bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse hepatic glutathione transferase isoenzymes and their differential induction by anticarcinogens. Specificities of butylated hydroxyanisole and bisethylxanthogen as inducers of glutathione transferases in male and female CD-1 mice
Authors: Benson A M , Hunkeler M J , York J L
Published: August 2015

GSH transferase isoenzymes of class Mu (two forms), class Pi (one form) and class Alpha (two forms) were purified from liver cytosols of female CD-1 mice pretreated with an anticarcinogenic inducer, 2(3)-t-butyl-4-hydroxyanisole. GSH transferases GT-8.7, GT-8.8a and GT-8.8b, GT-9.0, GT-9.3, GT-10.3 and GT-10.6 contained a minimum of six types of subunits distinguishable by structural, catalytic and immunological characteristics. H.p.l.c. analysis of the subunit compositions of affinity-purified GSH transferases from liver cytosols of induced and non-induced male and female CD-1 mice showed that two anticarcinogenic compounds, 2(3)-t-butyl-4-hydroxyanisole and bisethylxanthogen, differed markedly in their specificities as inducers of GSH transferase.
Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase)
Authors: Miller A D , Packman L C , Hart G J , Alefounder P R , Abell C , Battersby A R
Published: August 2015

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5′-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.
Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3α-hydroxysteroid dehydrogenase
Authors: Ricigliano J W , Penning T M
Published: August 2015

Two non-steroidal mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver have been synthesized: 1-(4′-nitrophenyl)-2-propen-1-ol (I), and 1-(4′-nitrophenyl)-2-propyn-1-ol (II). Both of these compounds inactivate homogeneous 3 alpha-HSD in a time- and concentration-dependent manner only in the presence of NAD+. Analysis of the pseudo-first-order inactivation data gave a Kd of 1.2 mM for the allylic alcohol and a t1/2 (time required to promote a 50% loss of enzyme activity) for the enzyme of less than 10 s at saturation. Similar inactivation studies with the acetylenic alcohol gave a Kd of 1.5 mM and a t1/2 for the enzyme of 9.9 min at saturation. The allylic alcohol and acetylenic alcohol are oxidized stereoselectively by the enzyme, yielding a Km of 2.0 mM and a Vmax. of 0.58 mumol/min per mg for the allylic alcohol and a Km of 0.75 mM and a Vmax. of 0.29 mumol/min per mg for the acetylenic alcohol. Effective partition ratios (kcat./kinact.) are low for both alcohols: for the allylic alcohol, 5.3; and for the acetylenic alcohol, 141. H.p.l.c. indicates that the Michael acceptors 1-(4′-nitrophenyl)-2-propen-1-one (III) and 1-(4′-nitrophenyl-2-propyn-1-one (IV) are the products of the enzymic oxidation of the corresponding alcohols. The latter compound (IV) was trapped as its monothioether adducts before h.p.l.c. analysis. The Michael acceptors III and IV inactivate the 3 alpha-HSD in the absence of NAD+ at a rate too high to accurately measure and titrate the enzyme in a stoichiometric manner. Enzyme inactivated by I and NAD+, II and NAD+, III or IV is not re-activated by gel filtration or dialysis, implying a stable covalent bond has been formed between the enzyme and the inactivators. A screen of five other HSDs, and two aliphatic alcohol dehydrogenases, indicates that alcohol I is a selective inactivator of rat liver 3 alpha-HSD. It is concluded that 3 alpha-HSD generates non-steroidal alkylating agents (III and IV) that potently inactivate the enzyme with low effective partition coefficients. This report of non-steroidal mechanism-based inactivators of 3 alpha-HSD may provide a precedent for the development of related compounds to act as suicide substrates of other HSDs.
Potassium channels in the luminal membrane of rabbit proximal straight tubule. Evidence from vesicle studies
Authors: Jacobsen C , Røigaard-Petersen H , Sheikh M I
Published: August 2015

The characteristics of 86Rb+ fluxes through K+ channels in luminal-membrane vesicles isolated from the pars recta of rabbit proximal tubule were studied. In KCl-loaded vesicles from the pars recta, transient accumulation of 86Rb+ is observed which is modestly inhibited by BaCl2 and blocked by CdCl2. The isotope accumulation is driven by an electrical diffusion potential, as shown in experiments using either these membrane vesicles loaded with different anions, or an outwardly directed Li+ gradient with a Li+ ionophore. The vesicles containing the channel show a cation selectivity with the order K+ greater than Rb+ greater than choline+ greater than or equal to Li+ greater than Na+. The CdCl2-sensitive 86Rb+ flux is dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake and at 1 microM-Ca2+ the CdCl2-sensitive isotope flux is nearly abolished.
The development of glutathione S-transferase subunits in rat liver. Sensitive detection of the major subunit forms of rat glutathione S-transferase by using an e.l.i.s.a. method
Authors: McCusker F M G , Boyce S J , Mantle T J
Published: August 2015

The development of the subunits of glutathione S-transferase in rat liver shows that there is a co-ordinated development of the Ya, Yb1, Yb2 and Yc subunits but that the Yf and Yk subunits show unique patterns of development. The Yk subunit is the only form that is expressed at relatively high levels during the foetal period as well as during the adult period. In contrast with all other forms, the Yf subunit in the rat declines rapidly during the last few days before parturition and is virtually undetectable in hepatocytes of adult animals. The expression of the Yf subunit in foetal liver presents a ‘patchy’ appearance that is similar to that induced by the administration of lead acetate and may reflect cell-cycle-associated regulation of expression.
Kinetic analysis of the interaction of alkyl glycosides with two human β-glucosidases
Authors: Gopalan V , Daniels L B , Glew R H , Claeyssens M
Published: August 2015

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic beta-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic beta-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl beta-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity beta-glucosidase compared with the placental glucocerebrosidase; for example, with octyl beta-D-glucoside the Ki values were 10 microM and 1490 microM for the cytosolic and lysosomal beta-glucosidases respectively. Furthermore the higher affinity of the cytosolic beta-glucosidase than glucocerebrosidase for the amphipathic alkyl beta-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [delta delta G0 (K,)/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic beta-glucosidase are discussed with regard to its potential physiological substrates.
Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase
Authors: Ellis J A , Cross A R , Jones O T G
Published: August 2015

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH-FAD-cytochrome b-245-O2.
A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells
Authors: Blomhoff R , Nenseter M S , Green M H , Berg T
Published: August 2015

Fluid-phase endocytosis was studied in isolated rabbit liver parenchymal cells by using 125I-poly(vinylpyrrolidone) (PVP) as a marker. First, uptake of 125I-PVP by cells was determined. Also, cells were loaded with 125I-PVP for 20, 60 and 120 min, and release of marker was monitored for 120-220 min. Then we used the Simulation, Analysis and Modeling (SAAM) computer program and the technique of model-based compartmental analysis to develop a mechanistic model for fluid-phase endocytosis in these cells. To fit all data simultaneously, a model with three cellular compartments and one extracellular compartment was required. The three kinetically distinct cellular compartments are interpreted to represent (1) early endosomes, (2) a prelysosomal compartment equivalent to the compartment for uncoupling of receptor and ligand (CURL) and/or multivesicular bodies (MVB), and (3) lysosomes. The model predicts that approx. 80% of the internalized 125I-PVP was recycled to the medium from the early-endosome compartment. The apparent first-order rate constant for this recycling was 0.094 min-1, thus indicating that an average 125I-PVP molecule is recycled in 11 min. The model also predicts that recycling to the medium occurs from all three intracellular compartments. From the prelysosomal compartment, 40% of the 125I-PVP molecules are predicted to recycle to the medium and 60% are transferred to the lysosomal compartment. The average time for recycling from the prelysosomal compartment to the medium was estimated to be 66 min. For 125I-PVP in the lysosomal compartment, 0.3%/min was transferred back to the medium. These results, and the model developed to interpret the data, predict that there is extensive recycling of material endocytosed by fluid-phase endocytosis to the extracellular environment in rabbit liver parenchymal cells.
Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa
Authors: Scully M F , Ellis V , Shah N , Kakkar V
Published: August 2015

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.
Cloning and expression of human salivary-gland kallikrein in Escherichia coli
Authors: Angermann A , Bergmann C , Appelhans H
Published: August 2015

CDNA clones for human kallikrein have been identified in a cDNA library constructed from mRNA of human salivary gland. The entire coding sequence for preprokallikrein and for the 5′- and 3′-untranslated regions were isolated by using a mixture of oligonucleotides corresponding to amino acids 51-56 of human urinary kallikrein and one oligonucleotide corresponding to amino acids 233-238 of human pancreatic kallikrein. The DNA sequence proved that, with the exception of two amino acid exchanges, kallikrein of the human salivary gland is identical with pancreatic kallikrein. Salivary gland and renal kallikrein was expressed in Escherichia coli from plasmid pKK223-3 under the control of the tac promoter. The protein was identified by Western-blot analysis and by demonstration of its specific proteolytic activity.
Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts
Authors: Dumas R , Joyard J , Douce R
Published: August 2015

Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.
Mitogen-induced phosphorylation of human B-lymphocyte proteins. Relationship to protein kinase C activation
Authors: Guy G R , Finney M , Michell R H , Gordon J
Published: August 2015

We have investigated the rapid phosphorylation of proteins in B-lymphocytes incubated with the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), anti-Ig and combinations of TPA and the Ca2+ ionophore ionomycin. Two-dimensional electrophoretic analysis was used to identify the proteins phosphorylated in cells preincubated with [32P]Pi. TPA induced a characteristic pattern of labelled proteins, four of which (pp85, pp76, pp66 and pp63) showed a dose-dependent incorporation of 32P on serine residues. The phosphorylation of pp63 and pp66, in particular, correlated with the mitogenic dose-response curve. Addition of the Ca2+ ionophore ionomycin to B-cells also stimulated a characteristic incorporation of 32P into proteins, which included pp63 and pp66. With combined doses of TPA and ionomycin, these two proteins show an enhanced phosphorylation, which correlated well with the synergistic enhancement of proliferation shown by this combination of agents. Protein kinase C (PKC) was partially purified from B-cells and separated into alpha and beta subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca2+ of 0.1 microM and 2 microM. For both forms of PKC, in particular the beta form, higher concentrations of Ca2+ shifted the dose-response curve for TPA to the left and increased the maximum activation. Anti-Ig, which stimulated B-cells by cross-linking surface immunoglobulin and causing hydrolysis of PtdIns(4,5)P2, also caused increased phosphorylation of several proteins, which again included pp63 and pp66. These data suggest that PKC, particularly the beta form, is involved in the early part of the proliferation cascade for human B-lymphocytes. It is most probably activated in a synergistic manner by the increased Ca2+ and diacylglycerol levels which result from the earlier hydrolysis of PtdIns(4,5)P2.
Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin
Authors: Singh V , Sairam M R
Published: August 2015

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.
The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex
Authors: Rutter G A , Denton R M
Published: August 2015

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.
Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase
Authors: Deistung J , Bray R C
Published: August 2015

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.
Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595
Authors: Tomlinson S , Taylor P W , Morgan B P , Luzio J P
Published: August 2015

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.
Translocation to rat liver mitochondria of phosphatidate phosphohydrolase
Authors: Freeman M , Mangiapane E H
Published: August 2015

When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.
Purification and characterization of an anticonvulsant-induced human cytochrome P-450 catalysing cyclosporin metabolism
Authors: Shaw P M , Barnes T S , Cameron D , Engeset J , Melvin W T , Omar G , Petrie J C , Rush W R , Snyder C P , Whiting P H , Wolf C R , Burke M D
Published: August 2015

A form of human hepatic microsomal cytochrome P-450 (P450hA7) with subunit Mr 50,400 has been purified from an epileptic who had been receiving long-term treatment with anticonvulsant drugs. P450hA7 metabolized the immunosuppressant drug cyclosporin A and the dihydropyridine calcium channel antagonist nifedipine, but did not metabolize a similar dihydropyridine drug, nicardipine, nor a series of alkoxyresorufin model substrates. The hepatic microsomal concentration of P450hA7 was higher in five individuals who had been receiving long-term anticonvulsant treatment than in any of 21 individuals who had not been similarly treated. The mean P450hA7 concentration in the treated individuals was 5-fold higher than the mean concentration in the untreated individuals. It is concluded that P450hA7 is a member of the cytochrome P450III family which is induced by anticonvulsant drugs in man.
Sex-related differences in the enhancing effects of perfluoro-octanoic acid on stearoyl-CoA desaturase and its influence on the acyl composition of phospholipid in rat liver. Comparison with clofibric acid and tiadenol
Authors: Kawashima Y , Uy-Yu N , Kozuka H
Published: August 2015

The effects of the peroxisome proliferators clofibric acid (p-chlorophenoxyisobutyric acid), tiadenol [2,2′-(decamethylenedithio)diethanol] and perfluoro-octanoic acid (PFOA) on hepatic stearoyl-CoA desaturation in male and female rats were compared. Treatment of male rats with the three peroxisome proliferators increased markedly the activity of stearoyl-CoA desaturase. Administration of clofibric acid or tiadenol to female rats increased greatly the hepatic activity of stearoyl-CoA desaturase, the extent of the increases being slightly less pronounced than those of male rats. In contrast with the other two peroxisome proliferators, however, PFOA did not change the activity of stearoyl-CoA desaturase in female rats. Hormonal manipulations revealed that this sex-related difference in the effect of PFOA on stearoyl-CoA desaturase activity is strongly dependent on testosterone. The increase in stearoyl-CoA desaturase activity by peroxisome proliferators was not accompanied by any notable increases in the microsomal content of cytochrome b5 or the activity of NADH: cytochrome b5 reductase. The administration of the peroxisome proliferators greatly altered the acyl composition of hepatic phosphatidylcholine and phosphatidylethanolamine (namely the proportions of C18:1 and C20:3,n-9 fatty acids increased in both phospholipids), and the alterations were partially associated with the increase in stearoyl-CoA desaturase activity.
Long-term maintenance of high rates of very-low-density-lipoprotein secretion in hepatocyte cultures. A model for studying the direct effects of insulin and insulin deficiency in vitro
Authors: Duerden J M , Bartlett S M , Gibbons G F
Published: August 2015

High rates of hepatic cellular triacylglycerol synthesis and very-low-density-lipoprotein (VLDL) triacylglycerol output were maintained in vitro for at least 3 days when hepatocytes were cultured in a medium lacking insulin but supplemented with 1 microM-dexamethasone, 10 mM-lactate, 1 mM-pyruvate and 0.75 mM-oleate (supplemented medium). Under these conditions VLDL output remained constant, whereas cell triacyglycerol content increased 10-fold over 3 days, suggesting that the secretory process was saturated. Insulin, present during the first 24 h period, enhanced the storage of cellular triacylglycerol by inhibiting the secretion of VLDL. This stored triacyglycerol was subsequently released into the medium as VLDL if insulin was removed. With the supplemented medium the increased rate of VLDL secretion after insulin removal exceeded that observed under ‘saturating’ conditions, suggesting that pre-treatment with insulin enhanced the capacity for VLDL secretion. In contrast with the short-term (24 h) effects of insulin, longer-term exposure (greater than 48 h) to insulin enhanced the secretion of VLDL compared with insulin-untreated cultures. Under these conditions, insulin increased the net rates of triacylglycerol synthesis. The results suggest that insulin affects the secretion of VLDL triacylglycerol by two distinct and opposing mechanisms: first, by direct inhibition of secretion; second by increasing triacylglycerol synthesis, which stimulates secretion. The net effect at any time depends upon the relative importance of each of these processes.
Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C
Authors: Wheeler-Jones C P D , Patel Y , Kakkar V V , Krishnamurthi S
Published: August 2015

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.
Comparison of effects of phorbol esters and glucose on protein kinase C activation and insulin secretion in pancreatic islets
Authors: Easom R A , Hughes J H , Landt M , Wolf B A , Turk J , McDaniel M L
Published: August 2015

The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces insulin secretion from isolated pancreatic islets, and this suggests a potential role for protein kinase C in the regulation of stimulus-secretion coupling in islets. In the present study, the hypothesis that the insulinotropic effect of TPA is mediated by activation of protein kinase C in pancreatic islets has been examined. TPA induced a gradual translocation of protein kinase C from the cytosol to a membrane-associated state which correlated with the gradual onset of insulin secretion. The pharmacologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not mimic this effect. TPA also induced a rapid time-dependent decline of total protein kinase C activity in islets and the appearance of a Ca2+- and phospholipid-independent protein kinase activity. Insulin secretion induced by TPA was completely suppressed (IC50 approximately 10 nM) by staurosporine, a potent protein kinase C inhibitor. Staurosporine also inhibited islet cytosolic protein kinase C activity at similar concentrations (IC50 approximately 2 nM). In addition, staurosporine partially (approximately 60%) inhibited glucose-induced insulin secretion at concentrations (IC50 approximately 10 nM) similar to those required to inhibit TPA-induced insulin secretion, suggesting that staurosporine may act at a step common to both mechanisms, possibly the activation of protein kinase C. However, stimulatory concentrations of glucose did not induce down-regulation of translocation of protein kinase C, and the inhibition of glucose-induced insulin release by staurosporine was incomplete. Significant questions therefore remain unresolved as to the possible involvement of protein kinase C in glucose-induced insulin secretion.
The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow
Authors: Crofts J N , Barritt G J
Published: August 2015

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.
Conformational analysis of PKI(5–22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase
Authors: Reed J , De Ropp J S , Trewhella J , Glass D B , Liddle W K , Bradbury E M , Kinzel V , Walsh D A
Published: August 2015

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.
Role of arginine residues in the stimulation of the smooth-muscle plasma-membrane Ca2+ pump by negatively charged phospholipids
Authors: Missiaen L , Raeymaekers L , Droogmans G , Wuytack F , Casteels R
Published: August 2015

Negatively charged phospholipids strongly stimulate the purified plasma membrane Ca2+ pump of erythrocytes [Enyedi, Flura, Sarkadi, Gardos & Carafoli (1987) J. Biol. Chem. 262, 6425-6430] and of smooth muscle [Missiaen, Raeymaekers, Wuytack, Vrolix, De Smedt & Casteels, (1989) Biochem. J. 263, 687-694]. We have investigated the role of arginine residues in the interaction of these acidic phospholipids with the smooth-muscle Ca2+ transport ATPase. The arginine-modifying reagent phenylglyoxal inhiibited the ATPase activity in a time-dependent fashion by decreasing the Vmax. of the Ca2(+)-activation curve. Low concentrations of PtdIns, PtdIns4P, PtdIns(4,5) P2, phosphatidylserine and phosphatidic acid partially prevented this inactivation. This protective effect was however not apparent at higher concentrations of PtdIns4P, PtdIns(4,5) P2 and phosphatidic acid, which may be related to the previously observed inhibition of the enzyme at higher concentrations of these phospholipids. These findings indicate that the functionally important interaction of the acidic lipids with the protein occurs at least partially via arginine residue(s).
Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line
Authors: Dadabay C Y , Pike L J
Published: August 2015

Transglutaminases are a family of Ca2(+)-dependent enzymes that catalyse the formation of isopeptide bonds between the side chains of glutamine and lysine residues. The enzymes have been hypothesized to be involved in a wide range of cellular processes, including growth and differentiation and stabilization of the cytoskeleton. The human epidermal carcinoma-cell line, A431 cells, have relatively high amounts of a cytosolic transglutaminase activity that varies upon treatment of the cells with epidermal growth factor. We demonstrate here that this cytosolic activity has the biochemical and immunological properties of a tissue transglutaminase. We also report the purification of this enzyme to apparent homogeneity by a protocol which involves a novel affinity-elution step. Polyclonal antibodies to the transglutaminase were raised and used to identify the enzyme by Western blotting. The availability of purified transglutaminase and antitransglutaminase antibodies will permit further study of the role of this enzyme in the growth of this hormone-responsive human tumour-cell line.
Types of oligosaccharide sulphation, depending on mucus glycoprotein source, corpus or antral, in rat stomach
Authors: Goso Y , Hotta K
Published: August 2015

Radiolabelled mucus glycoprotein was obtained from tissue and a culture medium each of the corpus and antrum of rat stomach incubated with [35S]sulphate in vitro. Gel-filtration analysis of oligosaccharides liberated by alkaline-borohydride treatment from glycoproteins indicated that 35S-labelled oligosaccharides from the corpus vary considerably with respect to chain length whereas those from antral mucus glycoprotein are composed of small oligosaccharides. Examination of the reduced radiolabelled products obtained by HNO2 cleavage of the hydrazine-treated oligosaccharides indicated sulphate esters of N-acetylglucosamine to be present at three locations on a carbohydrate unit: [35S]sulphated monosaccharide (2,5-anhydromannitol 6-sulphate), [35S]sulphated disaccharide [galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate] and [35S]sulphated trisaccharide [fucosyl(alpha 1-2)-galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate]. Sulphated disaccharide and trisaccharide, possibly originating from the N-acetyl-lactosamine and fucosyl-N-acetyl-lactosamine sequences respectively, were detected in the corpus, especially as large oligosaccharides, but were present in the antrum in only very small amounts. The sulphated monosaccharide, however, most probably originating from 6-sulphated N-acetylglucosamine residues at non-reducing termini, was present in all oligosaccharide fractions in both the corpus and antrum.
The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix
Authors: DenBesten P K , Heffernan L M , Treadwell B V , Awbrey B J
Published: August 2015

The developing enamel matrix contains mostly amelogenins, which are hydrophobic proline-rich proteins. During amelogenesis, the amelogenins are presumably hydrolysed and removed from the enamel. Recently a number of metalloproteinases that may be important in amelogenesis have been identified in zymograms of the developing enamel matrix. In the present study an antibody specific for the matrix metalloproteinase collagenase activator protein (CAP) was characterized and used to identify this metalloproteinase in enamel. Immunoblotting showed that the CAP proteinase was present in the enamel matrix. Immunohistochemistry confirmed that the proteinase is localized in the enamel matrix, most specifically along the dentino-enamel junction. Purified CAP was found to hydrolyse amelogenin protein. Possible functions of the proteinase in the enamel matrix are discussed.
Inhibition of the formation of the complement membrane-attack complex by a monoclonal antibody to the complement component C8 α subunit
Authors: Abraha A , Luzio J P
Published: August 2015

The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.
Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase
Authors: Yea C M , Cross A R , Jones O T G
Published: August 2015

The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2′,5′-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.
Mucus glycoproteins from ‘normal’ human tracheobronchial secretion
Authors: Thornton D J , Davies J R , Kraayenbrink M , Richardson P S , Sheehan J K , Carlstedt I
Published: August 2015

Mucous secretions were collected from tracheas of patients undergoing minor surgery under general anaesthesia with tracheal intubation, and mucus glycoproteins were isolated by using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. ‘Whole’ mucins were excluded from a Sepharose CL-2B gel, whereas subunits obtained after reduction were included. Trypsin digestion of subunits afforded high-Mr glycopeptides (T-domains), which were further included in the gel. The latter fragments are heterogeneous and comprise two or three populations, as indicated by gel chromatography and ion-exchange h.p.l.c. Rate-zonal centrifugation showed that the ‘whole’ mucins are polydisperse in size, with a weight-average Mr of (14-16) x 10(6). The macromolecules were observed by electron microscopy, as linear and apparently flexible thread-like structures. Subunits and T-domains had weight-average contour lengths of 490 nm and 160 nm respectively. It is concluded that mucus glycoproteins are present in secretions from the healthy lower respiratory tract. The ‘whole’ tracheal mucins are assembled from subunits, which in turn can be fragmented into high-Mr glycopeptides corresponding to the oligosaccharide domains typically found in mucus glycoproteins. The size and macromolecular architecture of the tracheal mucins is thus similar to that observed for mucins from human cervical mucus, chronic bronchitic sputum and pig stomach, providing yet another example of this general design of these macromolecules, i.e. subunits assembled end-to-end into very large linear and flexible macromolecules.
1H-n.m.r. evaluation of the ferricytochrome c-cardiolipin interaction. Effect of superoxide radicals
Authors: Soussi B , Bylund-Fellenius A C , Scherstén T , Ångström J
Published: August 2015

The interaction between ferricytochrome c and cardiolipin was investigated by 1H n.m.r. at 270 MHz. From the phospholipid-induced changes of the protein spectral features it is concluded that the first 2 equivalents of cardiolipin cause a conformational change at the lower part of the solvent-exposed haem edge, involving a rearrangement of the hydrogen-bond interactions of propionate 6, thus partly accounting for the lowered redox potential of cytochrome c in the presence of cardiolipin. The increased value for the pK of the alkaline isomerization of ferricytochrome c shows that cardiolipin stabilizes the native structure of the protein, indicating that the oxidized form assumes ferrocytochrome c-like properties. Peroxidation of cardiolipin by superoxide radical ions drastically decreases the protein binding to this phospholipid. The implications of this finding, and the likelihood of the ternary cytochrome c-cardiolipin-cytochrome c oxidase complex, for the binding of cytochrome c to cytochrome c oxidase in vivo, are discussed in relation to peroxidative damage following ischaemia and reperfusion.
Localization of the essential histidine and carboxylate group in d-xylose isomerases
Authors: Vangrysperre W , Van Damme J , Vandekerckhove J , De Bruyne C K , Cornelis R , Kersters-Hilderson H
Published: August 2015

D-Xylose isomerases from different bacterial strains were chemically modified with histidine and carboxylate-specific reagents. The active-site residues were identified by amino acid sequence analysis of peptides recognized by differential peptide mapping on ligand-protected and unprotected derivatized enzyme. Both types of modified residues were found to cluster in a region with consensus sequence: Phe-His-Asp-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly, conserved in all D-xylose isomerases studied so far. These results are consistent with the recently published X-ray data of the enzyme active centre from Streptomyces rubiginosus showing hydrogen bond formation between Asp-57 and His-54 which locks the latter in one tautomeric form. A study of the pH-dependence of the kinetic parameters suggests the participation of a histidine group in the substrate-binding but not in the isomerization process. Comparison of the N-terminal amino acid sequences of several D-xylose isomerases further revealed a striking homology among the Actinomycetaceae enzymes and identifies them as a specific class of D-xylose isomerases.