
Published: August 2015

The role of insulin to regulate protein turnover in fetal liver was investigated using primary cultures of fetal-rat hepatocytes. The basal rate of protein degradation (in the presence of insulin and amino acids) was the same in cultured fetal and adult hepatocytes (2.48 +/- 0.16 versus 2.46 +/- 0.06% of total protein degraded/h respectively). Incubation of cells in an unsupplemented media (without insulin or amino acids) resulted in a deprivation-induced increase in degradation in cells from both groups (P less than 0.05). Rates of proteolysis could be returned to their respective basal values by the addition of amino acids at 5 times their normal plasma concentrations. In adult cells, addition of insulin alone significantly inhibited protein degradation (P less than 0.05), whereas, in contrast, insulin was without effect on protein degradation in fetal hepatocytes. Both fetal and adults cells responded to dibutyryl cyclic AMP with an increase in protein degradation above that seen in the no-additions group. Results of experiments in which the effect of inhibitors of protein degradation (chloroquine, NH4Cl, amino acids and dinitrophenol) were tested suggested that lysosomes were responsible for 20-30% of total protein degradation in fetal hepatocytes. Impaired insulin processing in fetal hepatocytes was examined as a possible cause of the insulin-resistance in these cells. As determined by h.p.l.c. analysis, the same pattern of initial degradation products of insulin was found in fetal hepatocytes as had previously been found in adult hepatocytes. Incubation of cells with various doses of chloroquine resulted in an increase in cell-associated 125I-insulin and a decrease in insulin degradation in both fetal and adult cells. At the highest dose of chloroquine tested (500 microM), a slightly greater increase in insulin binding and a decrease in insulin degradation were observed in fetal cells as compared with adult cells. Rates of insulin internalization were also compared between fetal and adult cells. A 30% slower rate of insulin internalization was observed in fetal cells, as compared with adult cells. It was concluded that the absence of an effect of insulin on protein degradation in fetal hepatocytes is not the result of a major difference in insulin internalization and processing between fetal and adult hepatocytes.
Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues
Authors: Murray K J , England P J , Lynham J A , Mills D , Schmitz-Peiffer C , Reeves M L
Published: August 2015

1. The cyclic AMP-dependent protein kinase activity-ratio assay was investigated by comparing histone and a synthetic peptide, malantide [Malencik & Anderson (1983) Anal. Biochem. 132, 32-40], as substrates. 2. In several tissues the activity ratio was higher when assayed with histone as the substrate; this result was obtained in control tissues and also in those incubated with agents known to increase cyclic AMP. The effect of these agents to increase the activity ratio was more clearly demonstrated with malantide. 3. The higher activity ratios observed with histone are due to: (a) measurement of phosphorylation not catalysed by cyclic AMP-dependent protein kinase; (b) activation of cyclic AMP-dependent protein kinase by histone during the assay. 4. When tissues were homogenized in buffers without NACl, lower activity ratios were found, owing to the catalytic subunit being artifactually removed from the supernatant. 5. We conclude that the measured activity ratio more faithfully reflects that in the tissue when NaCl is included in the homogenization buffer and malantide is used in the assay. This was confirmed in experiments where cyclic AMP-dependent protein kinase was added to the tissue before homogenization, and no dissociation of the exogenous enzyme was observed.
Quantification of the α and β subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice
Authors: Bégin-Heick N
Published: August 2015

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.
Inhibition of Ca2+-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase
Authors: Halestrap A P , Davidson A M
Published: August 2015

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the ‘c’ conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.
Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats
Authors: Kotal P , Fevery J
Published: August 2015

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.
The oxidation of leucine in tumour-bearing rats
Authors: Argilés J M , López-Soriano F J
Published: August 2015

Rats bearing the Walker-256 carcinosarcoma showed significant changes in leucine metabolism compared with their non-tumour-bearing controls. After a single intravenous tracer dose of L-[1-14C]leucine in vivo, 14CO2 release by tumour-bearing rats was significantly elevated throughout the time course of administration. In addition, both the clearance and turnover rates of the tracer were significantly enhanced in these animals. Incubation of soleus muscles from control and tumour-bearing rats in the presence of L-[1-14C]leucine revealed an enhanced oxidation of the amino acid in the tumour-bearing group. Tumour tissue slices were also able to oxidize the tracer at a similar rate to that found in soleus muscles from control animals.
Preferential over-expression of the class alpha rat Ya2 glutathione S-transferase subunit in livers bearing aflatoxin-induced pre-neoplastic nodules. Comparison of the primary structures of Ya1 and Ya2 with cloned class alpha glutathione S-transferase cDNA sequences
Authors: Hayes J D , Kerr L A , Harrison D J , Cronshaw A D , Ross A G , Neal G E
Published: August 2015

Normal rat liver expresses Ya (Mr 25,500), Yc (Mr 27,500) and Yk (Mr 25,000) Class Alpha glutathione S-transferase (GST) subunits. The Ya-type subunit can be resolved into two separate polypeptides, designated Ya1 and Ya2, by reverse-phase h.p.l.c. In rat livers that possess aflatoxin B1-induced pre-neoplastic nodules, a marked increase is observed in the expression of Ya1, Ya2, Yc and Yk; of these subunits, Ya2 exhibited the greatest increase in concentration. The Ya1 and Ya2 subunits isolated from nodule-bearing livers were cleaved with CNBr, and the purified peptides were subjected to automated amino-acid-sequence analysis. Differences in the primary structures of the two Ya GST subunits were found at positions 31, 34, 107 and 117. These data demonstrate that Ya1 and Ya2 are distinct polypeptides and are the products of separate genes. The amino acid sequences obtained from Ya1 and Ya2 were compared with the cloned cDNAs pGTB 38 [Pickett, Telakowski-Hopkins, Ding, Argenbright & Lu (1984) J. Biol. Chem. 259, 4112-4115] and pGTR 261 [Lai, Li, Weiss, Reddy & Tu (1984) J. Biol. Chem. 259, 5182-5188], which encode rat Ya-type subunits. From these comparisons it appears probable that Ya1 represents the GST subunit encoded by pGTR 261, whereas Ya2 represents the subunit encoded by pGTB 38. It is likely that the over-expression of Ya1 and Ya2 in nodule-bearing livers is of major significance in the acquired resistance of nodules to aflatoxin B1, since previous work [Coles, Meyer, Ketterer, Stanton & Garner (1985) Carcinogenesis 6, 693-697] has shown that the Ya-type GST subunit has high activity towards aflatoxin B1 8,9-epoxide.
The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin
Authors: Paudel H K , Carlson G M
Published: August 2015

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, β- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, β- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only β-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.
Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters
Authors: Denis-Pouxviel C , Gauthier T , Daviaud D , Murat J C
Published: August 2015

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.
Regulation by a protein-free carbohydrate-rich diet of rat pancreatic mRNAs encoding trypsin and elastase isoenzymes
Authors: Dakka N , Puigserver A , Wicker C
Published: August 2015

The levels of mRNAs coding for trypsin and elastase isoenzymic forms were determined in the pancreatic tissue of rats fed a high-carbohydrate protein-free diet for a 0-5-day period. No change in the amounts of mRNAs coding for the two isoelastases was observed, although previous results showed that the biosynthesis of anionic elastase was markedly increased, whereas the biosynthesis of cationic elastase decreased, suggesting the existence of a translational-control mechanism in response to nutritional substrates. In contrast, the levels of mRNAs specific for the three isotrypsins were significantly enhanced, possibly as a result of transcriptional regulation and/or a change in messenger stability. In combination with earlier observations of an overall decrease in cationic trypsin biosynthesis during the same nutritional manipulation, these results suggest that formation of this enzyme is also subject to translational control.
Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes
Authors: Kersten P J , Kalyanaraman B , Hammel K E , Reinhammar B , Kirk T K
Published: August 2015

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.
An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation
Authors: Yu K T , Benjamin W B , Ramakrishna S , Khalaf N , Czech M P
Published: August 2015

Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf & Czech (1987) J. Biol. Chem. 262, 16677-16685]. The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 microM. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cells.
Structural analysis of sulphated glycoprotein 2 from amino acid sequence. Relationship to clusterin and serum protein 40,40
Authors: Tsuruta J K , Wong K , Fritz I B , Griswold M D
Published: August 2015

Sulphated glycoprotein 2 (SGP-2) is the major secreted protein product of rat Sertoli cells; likewise, clusterin is a major constituent of ram rete testis fluid. Isolation and sequencing of the intact subunits and peptides derived from clusterin show that it is the ram homologue of rat SGP-2. Human serum protein 40,40 (SP-40,40), a component of the SC5b-9 complex of complement, has recently been reported to be the human homologue of rat SGP-2. Analysis of the amino acid sequences of rat SGP-2 and human SP-40,40 show that both of these proteins have a significant relationship to the heavy chain of myosin. The regions of highest sequence similarity correspond to the major amphipathic domains in SGP-2/SP-40,40 and the long alpha-helical-tail domain of myosin, which forms a rod-like structure. SGP-2 has anomalous sedimentation behaviour which indicates that it probably exists in an extended conformation. A putative dinucleotide-binding structure has been identified in the longest stretch of identity between SGP-2 and SP-40,40. Elucidation of these features of SGP-2 and SP-40,40 may help to direct future studies into the role of these proteins in the reproductive and complement systems.
The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat
Authors: Shaw S , Jayatilleke E
Published: August 2015

Hepatic lipid peroxidation has been implicated in the pathogenesis of alcohol-induced liver injury, but the mechanism(s) by which ethanol metabolism or resultant free radicals initiate lipid peroxidation is not fully defined. The role of the molybdenum-containing enzymes aldehyde oxidase and xanthine oxidase in the generation of such free radicals was investigated by measuring alkane production (lipoperoxidation products) in isolated rat hepatocytes during ethanol metabolism. Inhibition of aldehyde oxidase and xanthine oxidase (by feeding tungstate at 100 mg/day per kg) decreased alkane production (80-95%), whereas allopurinol (20 mg/kg by mouth), a marked inhibitor of xanthine oxidase, inhibited alkane production by only 35-50%. Addition of acetaldehyde (0-100 microM) (in the presence of 50 microM-4-methylpyrazole) increased alkane production in a dose-dependent manner (Km of aldehyde oxidase for acetaldehyde 1 mM); menadione, an inhibitor of aldehyde oxidase, virtually inhibited alkane production. Desferrioxamine (5-10 microM) completely abolished alkane production induced by both ethanol and acetaldehyde, indicating the importance of catalytic iron. Thus free radicals generated during the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism in the initiation of alcohol-induced liver injury.
Glycation of the human erythrocyte glucose transporter in vitro and its functional consequences
Authors: Bilan P J , Klip A
Published: August 2015

Glycation of human erythrocyte membrane proteins was induced by incubation in vitro with high concentrations (80 mM or 200 mM) of D-glucose for 3 or 6 days. The extent of glycation was quantified from the covalent incorporation of 3H by reduction of the glucose glycation products with NaB3H4. For membranes incubated for 3 days with 80 mM-D-glucose, glycation in vitro of Band 4.5 (containing the glucose transporter) was equivalent to 0.11 mol of glucose/mol of glucose transporter, compared with 3H labelling in 3-day-incubated control membranes of 0.055 mol of glucose/mol of glucose transporter. In membranes incubated for 6 days with 200 mM-D-glucose, glycation increased to 0.21 mol of glucose/mol of glucose transporter, whereas the controls without glucose had 0.11 mol of glucose/mol of glucose transporter. Glycation in vitro was accompanied by a fall in the Bmax of binding of [3H]cytochalasin B (a competitive inhibitor of glucose transport), without any change in the binding affinity. The data suggest that glycated glucose transporters have decreased ability to bind cytochalasin B. It is proposed that glycation can alter glucose transporter activity.
Distinct ryanodine- and inositol 1,4,5-trisphosphate-binding sites in hepatic microsomes
Authors: Shoshan-Barmatz V , Zhang G H , Garretson L , Kraus-Friedmann N
Published: August 2015

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca2+ sequestration. Ca2(+)-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca2+ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca2+ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-trisphosphate (IP3) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP3 by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP3 binding, whereas heparin inhibited IP3 binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to Mr values of (x 10(-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca2(+)-release processes.
Studies on the incorporation of a covalently bound disubstituted phosphate residue into Azotobacter vinelandii flavodoxin in vivo
Authors: Boylan M H , Edmondson D E
Published: August 2015

Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson & James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory.