bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of mevalonate 5-pyrophosphate decarboxylase in isolated cells from chick intestinal epithelium
Authors: Iglesias J , Gonzalez-Pacanowska D , Marco C , Garcia-Peregrin E
Published: August 2015

The present studies were undertaken to determine whether mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) is subject to physiological regulation in the intestinal mucosa. Activity was determined in epithelial cells isolated in a villus-to-crypt gradient from chicks fed on different diets in order to vary the sterol flux across the intestinal epithelium. When animals were fed on cholesterol, decarboxylase activity was decreased in all the cell fractions studied, although percentages of inhibition were maximum in crypts of jejunum and ileum. In contrast, decreased sterol flux as a consequence of cholestyramine feeding stimulated decarboxylase activity, especially in villi of the duodenum, where values increased 3-fold with respect to controls. On the other hand, the total cellular sterol content was significantly increased by the cholesterol diet. In duodenum and jejunum, 20-30% of the total cholesterol was in the esterified form under these conditions. However, dietary cholestyramine did not significantly affect amounts of total cellular cholesterol in any of the cell fractions. These results demonstrate that mevalonate 5-pyrophosphate decarboxylase activity changes considerably under different dietary situations and that the existence of secondary sites in the physiological regulation of sterol synthesis in the intestinal mucosa should be considered.
The role of N-linked glycosylation in the regulation of activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and proliferation of SV40-transformed 3T3 cells
Authors: Larsson O , Engström W
Published: August 2015

The effects of glycosylation inhibitors on the proliferation of SV40-transformed 3T3 cells (SV-3T3) were examined in vitro. Whereas swainsonine and castanospermine, which inhibit distal steps in the glycosylational processing, exerted marginal or no effects on cell proliferation, a proximal inhibitor, tunicamycin, efficiently decreased the rate of DNA synthesis and also inhibited the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. The inhibitory effects of tunicamycin on cell proliferation could be partially reversed by addition of dolichol, a metabolite in the pathway regulated by HMG-CoA reductase. This finding suggests that tunicamycin exerts at least one of its effects on cell proliferation by modulating the activity of HMG-CoA reductase.
Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver
Authors: Hirashima Y , Farooqui A A , Horrocks L A
Published: August 2015

We developed a continuous spectrofluorimetric assay of lysoplasmalogenase activity with the use of horse liver alcohol dehydrogenase as a coupling enzyme. In this method the disappearance of NADH is measured spectrofluorimetrically. The excitation and emission monochromators were set at 340 and 460 nm respectively. The assay is 10 times as sensitive as the previous u.v. spectrophotometric method. We could detect approx. 0.02 nmol of aldehyde produced/min per ml.
Effects of penicillins and 6-aminohexanoic acid on the kinetics of human plasmin
Authors: Higazi A A A , Mayer M
Published: August 2015

Human plasmin activity is inhibited by various penicillins in a dose-dependent manner. Ampicillin and cloxacillin produce a 50% inhibition of the globinolytic activity of plasmin at 4.5 and 5.3 mM respectively. A lower inhibitory capacity is displayed by carbenicillin. Assay of plasmin by its amidolytic activity on D-valyl-L-leucyl-L-lysine p-nitroanilide dihydrochloride showed that ampicillin at a concentration producing half-maximal inhibition converted the hyperbolic activity-substrate concentration curve into a sigmoidal curve. A similar conversion occurred in the presence of ampicillin when plasmin was assayed with an alternative chromogenic substrate, L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride 6-Aminohexanoic acid at 7.5 microM abolished the inhibition of plasmin induced by ampicillin. The present observations suggest that ampicillin interacts with plasmin at a regulatory site different from the active site of the enzyme. The effect of 6-aminohexanoic acid indicates that the lysine-binding site may be part of a regulatory site. It is possible that modulation of plasmin activity by ligands plays a role in the control of fibrinolysis.
Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon
Authors: Buck D , Guest J R
Published: August 2015

The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.
Effect of Sarkosyl and heparin on single-step addition reactions catalysed by wheat-germ RNA polymerase II–poly[d(A–T)]transcription complexes
Authors: De Mercoyrol L , Job C , Job D
Published: August 2015

Incubation of purified wheat-germ RNA polymerase II with poly[d(A-T)] template, Mn2+, U-A dinucleoside monophosphate primer and UTP substrate resulted in catalytic formation of the trinucleoside diphosphate U-A-U, in accordance with the results of previous studies. Both Sarkosyl and heparin inhibited completely and immediately (within less than 1 min) U-A-U synthesis, if either of these compounds was added to the assays during the progress of the reaction. This behaviour is in marked contrast to that reported for single-step addition reactions catalysed by Escherichia coli RNA polymerase on the same template [Sylvester & Cashel (1980) Biochemistry 19, 1069-1074]. However, treatment of the transcription complexes with Sarkosyl or heparin for periods sufficient to abolish U-A-U formation completely did not suppress completely the ability of such complexes to elongate RNA chains. Hence, the effect of Sarkosyl or heparin on the rate of U-A-U synthesis was predominantly due to change in the rate (or in the mechanism) of trinucleotide product release by the transcription complexes. Furthermore, once U-A-U synthesis has begun on the poly[d(A-T)] template, the transcription complexes became resistant to the action of a competitor DNA such as poly[d(G-C)]. The results are consistent with a model where at least a sizeable fraction of the enzyme molecules remains associated with the DNA template upon formation of a single phosphodiester bond.
Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme
Authors: Wallin R , Patrick S D , Martin L F
Published: August 2015

The antidotal effect of vitamin K in overcoming poisoning by coumarin anticoagulant drugs is mediated by a vitamin K-reducing enzyme of the endoplasmic reticulum [Wallin & Martin (1987) Biochem. J. 241, 389-396]. With microsomes obtained from human liver biopsies, we have investigated the localization and the transverse orientation of this enzyme in the endoplasmic reticulum and compared its orientation to that of the other enzymes of the vitamin K-dependent carboxylation system. All enzymes were protected by the microsomal membrane and thus appear to have a luminal orientation in the endoplasmic reticulum, consistent with their role in the vitamin K-dependent modification of secretory glycoproteins. Separation of rough and smooth microsomes showed that vitamin K-dependent carboxylase activity was 6-fold higher in rough than in smooth microsomes. Vitamin K1 reduction by the coumarin-drug-sensitive (pathway I) and -insensitive (pathway II) enzymes of the vitamin K-dependent carboxylation system was the same in rough and smooth microsomes. The data suggest a close association between the pathway I and II enzymes in the endoplasmic reticulum. These pathways may be partial reactions of multienzyme complex which carries out the various activities associated with the vitamin K-dependent carboxylation system.
Steady-state kinetic analysis of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans
Authors: Davidson V L
Published: August 2015

A steady-state kinetic analysis was performed of the reaction of methylamine and phenazine ethosulphate (PES) with the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. Experiments with methylamine and PES as varied-concentration substrates produced a series of parallel reciprocal plots, and when the concentrations of these substrates were varied in a constant ratio a linear reciprocal plot of initial velocity against PES concentration was obtained. Nearly identical values of V/Km of PES were obtained with four different n-alkylamines. These data suggest that this reaction proceeds by a ping-pong type of mechanism. The enzyme reacted with a variety of n-alkylamines but not with secondary, tertiary or aromatic amines or amino acids. The substrate specificity was dictated primarily by the Km value exhibited by the particular amine. A deuterium kinetic isotope effect was observed with deuterated methylamine as a substrate. The enzyme exhibited a pH optimum for V at pH 7.5. The absorbance spectrum of the pyrroloquinoline quinone prosthetic group of this enzyme was also effected by pH at values greater than 7.5. The enzyme was relatively insensitive to changes in ionic strength, and exhibited a linear Arrhenius plot over a range of temperatures from 10 degrees C to 50 degrees C with an energy of activation 46 kJ/mol (11 kcal/mol).
Primary structure of a novel 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax
Authors: Jentsch T J , Garcia A M , Lodish H F
Published: August 2015

Polyclonal rabbit antibodies were raised against 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.
d-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes
Authors: Zaar K , Völkl A , Fahimi H D
Published: August 2015

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.
Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver
Authors: Tozuka M , Fidge N
Published: August 2015

The liver plays a major role in the metabolism of plasma high-density lipoprotein (HDL). Several groups have postulated, but others refuted, the existence of a classical membrane receptor which recognizes HDL. In the present study, we identified and purified two liver HDL-binding proteins of 120 kDa (HB1) and 100 kDa (HB2), with apparent specificity for HDL3 devoid of E apolipoprotein. The plasma membrane was the richest source of the HDL-binding protein. Both proteins bound A-I and A-II apolipoproteins and retained HDL-binding activity after final purification. HB1 activity, but not that of HB2, was lost after treatment with beta-mercaptoethanol, but reduction did not change the apparent molecular mass of either protein. Antibodies against HB1 or HB2 did not cross-react, and preliminary structural investigations provide evidence to suggest that HB1 and HB2 are not structurally related. We thus provide evidence for at least two liver plasma-membrane proteins which bind HDL apolipoproteins, suggesting that protein-protein interaction participates to some degree in the mechanism of HDL recognition by cells.
Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity
Authors: Litosch I
Published: August 2015

In cerebral-cortical membranes, hydrolysis-resistant guanine nucleotides exert a dual regulatory effect on phospholipase C activity. Nanomolar concentrations of guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) or guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited basal phospholipase C activity, with a maximum inhibition of 30% at 10 nM. Increasing the concentration of p[NH]ppG or GTP[S] to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of phospholipase C activity. These inhibitory effects were blocked by 100 microM-guanosine 5′-[beta-thio]diphosphate. GTP was relatively ineffective in producing either stimulation or inhibition of phospholipase C activity. Similarly, ATP, adenosine 5′-[beta gamma-imido]triphosphate and GDP were also ineffective. Expression of the dual effects of guanine nucleotides was affected by the Mg2+ concentration. At 0.3 mM-Mg2+, both the inhibitory and the stimulatory components of p[NH]ppG action were evident. At 2.5 mM-Mg2+, only p[NH]ppG stimulation was observed. Pertussis-toxin treatment blocked the p[NH]ppG-mediated inhibition of phospholipase C activity. These results demonstrate that non-hydrolysable guanine nucleotides exert both a stimulatory and an inhibitory effect on membrane phospholipase C activity. These effects may be mediated through distinct GTP-binding proteins.
Bromodeoxyuridine amplifies free-radical-mediated DNA damage
Authors: Poot M , Rabinovitch P S , Hoehn H
Published: August 2015

Elevated oxygen concentrations and paraquat, a superoxide-generating compound, induce an arrest of cells in the G2 phase of the cell cycle, which can be enhanced by adding bromodeoxyuridine (BrdU) to the culture medium. Experiments with the lipophilic peroxide cumene hydroperoxide and the free-radical scavenger vitamin E demonstrate that the BrdU-dependent G2 arrest is not mediated by lipid peroxidation. The BrdU-dependency of arrest in the G2 phase can be used as a sensitive cell biological assay to detect DNA damage elicited by oxygen free radicals.
Lipocortin-independent effect of dexamethasone on phospholipase activity in a thymic epithelial cell line
Authors: Piltch A , Sun L , Fava R A , Hayashi J
Published: August 2015

In the cloned rat thymic endocrine epithelial cell line TEA3A1, treatment with dexamethasone leads to decreased levels of prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2. Dexamethasone treatment also leads to a decrease of both calcium-dependent and calcium-independent phospholipase A2 activity measured in a cell-free assay. Dexamethasone-treated cells also have increased levels of lipocortin-I, a putative modulator of phospholipase A2 activity. The property of calcium-dependent binding of lipocortin to the particulate fraction was used to prepare cytosolic and particulate subcellular fractions which contained phospholiphase A2 activity but no lipocortin-I. Dexamethasone decreased phospholipase A2 activity in both cytosolic and particulate fractions even in the absence of lipocortin, suggesting the presence of a lipocortin-independent mechanism.
Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis
Authors: Takazawa K , Passareiro H , Dumont J E , Erneux C
Published: August 2015

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.
The synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage
Authors: Melching L I , Roughley P J
Published: August 2015

Non-aggregating dermatan sulphate proteoglycans can be extracted from both fetal and adult human articular cartilage. The dermatan sulphate proteoglycans appear to be smaller in the adult, this presumably being due to shorter glycosaminoglycan chains, and these chains contain a greater proportion of their uronic acid residues as iduronate. Both the adult and fetal dermatan sulphate proteoglycans contain a greater amount of 4-sulphation than 6-sulphation of the N-acetylgalactosamine residues, in contrast with the aggregating proteoglycans, which always show more 6-sulphation on their chondroitin sulphate chains. In the fetus the major dermatan sulphate proteoglycan to be synthesized is DS-PGI, though DS-PGII is synthesized in reasonable amounts. In the adult, however, DS-PGI synthesis is barely detectable relative to DS-PGII, which is still synthesized in substantial amounts. Purification of the dermatan sulphate proteoglycans from adult cartilage is hampered by the presence of degradation products derived from the large aggregating proteoglycans, which possess similar charge, size and density properties, but which can be distinguished by their ability to interact with hyaluronic acid.
Uptake, production and metabolism of cysteinyl leukotrienes in the isolated perfused rat liver. Inhibition of leukotriene uptake by cyclosporine
Authors: Hagmann W , Parthé S , Kaiser I
Published: August 2015

1. The isolated perfused rat liver efficiently takes up cysteinyl leukotrienes (LTs) C4, D4, E4 and N-acetyl-LTE4 from circulation. More than 70% of these cysteinyl LTs are excreted from liver into bile within 1 h of onset of a 5 min infusion, while about 5% remain in the liver. About 20% of infused N-acetyl-LTE4 escapes hepatic first-pass extraction under our conditions. 2. Metabolites of LTC4 appearing in bile within 20 min of the onset of infusion include mainly LTD4 and N-acetyl-LTE4, but also omega-hydroxy-N-acetyl-LTE4 and omega-carboxy-N-acetyl-LTE4. Metabolites generated from omega-carboxy-N-acetyl-LTE4 by beta-oxidation from the omega-end represent the major biliary LTs secreted at later times. 3. Stimulation of the isolated perfused liver by the combined infusion of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the Ca2+ ionophore A23187 results in a transient increase of endogenous cysteinyl LT production, which is independent of extrahepatic cells. 4. The immunosuppressive drug cyclosporine causes a dose-dependent inhibition of hepatobiliary cysteinyl LT excretion, probably by interference with the sinusoidal uptake system for cysteinyl LTs.
Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin
Authors: Satyanarayana K , Rao M R S
Published: August 2015

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5′-phosphoryl and 3′-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.
Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes
Authors: Weaver J , Pollack S
Published: August 2015

Guinea pig reticulocytes were pulse-labelled with 59Fe bound to transferrin. Haemolysates prepared from these reticulocytes were subjected to rapid (NH1)2SO1 precipitation and then chromatography on an anion-exchange resin. ATP-bound 59Fe was the dominant species in the reticulocyte cytosol; 2,3-bisphosphoglycerate and GTP iron complexes were not detected despite the fact that these were stable with (NH1)2SO1 precipitation and readily detected with anion-exchange chromatography. AMP-bound Fe was a minor component of the cytosol following rapid (NH1)2SO4 precipitation, and the major component when iron was released from transferrin by haemolysates. We speculate that ATP-Fe may be degraded in the cell to permit utilization of its iron for haem synthesis.
Cumene hydroperoxide-dependent oxidation of NNN'N'-tetramethyl-p-phenylenediamine and 7-ethoxycoumarin by cytochrome P-450. Comparison between the haemoproteins from liver and olfactory tissue
Authors: Reed C J , De Matteis F
Published: August 2015

The interaction of cytochromes P-450 of the liver and olfactory epithelium of male hamsters with cumene hydroperoxide (CHP) has been characterized with regard to the ability of CHP to (1) support 7-ethoxycoumarin-O-de-ethylase (ECOD) activity, (2) support the oxidation of NNN'N'-tetramethyl-p-phenylenediamme (peroxidase activity) and (3) cause inactivation of cytochrome P-450. In the liver, CHP was found to support both ECOD and peroxidase activities while causing only minimal inactivation of cytochrome P-450. In contrast, in the olfactory epithelium CHP was virtually unable to support ECOD activity, peroxidase activity was 4-fold greater than in the liver, and extensive inactivation of cytochrome P-450 occurred. The reasons for these differences have been investigated with particular reference to the mode of cytochrome P-450-catalysed decomposition of CHP, that is, via homolytic or heterolytic cleavage of the hydroperoxide dioxygen bond. In both tissues, cumenol (2-phenylpropan-2-ol) was the major product of CHP decomposition detected. The radical scavenger nitrosobenzene inhibited cumenol formation by 84% in the olfactory epithelium, but by only 38% in the liver. This may indicate that dioxygen-bond scission occurs predominantly homolytically in the nasal tissue, whereas there is a balance between homolysis and heterolysis in the liver. It is suggested that the inability of CHP to support ECOD activity in the olfactory epithelium and the extensive inactivation of cytochrome P-450 that it causes both stem from decomposition of the hydroperoxide occurring homolytically rather than heterolytically in this tissue.
Transforming growth factor β1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s)
Authors: Howe P H , Leof E B
Published: August 2015

Transforming growth factor beta (TGF beta 1) is a potent regulator of DNA synthesis and cellular proliferation. In this study, we investigated whether the growth stimulatory signal of TGF beta 1 is transduced intracellularly by guanine nucleotide regulatory proteins (G-proteins). In plasma membranes from AKR-2B cells, TGF beta 1 increased binding of the radiolabelled, non-hydrolysable GTP analogue, guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]), in a dose-dependent manner. Maximal effects occurred between 0.4 and 1.0 nM-TGF beta 1. Specific binding of GTP[35S] occurred with a Kd of 3.2 x 10(-8) M which was not affected by addition of TGF beta 1. Instead, TGF beta 1 increased the number of available binding sites for GTP[35S] from 16.2 +/- 1.2 to 21.6 +/- 2.1 pmol/mg of protein. GTP[35S] binding was both nucleotide- and growth-factor-specific. Only guanine nucleotides were able to compete for binding, and of the growth factors tested (epidermal growth factor, platelet-derived growth factor, insulin, TGF beta 1 and TGF beta 2) only TGF beta 1 affected GTP[35S] binding. TGF beta 1 increased GTPase activity, as determined by the release of 32PO4(3-) from GTP gamma[32P], from 116 +/- 5.5 to 175 +/- 4.3 pmol/mg of protein following a 15 min incubation. Pretreatment of the membranes with pertussis toxin inhibited both TGF beta 1-stimulated binding of GTP[35S] as well as TGF beta 1-stimulated GTPase activity. These inhibitory actions of pertussis toxin were associated with toxin-induced ADP-ribosylation of a 41 kDa protein. Furthermore, the stimulatory effects of TGF beta 1 on c-sis mRNA expression were shown to be pertussis-toxin sensitive and could be mimicked by direct activation of G-proteins with AIF4-. These results demonstrate that in AKR-2B cells a pertussis-toxin-sensitive guanine nucleotide regulatory protein(s) is coupled to TGF beta 1 receptor binding.
The phorbol ester TPA stimulates the expression of functional β-adrenoceptors in human T lymphoblasts Molt 3
Authors: Robberecht P , De Neef P , Buscail L , Christophe J
Published: August 2015

In crude membranes from human T lymphoblasts Molt 3 cultured under standard conditions, the adenylate cyclase system was stimulated by GTP, its beta gamma-imido analogue (p[NH]ppG,) NaF and forskolin, but not by isoprenaline, prostaglandin E1 and vasoactive intestinal peptide. TPA (tumour-promoting agent phorbol ester) added at low concentration (3.2 nM) to the culture medium induced a marked increase in functional beta 2-adrenoceptors. Competition curves of [125I]cyanopindolol with the antagonist ICI 118.551 and four beta-adrenergic agonists indicated that the emergence of functional beta 2-adrenoceptors corresponded to one class of binding sites, shifting from a high-affinity state for agonists to a low-affinity state in the presence of p[NH]ppG. This expression of beta 2-adrenoceptors after a 4 h lag period depended on newly formed mRNA and protein synthesis as judged by the inhibitory effects of actinomycin D and cycloheximide. Further effects of TPA included alterations of the stimulatory G-protein Gs and/or the catalytic unit of adenylate cyclase.
The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes
Authors: Alexson S E , Osmundsen H , Berge R K
Published: August 2015

The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH.
Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning
Authors: Gierow P , Jergil B
Published: August 2015

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.
Induction of cytochrome P-450 in cultured rat hepatocytes. The heterogeneous localization of specific isoenzymes using immunocytochemistry
Authors: Bars R G , Mitchell A M , Wolf C R , Elcombe C R
Published: August 2015

Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of ‘induced’ cells. However, whereas maximal concentrations of beta-naphthoflavone resulted in virtually all cells containing induced levels of MC1b, a maximal concentration of phenobarbitone resulted in only 30% of the cells containing induced levels of PB3a/PB3b. These results are discussed in relation to the heterogeneous distribution and induction of cytochrome P-450 in the intact liver.
Desensitization of calcium mobilization and cell function in human neutrophils
Authors: Lee G H D , Kaptein J S , Scott S J , Niedzin H , Kalunta C I , Lad P M
Published: August 2015

Neutrophils pretreated with the chemoattractant formylmethionyl-leucyl-phenylalanine become unresponsive when re-exposed to the same ligand, a process termed desensitization. We have examined whether desensitization of transduction (Ca2+ mobilization) or of other cell functions (superoxide generation, enzyme release, or aggregation) occurs synchronously. Simultaneous studies of Ca2+ mobilization and aggregation by using Fura-2-loaded cells indicate that, under conditions where the aggregation response is abolished, most of the Ca2+ mobilization is unaltered. Further studies were then carried out to ascertain whether desensitization of Ca2+ mobilization could in fact be induced. Desensitization was observed, and was dependent on the number of exposures of the cells to the ligand, the concentration of the ligand, and whether the ligand was left in the medium or was removed. The pattern of resensitization was dependent on the experimental design. Under conditions where ligand was continuously present, no recovery of the Ca2+-mobilization response was seen with subsequent challenges. In contrast, on removal of ligand, this response showed partial recovery. Whereas complete desensitization of aggregation was noted, enzyme release showed a markedly lesser degree of desensitization and required more frequent exposures to the ligand before it was observed. Little or no desensitization of superoxide generation was observed regardless of the conditions utilized. Studies using phorbol myristate acetate as the ligand showed that Ca2+ mobilization and aggregation could be simultaneously inhibited. Our results suggest that discrete mechanisms of desensitization are possible in human neutrophils, and that desensitization of one particular function (aggregation) does not imply concomitant desensitization of other functions.
Inhibition of IMP-specific cytosolic 5′-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5′-deoxy-5′-isobutylthio derivatives of adenosine and inosine
Authors: Skladanowski A C , Sala G B , Newby A C
Published: August 2015

1. The partially purified IMP-specific cytosolic 5′-nucleotidases from rat liver, polymorphonuclear leucocytes and heart were inhibited by 50% by 2-6 mM-5′-deoxy-5′-isobutylthioadenosine (IBTA) or 7-10 mM-5′-deoxy-5′-isobutylthioinosine (IBTI). IBTA and IBTI inhibited the rat liver and polymorphonuclear-leucocyte enzymes non-competitively. IBTA, but not IBTI, also inhibited the ecto-5′-nucleotidase of polymorphonuclear leucocytes. IBTI was, by contrast, a more potent inhibitor than IBTA of the AMP-specific soluble 5′-nucleotidase from pigeon heart. 2. During 2-deoxyglucose-induced ATP-catabolism in rat polymorphonuclear leucocytes, adenosine formation was inhibited by approx. 80% by 3 mM-IBTA and by approx. 70% by 7 mM-IBTI. 3. The results show that 5′-modified nucleosides are inhibitors of cytosolic 5′-nucleotidases and that they penetrate to inhibit their target enzymes in intact cells. Such inhibitors may be useful to clarify the mechanisms of adenosine formation and to prevent mononucleotide hydrolysis during ATP breakdown.
Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin
Authors: Bray B , Lane D A , Freyssinet J M , Pejler G , Lindahl U
Published: August 2015

The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.
Identification and immunochemical characterization of the human erythrocyte membrane glycoproteins that carry the Xga antigen
Authors: Herron R , Smith G A
Published: August 2015

When the membrane components of Xg(a+) erythrocytes were separated by electrophoresis and immunoblotted with an anti-Xga of human origin, two diffuse bands of approximate Mr 22,000-25,000 and 26,500-29,000 were stained. These reactive components were not evident in membranes from proteinase-treated Xg(a+) erythrocytes. Neuraminidase treatment of erythrocytes before immunoblotting resulted in one diffuse Xga-reactive band, the leading edge of which had a slightly increased mobility corresponding to a decrease in Mr of approx. 1500. These results suggest that the membrane components that carry Xga are sialoglycoproteins. A genetic relationship exists between Xga and the antigen recognized by the monoclonal antibody 12E7. An immunochemical comparison of the structures that carry the Xga and 12E7 antigens demonstrated that they differ in Mr and in the way in which they are modified by neuraminidase. This is in accord with evidence that Xga and 12E7 are the products of two separate structural loci.
Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters
Authors: Hancock J T , Maly F E , Jones O T
Published: August 2015

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.
Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3
Authors: Mitchell F M , Griffiths S L , Saggerson E D , Houslay M D , Knowler J T , Milligan G
Published: August 2015

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.
Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum
Authors: Lanoix J , Roy L , Paiement J
Published: August 2015

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5′-[gamma-thio]triphosphate or guanosine 5′-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.
Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence
Authors: Knudsen J , Højrup P , Hansen H O , Hansen H F , Roepstorff P
Published: August 2015

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.
Effects of re-feeding after prolonged starvation on pyruvate dehydrogenase activities in heart, diaphragm and selected skeletal muscles of the rat
Authors: Sugden M C , Holness M J
Published: August 2015

We investigated the capacity for pyruvate oxidation in skeletal muscle, diaphragm and heart after starvation and re-feeding. Starvation for 48 h decreased pyruvate dehydrogenase (PDH) activity in soleus (by 47%), extensor digitorum longus (64%), gastrocnemius (86%), diaphragm (87%), adductor longus (90%), tibialis anterior (92%) and heart (99%). Chow re-feeding increased PDH activity in all muscles to 43-78% of the fed value within 2 h. However, complete re-activation was not observed for at least 4-6 h, during which time hepatic glycogen was replenished. We discuss the importance of muscle PDH activity in relation to sparing carbohydrate for hepatic glycogen synthesis.