bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of carbohydrate moieties in peanut (Arachis hypogaea) peroxidases
Authors: Hu C F , van Huystee R B
Published: August 2015

The activities of a cationic (C.PRX) and an anionic peroxidase isolated from peanut (Arachis hypogaea)-cell suspension culture were drastically reduced when they were deglycosylated with glycopeptidase F or oxidized by 10 mM-periodate. In contrast with the controls, the deglycosylated or the oxidized peroxidases were much more susceptible to proteolytic degradation. In radiolabelling experiments with [35S]methionine, the non-glycosylated C.PRX was synthesized in the tunicamycin-treated cultures and secreted into the medium. Examination of the C.PRX polypeptides by SDS/polyacrylamide-gel electrophoresis followed by fluorography showed that the non-glycosylated form had an Mr of approx. 31,000, which is about 78% of that of the glycosylated form. Our results suggest that carbohydrates may not be essential for peroxidase secretion, but that stabilization of the peroxidase molecules and acquisition by these isoenzymes of a catalytically active conformation is linked directly or indirectly to glycosylation.
Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver
Authors: Shaw L , Schauer R
Published: August 2015

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.
Fish skeletal muscle contains a novel serine proteinase with an unusual subunit composition
Authors: Folco E J E , Busconi L , Martone C B , Sánchez J J
Published: August 2015

Proteinase I, an enzyme previously shown to be able to degrade contractile and cytoskeletal elements of white-croaker (Micropogon opercularis) myofibrils, was purified to apparent homogeneity by chromatography on DEAE-Sephacel, octyl-Sepharose CL 4B and arginine-Sepharose 4B. Its Mr was determined to be 269,000 by Sephacryl S-300 gel filtration. Under denaturing conditions, the enzyme dissociated into two subunits with Mr 20,000 and 15,500, in a molar ratio of 1.8:1. Proteinase I showed a pH optimum of 8.5. The enzyme was strongly inhibited by several serine proteinase inhibitors, whereas inhibitors of the other types of proteinases did not affect, or only scarcely affected, its activity. Several N-terminal-blocked 4-methyl-7-coumarylamide substrates having either arginine or lysine residues adjacent to the fluorogenic group were efficiently hydrolysed by the enzyme. These results indicate that proteinase I is a trypsin-like serine proteinase. The enzyme appears to be distinct from other proteinases previously described in skeletal muscle, and might be involved in the catabolism of myofibrillar proteins.
Inhibition of biliary cholesterol and phospholipid secretion during cyclobutyrol-induced hydrocholeresis
Authors: Monte M J , Cava F , Esteller A , Jimenez R
Published: August 2015

The effects of sodium cyclobutyrate, a synthetic hydrocholeretic drug, on biliary lipid secretion and on the biliary outputs of several plasma-membrane enzymes were investigated in anaesthetized rats. Administration of a single oral dose of cyclobutyrol (0.72 mmol/kg body wt.) reduced biliary concentration and output of cholesterol and phospholipid. However, bile acid secretion was not significantly modified. This uncoupling effect of lipid secretion remained even when the choleretic response to the drug had ceased. It additionally led to a statistically significant decrease in the cholesterol/bile acid and phospholipid/bile acid molar ratios and in the lithogenic index of the bile. The biliary outputs of the plasma-membrane enzymes alkaline phosphatase and gamma-glutamyltransferase were markedly reduced by the drug. When cyclobutyrol was administered to rats which had been previously fed with a high-cholesterol diet, the effects of cyclobutyrol persisted, but were less marked. Our results demonstrate that the bile acid-independent choleresis induced by cyclobutyrol (related to its pharmacokinetic effect) is accompanied by a pharmacodynamic action that selectively reduces the secretion of biliary lipids. This is due to an uncoupling of the secretion of cholesterol and phospholipids from that of bile acids. Possible explanations for the biliary response to cyclobutyrol are discussed.
Indirect immunofluorescence localization of β-adrenergic receptors and G-proteins in human A431 cells
Authors: Wang H Y , Berrios M , Malbon C C
Published: August 2015

Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters.
The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils
Authors: Cockcroft S , Stutchfield J
Published: August 2015

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5′-[gamma-thio]triphosphate (ATP[S]), adenosine 5′-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.
Effects of oxyradicals on oxymyoglobin. Deoxygenation, haem removal and iron release
Authors: Prasad M R , Engelman R M , Jones R M , Das D K
Published: August 2015

We have examined the effects of O2-derived free radicals on oxymyoglobin, the myocardial intracellular protein involved in the storage and transport of O2. The oxyradicals generated by the xanthine/xanthine oxidase system decreased the concentration of oxymyoglobin. Based on the decreases in absorbance peaks at 581 nm and 415 nm it is estimated that out of a 10 nmol decrease in oxymyoglobin, 5 nmol appears to be oxidized to ferrimyoglobin (deoxygenation), while haem was removed from the other 5 nmol of haem protein. These processes were inhibited by both catalase alone and superoxide dismutase in combination with catalase, but not by either superoxide dismutase alone or deferoxamine. These results suggest that among H2O2, OH. and O2.-, only H2O2 causes the removal of haem and the oxidation of oxymyoglobin. Furthermore, the oxyradicals also released 3 microM free iron from oxymyoglobin, which is at least 5-fold less than the 15 nmol loss of oxymyoglobin. The loss of oxymyoglobin also preceded the release of free iron. These results indicate that oxymyoglobin oxidation and haem removal occur before the removal of free iron. Thus myoglobin appears to be highly susceptible to free radical attack, and this may represent yet another mechanism of free radical-mediated cellular injury.
Inhibition of adenylate cyclase in rat brain synaptosomal membranes by GTP and phenylisopropyladenosine is enhanced in hypothyroidism
Authors: Mazurkiewicz D , Saggerson E D
Published: August 2015

1. Synaptosomal membranes were isolated from rats made hypothyroid by treatment with propylthiouracil and a low iodine diet. 2. When assayed in the presence of 100 mM-Na+, inhibition of forskolin-stimulated adenylate cyclase by GTP was enhanced in membranes from hypothyroid animals. 3. Hypothyroidism also enhanced inhibition of adenylate cyclase by phenylisopropyladenosine (with 100 mM-Na+ and 10 microM-GTP present). 4. Hypothyroidism did not increase binding of the A1 adenosine receptor agonist phenylisopropyladenosine to synaptosomal membranes; rather, the maximum binding was slightly decreased without any change in the KD. 5. The effect of GTP in modifying the displacement of the antagonist [3H]diethylphenylxanthine from synaptosomal membranes by unlabelled phenylisopropyladenosine was more pronounced in the hypothyroid state. 6. These findings are consistent with hypothyroidism causing modification of the brain adenylate cyclase system at the level of the coupling protein Gi.
Enantiomeric selectivity of adenosine transport systems in mouse erythrocytes and L1210 cells
Authors: Gati W P , Dagnino L , Paterson A R P
Published: August 2015

In mediating the entry of adenosine into mouse erythrocytes and mouse leukaemia L1210 cells, nucleoside transport systems were stereoselective, showing a marked preference for the D-enantiomer of adenosine (D-Ado). Inward zero-trans fluxes of the mirror-image isomer, L-adenosine (L-Ado), in those cells were slow relative to those of D-Ado. Contributing to L-Ado fluxes in both cell types were (i) a transporter-mediated process of high nitrobenzylthioinosine-sensitivity and (ii) simple diffusion.
Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12
Authors: Alwan A F , Mgbeje B I A , Jordan P M
Published: August 2015

The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E. coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type. The enzyme was purified to homogeneity from these strains in milligram amounts. The enzyme is a monomer of Mr 28,000 with an isoelectric point of 5.2 and a pH optimum of 7.8. The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 microM. The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence. The enzyme contains four 5,5′-dithiobis-(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity. The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction.
Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans
Authors: Guest P C , Pipeleers D , Rossier J , Rhodes C J , Hutton J C
Published: August 2015

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.
Epidermal growth factor stimulates rat cardiac adenylate cyclase through a GTP-binding regulatory protein
Authors: Nair B G , Rashed H M , Patel T B
Published: August 2015

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5′-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5′-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.
Phospholipase D activation is functionally linked to superoxide generation in the human neutrophil
Authors: Bonser R W , Thompson N T , Randall R W , Garland L G
Published: August 2015

Neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) in the presence of butanol and ethanol formed phosphatidyl alcohols through a phospholipase D mechanism. The alcohols inhibited phosphatidic acid and diradylglycerol (DRG) formation, but did not block inositol 1, 4, 5-trisphosphate release. fMet-Leu-Phe-stimulated superoxide production was inhibited by alcohol concentrations which blocked DRG formation, whereas opsonized-zymosan-stimulated superoxide production was only partially decreased. These results suggest that phospholipase D activation is functionally linked to superoxide production in the human neutrophil.
Studies on the active site of pig plasma amine oxidase
Authors: Collison D , Knowles P F , Mabbs F E , Rius F X , Singh I , Dooley D M , Cote C E , McGuirl M
Published: August 2015

Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity.
Kinetics of the reaction of streptokinase-plasmin complex with purified human and mouse α2-macroglobulin. Implications for mechanism
Authors: Anonick P K , Vetter W H , Gonias S L
Published: August 2015

Streptokinase-human plasmin complex (Sk-hPm) reacted rapidly with purified mouse alpha 2-macroglobulin (m alpha 2M) in vitro at 37 degrees C. Approx. 98% of the plasmin in Sk-hPm bound covalently to at least one m alpha 2M subunit. Most of the streptokinase dissociated (95%). The rate of Sk-hPm inactivation clearly depended on the m alpha 2M concentration. With 1.2 microM-m alpha 2M, 50% of the Sk-hPm (0.02 microM) reacted in less than 50 s. A double-reciprocal plot comparing pseudo-first-order rate constants (kapp.) and m alpha 2M concentration yielded a second-order rate constant of 2.3 x 10(4) M-1.s-1 (r = 0.97). This value is an approximation, since Sk-hPm preparations are heterogeneous. Sk-hPm reacted with human alpha 2M (h alpha 2M), forming alpha 2M-plasmin complex (98% covalent). More than 99% of the streptokinase dissociated. The rate of reaction of Sk-hPm with h alpha 2M did not clearly depend on inhibitor concentration. The kapp. values determined with 0.6-1.2 microM-h alpha 2M were decreased 10-20-fold compared with m alpha 2M. In order to study the effect of Sk-hPm heterogeneity on the reaction with alpha 2M, the proteinase was incubated for various amounts of time at 37 degrees C before addition of inhibitor. The enzyme amidase activity was maximal within 5 min; however, reaction of Sk-hPm with m alpha 2M or h alpha 2M was most extensive after 20 min and 2 h respectively. After incubation for more than 1 h, Sk-hPm acquired fibrinogenolytic activity, suggesting plasmin dissociation. Therefore the enhanced reaction of h alpha 2M with ‘older’ Sk-hPm preparations may have resulted in part from dissociated plasmin or ‘plasmin-like’ species. By contrast, the reaction of Sk-hPm with m alpha 2M was most rapid when the proteinase preparation was free of plasmin, indicating direct reaction of Sk-hPm with m alpha 2M as the only major mechanism. Finally, streptokinase-cat plasminogen complex reacted more extensively with m alpha 2M than with h alpha 2M, suggesting that m alpha 2M may be a superior inhibitor with this class of plasminogen activators in general.
Antibodies against the non-muscle isoform of the endoplasmic reticulum Ca2+-transport ATPase
Authors: Wuytack F , Eggermont J A , Raeymaekers L , Plessers L , Casteels R
Published: August 2015

We report here the production of a polyclonal antiserum which specifically recognizes an epitope confined to the ultimate 12-residue-long C-terminus of an alternatively spliced transcript of gene 2 encoding the sarcoplasmic reticulum Ca2+ pump in slow skeletal and cardiac muscle. This alternatively spliced transcript was shown to be mainly represented in non-muscle tissues. These antibodies have enabled us to show the presence of the unique C-terminus of this type of Ca2+ pump, as predicted from the cDNA sequence, in the endoplasmic reticulum of vascular and gastric smooth muscle, liver and kidney.
Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans
Authors: Schmidtchen A , Carlstedt I , Malmström A , Fransson L Å
Published: August 2015

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ‘matrix’, and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).
Compartmentation of acetyl CoA studied by analysis of tricarboxylic acid cycle acids and 3-hydroxybutyrate in bile of rats given [2,2,2-2H3]ethanol
Authors: Norsten C , Cronholm T
Published: August 2015

Acetate, 3-hydroxybutyrate, pyruvate, lactate, citrate, 2-oxoglutarate, succinate, fumarate and malate were analysed in rat bile by gas chromatography and gas chromatography/mass spectrometry of their O-melthyloxime-t-butyldimethylsilyl derivatives. The concentration of acetate increased to about 1.8 mmol/l after administration of [2,2,2-2H3]ethanol. Acetate was formed from ethanol to an extent of about 82% and retained all of the 2H at C-2, whereas 15% of the 2H had been lost in the tricarboxylic acid cycle intermediates and 24% in 3-hydroxybutyrate. Thus the exchange of 2H for 1H takes place after formation of acetyl CoA. For citrate and 3-hydroxybutyrate, 41% and 11% respectively was formed from [2,2,2-2H3]ethanol. These results indicate that different pools of acetyl CoA are used for the synthesis of ketone bodies and citrate, with the latter being derived from ethanol to a much larger extent. Smaller fractions of 2-oxoglutarate (16%) and succinate (5%) were derived from [2,2,2--2H3]ethanol, indicating significant contributions from amino acids.
Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells
Authors: Socorro L , Alexander R W , Griendling K K
Published: August 2015

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.
Use of a genetic variant to study the hexose transport properties of human skin fibroblasts
Authors: Mesmer O T , Gordon B A , Rupar C A , Lo T C Y
Published: August 2015

Human skin fibroblasts from ‘normal’ subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the ‘normal’ and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.
Hepatitis B surface antigen. Role of lipids in maintaining the structural and antigenic properties of protein components
Authors: Gavilanes F , Gomez-Gutierrez J , Aracil M , Gonzalez-Ros J M , Ferragut J A , Guerrero E , Peterson D L
Published: August 2015

Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.
The effect of fasting on the activation in vivo of the insulin receptor kinase
Authors: Contreras I , Dohm G L , Abdallah S , Wells J A , Mooney N , Rovira A , Caro J F
Published: August 2015

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.
Characterization and localization of progesterone 5α-reductase from cell cultures of foxglove (Digitalis lanata EHRH)
Authors: Wendroth S , Seitz H U
Published: August 2015

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum.
Cathepsin G binding to human platelets. Evidence for a specific receptor
Authors: Selak M A , Smith J B
Published: August 2015

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.
Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus
Authors: Merryweather A T , Crampton J M , Townson H
Published: August 2015

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.
Expression of extracellular superoxide dismutase by human cell lines
Authors: Marklund S L
Published: August 2015

Extracellular superoxide dismutase (EC-SOD) is the major SOD isoenzyme in extracellular fluids, but occurs also in tissues. The sites and characteristics of the synthesis of the enzyme are unknown. The occurrence of EC-SOD in cultures of a large panel of human cell lines was assayed by means of an e.l.i.s.a. Unlike the situation for the intracellular isoenzymes CuZn-SOD and Mn-SOD, expression of EC-SOD occurs in only a few cell types. None of the ten investigated suspension-growing cell lines produced EC-SOD. Among normal diploid anchorage-dependent cell lines, expression was found in all 25 investigated fibroblast cell lines, in the two glia-cell lines, but not in six endothelial-cell lines, two epithelial-cell lines or in two amnion-derived lines. Among neoplastic anchorage-dependent cell lines expression was found in 13 out of 29. EC-SOD was secreted into the culture medium by cell lines expressing the enzyme. The rate of EC-SOD synthesis varied by nearly 100-fold among the fibroblast lines and remained essentially constant in the individual lines during long-term culture. In the nine investigated cases, the secreted EC-SOD was of the high-heparin-affinity C type. It is suggested that tissue EC-SOD is secreted by a few well-dispersed cell types, such as fibroblasts and glia cells, to diffuse subsequently around and reversibly bind to heparan sulphate proteoglycan ligands in the glycocalyx of the surface of most tissue cell types and in the interstitial matrix.
Inhibition of the α-l-arabinofuranosidase III of Monilinia fructigena by 1,4-dideoxy-1,4-imino-l-threitol and 1,4-dideoxy-1,4-imino-l-arabinitol
Authors: Axamawaty M T H , Fleet G W J , Hannah K A , Namgoong S K , Sinnott M L
Published: August 2015

1. 1,4-Dideoxy-1,4-imino-L-threitol was synthesized and the synthesis of 1,4-dideoxy-1,4-imino-L-arabinitol was improved. 2. Both compounds are competitive inhibitors of Monilinia fructigena alpha-L-arabinofuranosidase III, the additional hydroxymethyl group in the arabinitol contributing about 17.8 kj/mol (4.25 kcal/mol) to the Gibbs free energy of binding. 3. The affinities (1/Ki) of both compounds vary with pH in a classical bell-shaped way, the pKa value being that of the acid-catalytic group on the enzyme [5.9; Selwood & Sinnott (1988) Biochem. J. 254, 899-901] and the pKb values being those of the free inhibitors, 7.6 and 7.8 respectively. 4. On the basis of these and literature data we suggest that efficient inhibition of a glycosidase at its pH optimum by an appropriate iminoalditol will be found when the pKa of the iminoalditol is below that of the acid-catalytic group of the target enzyme.
A highly conserved surface loop in the C-terminal domain of ovotransferrin (residues 570-584) is remote from the receptor-binding site
Authors: Mason A B , Brown S A , Church W R
Published: August 2015

A peptide corresponding to a surface loop in the C-terminal domain of chicken ovotransferrin (residues 570-584) was made by solid-phase synthesis and used to immunize rabbits. A 15-amino acid-residue disulphide-linked loop occurs in both domains of all five transferrins for which the sequence is available and lies on the opposite side of the iron-binding site from the interdomain cleft. Polyclonal antibodies to the peptide were specific for non-reduced holo-ovotransferrin and the C-terminal domain, as shown by e.l.i.s.a. and immunoblotting. The antibody did not inhibit binding of ovotransferrin to receptors on chick-embryo reticulocytes but was able to bind ovotransferrin bound to the cellular receptors at 0 degree C. The loop composed of residues 570-584 appears to be remote from the transferrin receptor-binding site.
Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159)
Authors: Fears R , Dodd I , Ferres H , Robinson J H
Published: August 2015

The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for ‘Lys-plasminogen’ (human plasminogen with N-terminal lysine) and ‘Glu-plasminogen’ (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.
Release of purine and pyrimidine nucleosides and their catabolites from the perfused rat hindlimb in response to noradrenaline, vasopressin, angiotensin II and sciatic-nerve stimulation
Authors: Clark M G , Richards S M , Hettiarachchi M , Ye J M , Appleby G J , Rattigan S , Colquhoun E Q
Published: August 2015

Uric acid and uracil were released at constant rates (0.95 and 0.4 nmol/min per g respectively) by the perfused rat hindlimb. Noradrenaline, vasopressin or angiotensin II further increased the release of these substances 2-5-fold, coinciding with increases in both perfusion pressure (vasoconstriction) and O2 uptake. The hindlimb also released, but in lesser amounts, uridine, hypoxanthine, xanthine, inosine and guanosine, and all but hypoxanthine and guanosine were increased during intense vasoconstriction. Uric acid and uracil releases were increased by noradrenaline in a dose-dependent manner. However, the release of these substances did not fully correspond with the dose-dependent increase in O2 uptake and perfusion pressure, where changes in the latter occurred at lower doses of noradrenaline. Sciatic-nerve stimulation (skeletal-muscle contraction) did not increase the release of uracil, uric acid or uridine, but instead increased the release of inosine (7-fold) and hypoxanthine (2-fold). Since the UTP content as well as the UTP/ATP ratio are higher in smooth muscle than in skeletal muscle, it is proposed that release of uric acid and uracil arises from increased metabolism of the respective adenosine and uridine nucleotides during intense constriction of smooth muscle.
Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin
Authors: Bygrave F L , Lenton L , Altin J G , Setchell B A , Karjalainen A
Published: August 2015

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.