bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms
Authors: Williams R A , Andrews P
Published: August 2015

The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.
Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor
Authors: Pierce E J , Davison M D , Parton R G , Habig W H , Critchley D R
Published: August 2015

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.
Incorporation and metabolism of exogenous GM1 ganglioside in rat liver
Authors: Ghidoni R , Trinchera M , Venerando B , Fiorilli A , Sonnino S , Tettamanti G
Published: August 2015

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide.
The association behaviour of β-lactamases. Sedimentation equilibrium studies in ammonium sulphate solutions
Authors: Braswell E H , Knox J R , Frère J M
Published: August 2015

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 × 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 × 10(4) and 3.3 × 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 × 10(3) and 1.7 × 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 × 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.
Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine
Authors: Mauro J M , Lewis R V , Barden R E
Published: August 2015

A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.
NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions
Authors: Murakami M , Nakamura M , Minakami S
Published: August 2015

The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme.
Induction of haem synthesis in Hep G2 human hepatoma cells by dimethyl sulphoxide. A transcriptionally activated event
Authors: Galbraith R A , Sassa S , Kappas A
Published: August 2015

Exposure of cultured human hepatoma cells (Hep G2) to medium containing 2% (v/v) dimethyl sulphoxide resulted in an approximate doubling in the activity of delta-aminolaevulinate dehydratase, an increase in the haem content and a decreased growth rate; induced enzyme activity was decrease by 50% after treatment with alpha-amanitin. The findings are strikingly similar to those seen in murine Friend-virus-transformed erythroleukaemia cells.
Isolation, characterization and metal-ion-binding properties of the α-subunit from S-100a protein
Authors: Leung I K M , Mani R S , Kay C M
Published: August 2015

The brain-specific S-100 protein is a mixture of two predominant components, S-100a and S-100b, with subunit compositions of alpha beta and beta beta respectively. In the present study, the alpha-subunit, isolated from S-100a by using anion-exchange chromatography in the presence of 8 M-urea, was homogeneous by the criteria of SDS/polyacrylamide-gel, urea/SDS/polyacrylamide-gel and non-SDS/polyacrylamide-gel electrophoresis. The alpha-subunit underwent a conformational change upon binding Ca2+ and Zn2+ at pH 7.5, as revealed by u.v. difference spectroscopy, c.d. and fluorescence measurements. Far-u.v. c.d. studies indicated the apparent alpha-helical content to fall when the protein bound either Ca2+ or Zn2+. Addition of Ca2+ to the alpha-subunit resulted in exposing to the solvent the single tryptophan residue and one or more tyrosine and phenylalanine residues. Zn2+ induced only a small conformational change, and among the aromatic chromophores only tyrosine residues were affected to a small extent. Ca2+ was able to bind to the alpha-subunit in the presence of Zn2+, and the two metal-ion-binding sites appeared to be different. When the apoprotein was excited at 280 nm, the fluorescence emission maximum was located at 337 nm. In the presence of Ca2+, the emission maximum occurred at 340 nm and was accompanied by a nearly 25% increase in fluorescence intensity. Fluorescence titration with Ca2+ at pH 7.5 revealed only one class of binding site, with a Kd value of 1.26 × 10(-4) M. The effect of K+ on the protein was slightly antagonistic to that of Ca2+, as indicated by u.v. difference spectroscopy and fluorescence titration.
The use of inhibitors to identify early events during Bacillus megaterium KM spore germination
Authors: Foster S J , Johnstone K
Published: August 2015

The germination response of spores of Bacillus megaterium KM, as measured by loss of A600, is more than 95% inhibited by 1 mM-HgCl2. Two Hg2+-sensitive sites (referred to as ‘sites I and II’) have been identified during germination. Site I represents a pre-commitment event and can be protected from HgCl2 by 50 mM-D-alanine, whereas site II represents a post-commitment event and is not D-alanine-protectable. At 1 mM-HgCl2, 25% of the spore population becomes committed to germinate, but an A600 loss of less than 5% occurs. In this system, loss of heat resistance was associated with commitment, whereas selective cortex hydrolysis, release of pyridine-2,6-dicarboxylic acid, Zn2+ and soluble peptidoglycan, as well as loss of refractility, were identified as post-commitment events. The commitment event was reversibly inhibited by several proteinase inhibitors and a membrane bulking agent. A model of spore germination based on these results is presented.
Role of a pertussis toxin substrate in the control of lectin-induced cap formation in human neutrophils
Authors: Lad P M , Olson C V , Grewal I S
Published: August 2015

We have examined the role of GTP-binding proteins and the associated cyclic AMP- and calcium-related transduction mechanisms in the regulation of capping in human neutrophils. Pertussis toxin (PT), a probe for the GTP-binding protein Ni, abolished capping induced by fluorescein isothiocyanate-conjugated concanavalin A (Con-A), whereas cholera toxin, a probe for the GTP-binding protein Ns, was without effect. Consistent with the latter finding, ligands acting at receptors associated with the Ns protein, namely the prostaglandin E1 and beta-adrenergic agonists, were without effect on the capping reaction. The possible role of mobilization of internal calcium was evaluated by using Quin2-loaded cells. Calcium mobilization was observed at concentrations of Con-A which yielded optimal capping (10 micrograms/ml). Treatment with PT, phorbol myristrate acetate or 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) abolished both calcium mobilization and capping. Colchicine, which substantially enhanced capping, had no effect on calcium mobilization. At concentrations of the lectin above those required for capping, superoxide generation and enzyme release were noted. These reactions were less susceptible to inhibition by PT, effects being observed only on the Kact. for Con-A-mediated superoxide generation with little effect on the Vmax. The degree of PT-mediated inhibition for enzyme release with Con-A was much lower than that observed with fMet-Leu-Phe. Our results imply that a step involving Ni-mediated calcium mobilization, sensitive to phorbol myristate acetate, is essential to the regulation of capping; a distinct mechanism may be involved in colchicine-mediated enhancement of capping; and Ni may play a relative minor role in the regulation of lectin-mediated exocytosis.
Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation
Authors: Saggerson E D
Published: August 2015

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.
Dinitrophenyl glutathione efflux from human erythrocytes is primary active ATP-dependent transport
Authors: LaBelle E F , Singh S V , Srivastava S K , Awasthi Y C
Published: August 2015

Dinitrophenyl S-glutathione is accumulated by inside-out vesicles made from human erythrocytes in a process totally dependent on ATP and Mg2+. The vesicles were shown to accumulate dinitrophenyl S-glutathione against a concentration gradient. The vesicles were able to concentrate this glutathione derivative even in the absence of membrane potential. This indicated that the ATP-dependent uptake of dinitrophenyl S-glutathione by inside-out vesicles represented an active transport process. Neither extravesicular EGTA nor intravesicular ouabain inhibited the transport process, indicating that neither the Ca2+-ATPase nor the Na+, K+-ATPase were involved. These results indicated that dinitrophenyl S-glutathione uptake by inside-out vesicles probably represented primary active transport. The uptake of dinitrophenyl S-glutathione was a linear function of time (up to 5 h) and vesicle protein. The rate of uptake was optimal between pH 7.0 and 8.0 and at 37 degrees C. The Km values determined for dinitrophenyl S-glutathione and ATP were 0.29 mM and 1 mM, respectively. The transport process was completely inhibited by vanadate and by p-hydroxymercuribenzene sulphonate and inhibited to a lesser extent by N-ethylmaleimide. GTP could efficiently substitute for ATP as an energy source for the transport process, but CTP and UTP were comparatively much less effective.
The radiosensitivity of rat thymocytes
Authors: Ord M G , Stocken L A
Published: August 2015

gamma-Irradiation in vitro apparently blocked a plasma-membrane associated, superoxide-producing, NADPH oxidase in rat thymocytes. Differential centrifugation of the mixed thymocytes indicated the smaller lymphocytes (approx. 6 microns diameter) to be the radiosensitive population. The oxidase system co-isolated in part with thymus nuclei and could be solubilized by detergent treatment [Bellavite, Jones, Cross, Papini & Rossi (1984) Biochem. J. 223, 639-648]. Endogenous NADPH was the rate-limiting component for superoxide formation in vitro. The level of NADPH was lowered by gamma-irradiation, an effect mimicked by GSSG in the presence of 50 microM-ZnCl2 to inhibit GSSG reductase. These findings are suggested as the metabolic basis for interphase death of small lymphocytes exposed to ionizing radiation.
Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone
Authors: Davis J S , Weakland L L , West L A , Farese R V
Published: August 2015

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in ‘second messengers’ derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.
Blood-group-related carbohydrate antigens are expressed on human milk galactosyltransferase and are immunogenic in rabbits
Authors: Childs R A , Berger E G , Thorpe S J , Aegerter E , Feizi T
Published: August 2015

Immunochemical evidence is presented for the presence of blood-group-related carbohydrate structures on human milk galactosyltransferase and for the occurrence of the corresponding specificities among rabbit antibodies to this enzyme. Although these carbohydrate specificities constitute minor populations among antisera and affinity-purified antibodies to galactosyltransferase, their presence is important in the immunohistochemical approach to enzyme localization, since they give rise to strong reactivities with epithelial cells of the gastrointestinal tract.
Binuclear platinum (II)-terpyridine complexes. A new class of bifunctional DNA-intercalating agent
Authors: McFadyen W D , Wakelin L P G , Roos I A G , Hillcoat B L
Published: August 2015

A series of binuclear DNA-binding ligands was prepared by linking two (2,2′:6′,2″-terpyridine)platinum(II) moieties via alpha omega-dithiols of the type HS-[CH2]n-SH where n = 4-10. A monomeric analogue was also synthesized. Compounds were characterized by elemental analysis and electronic and n.m.r. spectroscopy. Viscometric measurements with sonicated rod-like DNA fragments and covalently closed circular DNA were performed to investigate the mode of binding of these agents. The ligands with n = 5 and 6 function as bis intercalators and form a single ‘base-pair sandwich’ in violation of neighbour-exclusion binding. Bifunctional reaction occurs for the ligand with n = 7, whereas the ligands with n = 8 and 10 show a preference for mixed monofunctional/bifunctional binding. The data do not permit definitive assignment of the binding mode of the ligands with n = 4 and 9. All compounds are growth-inhibitory against mouse leukaemia L1210 cells in culture with IC50 values in the range 2-14 microM.
Fat synthesis in adipose tissue. An examination of stoichiometric constraints
Authors: Fell D A , Small J R
Published: August 2015

The requirement for net balance of synthesis, degradation and transport for all intermediates in the pathways from glucose to fat imposes constraints on the balance of fluxes between different pathways. Linear programming has been used to examine the interactions between these constraints on metabolism in adipocytes and the requirement for efficiency in the conversion of glucose into fat. The circumstances under which excessive ATP synthesis would accompany this conversion have been investigated.
Action in vivo and in vitro of 2-tetradecylglycidic acid, 2-tetradecylglycidyl-CoA and 2-tetradecylglycidylcarnitine on hepatic carnitine palmitoyltransferase
Authors: Brady P S , Brady L J
Published: August 2015

The effects of 2-tetradecylglycidic acid (TDGA), TDGA-CoA and TDGA-carnitine were examined in purified hepatic CPT (carnitine palmitoyltransferase) and in hepatic mitochondria and inverted submitochondrial vesicles derived from Sprague-Dawley rats. Since TDGA has been reported as a specific inhibitor of carnitine palmitoyltransferase-A (CPT-A), the focus was on kinetics and inhibition of CPT-A, and the relationship of this key enzyme to beta-oxidation. After administration of TDGA in vivo to overnight-starved rats, the Vmax. of CPT in intact mitochondria and in inverted vesicles (CPT-B) was depressed by 66%. The S0.5 for palmitoyl-CoA and Km for carnitine were unchanged. The I50 (concn. giving 50% inhibition) for malonyl-CoA was significantly increased from 20 to 141 microM in intact mitochondria, but unchanged (199 versus 268 microM) in inverted vesicles. The addition in vitro of TDGA-CoA (0-1.0 microM) gave I50 values of 0.29 and 0.27 microM (S.E.M. = 0.19) in intact mitochondria from fed and 48 h-starved rats, and 0.81 and 1.57 microM (S.E.M. = 0.29) for inverted vesicles derived from fed and starved rats. Addition in vitro of TDGA-carnitine to mitochondria from starved rats yielded an I50 value of 27.7 mM (S.E.M. = 12.2) for L-[methyl-14C]carnitine release from palmitoyl-L-[methyl-14C]carnitine and 0.64 mM (S.E.M. = 0.07) for palmitoyl-L-[methyl-14C]carnitine formation from L-[methyl-14C]carnitine in intact mitochondria. Inverted vesicles were not measurably sensitive to TDGA-carnitine up to 500 microM for the assay of L-[methyl-14C]carnitine release, but were as sensitive as intact mitochondria when inhibition was determined in the direction of palmitoyl-L-[methyl-14C]carnitine formation (I50 = 0.54 +/- 0.07 microM). When TDGA-CoA was added to intact mitochondria, then incubated for 5 min at room temperature and subsequently washed out, Vmax. of CPT decreased from 5.8 to 3.5 (S.E.M. = 0.6) in intact mitochondria, and from 17.2 to 6.3 (S.E.M. = 4.8) in inverted vesicles. The Km for L-carnitine and the S0.5 for palmitoyl-CoA increased 2-fold with TDGA-CoA pretreatment in both intact mitochondria and inverted vesicles. Detergent solubilization (0.05% Triton X-100) resulted in a complete loss of TDGA-CoA sensitivity (up to 1.0 microM measured). Sonicated mitochondria exhibited an I50 of 0.72 +/- 0.03 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Abnormal regulation of insulin secretion in the genetically obese (ob/ob) mouse
Authors: Black M , Heick H M , Bégin-Heick N
Published: August 2015

To determine whether the abnormal insulin-secretory activity encountered in obese mice is due to an anomaly in the production of cyclic AMP, islets of lean and obese mice were incubated with forskolin under various conditions. Our data show that, in addition to the well-known quantitative differences in insulin-secretory activity between islets of lean and obese mice, there are important qualitative differences. The islets of obese mice accumulated less cyclic AMP than did those of lean mice in response to given doses of forskolin, yet their insulin secretion was enhanced to much higher values. In the islets of obese mice, but not in those of lean mice, the stimulatory effect of forskolin on insulin secretion was evident even at non-stimulatory concentrations of glucose or in Ca2+-deprived incubation media, showing that the islet of the obese mouse is less dependent on a primary stimulus (glucose) and on the provision of normal concentrations of Ca2+ in the bathing medium than is the islet of the lean mouse.
Short term memory in the calcium messenger system. Evidence for a sustained activation of protein kinase C in adrenal glomerulosa cells
Authors: Barrett P Q , Kojima I , Kojima K , Zawalich K , Isales C M , Rasmussen H
Published: August 2015

If adrenal glomerulosa cells are treated with angiotensin II for a period of 20-30 min, their subsequent response to either a rechallenge with the same concentration of angiotensin II or treatment with BAY K 8644, a calcium channel agonist, differs from the responses of control cells. Perifusion of control cells with 10 nM-angiotensin II leads to an increase in aldosterone secretory rate from 44 +/- 7 to 166 +/- 9 pg/min per 10(6) cells, but perifusion of cells pretreated for a 20 min period with angiotensin II leads to an increase in secretory rate from 51 +/- 9 to 209 +/- 18 pg/min per 10(6) cells. Likewise, treatment of control cells with 10 nM-BAY K 8644 leads to no significant increase in aldosterone secretory rate, but treatment of previously exposed cells to angiotensin II leads to an increase in rate from 51 +/- 9 to 130 +/- 11 pg/min per 10(6) cells. This memory effect is time-dependent in two ways: cells must be exposed to angiotensin II for 20 min or more before it is apparent; the longer the time between removal of angiotensin II and the rechallenge, the less effect these agents have on aldosterone secretory rate. When cells are exposed to angiotensin II for 20 min and then treated with [Sar1,Ala8]angiotensin II, a competitive antagonist of angiotensin II action, the aldosterone secretory rate falls to basal with a half time of 5-7 min. If BAY K 8644 is added simultaneously with [Sar1,Ala8]angiotensin II, the secretory rate falls with a halftime of 35-60 min. BAY K 8644 increases Ca2+ influx rate to the same extent in the presence or absence of [Sar1,Ala8]angiotensin II, and does not alter the effect of either angiotensin II or [Sar1,Ala8]angiotensin II on the production of inositol tris-, bis-, or mono-phosphate. In cells treated with 10 nM-angiotensin II for either 20, 30 or 45 min, the extent of phosphorylation of four cellular proteins is increased. If cells treated for 20 min with angiotensin II are then treated with [Sar1,Ala8]angiotensin II, and examined 15 min later (35 min), there is no longer an increase in the extent of phosphorylation of any of the four proteins. If such cells are then treated with 10 nM-BAY K 8644 and re-examined 5 min later (40 min), all four patients show an increase in the extent of phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Monoclonal antibodies against rat kidney γ-glutamyl transpeptidase show species and tissue specificity
Authors: Green J A , Cook N D , Manson M M
Published: August 2015

Monoclonal antibodies have been raised against rat kidney gamma-glutamyl transpeptidase (GGT). All five antibodies immunoprecipitate enzyme activity from solubilized kidney brush-border membranes, but not from hepatocellular carcinoma membranes. Three of the antibodies react immunohistochemically with brush-border membranes in sections of adult rat kidney, but none of the antibodies cross-react with sections of guinea-pig, mouse or marmoset kidney or with untreated or carcinogen-treated rat liver. The antibodies do not recognize GGT in foetal-rat kidney and react poorly with kidney from 2-year-old rat. They do react with tubules trapped within mesenchymal kidney tumours induced by dimethylnitrosamine, but epithelial tumours, which are GGT-positive (although much less so than normal kidney), are not immunoreactive.
Electron microscopy of cervical, gastric and bronchial mucus glycoproteins
Authors: Sheehan J K , Oates K , Carlstedt I
Published: August 2015

Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.
Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria
Authors: Denyer G S , Kerbey A L , Randle P J
Published: August 2015

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.
The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method
Authors: Itoh S , Yamakawa T , Onishi S , Isobe K , Manabe M , Sasaki K
Published: August 2015

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.
Molecular-size standards for use in radiation-inactivation studies on proteins
Authors: Nugent J H A
Published: August 2015

The accuracy of the radiation-inactivation technique for estimating molecular size was investigated with a range of proteins of known molecular mass. With the use of irradiation with a 16 MeV electron beam, inactivation was examined both in frozen samples at 77 K and in freeze-dried samples at room temperature. The effect of the presence of detergents and chloroplast membrane preparations was also measured. It was demonstrated that proteins added as internal standards, including malate dehydrogenase, glucose-6-phosphate dehydrogenase and cytochrome c, can provide an accurate calibration of molecular size. However, a disadvantage of the technique was that the target size of oligomeric enzymes could be that of either the monomers, dimers or higher oligomers. The detergent Triton X-100 increased the rate of inactivation of the proteins investigated.
Trypsin-induced increase in cyclic AMP concentration in rat thymocytes An effect independent of calcium and calmodulin
Authors: Segal J
Published: August 2015

Trypsin produces a dose-related increase in cellular cyclic AMP concentration in rat thymocytes [Shneyour, Patt & Trainin (1976) J. Immunol. 117, 2143-2149; Segal & Ingbar (1983) Clin. Res. 31, 277A]. In the present study, I examined whether this effect of trypsin requires Ca2+ and whether it is modified by calmodulin. In fresh thymocytes suspended in standard medium (containing 1 mM-Ca2+), trypsin produced a concentration-dependent increase in cytoplasmic free Ca2+ concentration, which was evident at a concentration of 50 micrograms of trypsin/ml and reached maximal values at about 1 mg/ml. This effect of trypsin was very prompt in onset, almost immediate, and reached maximal values within 2-3 min. But in cells suspended in essentially Ca2+-free medium (6 nM free Ca2+), trypsin had no effect on cytoplasmic free Ca2+ concentration, which indicates that trypsin acted by increasing Ca2+ uptake rather than Ca2+ release from an intracellular pool. However, the increase in thymocyte cyclic AMP concentration produced by trypsin was independent of extracellular Ca2+ and was not influenced by calmodulin, because it was the same in the presence or absence of Ca2+ and was not changed by the calmodulin inhibitor trifluoperazine. I therefore suggest that in rat thymocytes the trypsin-induced increase in cyclic AMP concentration does not require Ca2+ and is not influenced by calmodulin.
Inhibition of microsomal oxidation of ethanol by pyrazole and 4-methylpyrazole in vitro Increased effectiveness after induction by pyrazole and 4-methylpyrazole
Authors: Feierman D E , Cederbaum A I
Published: August 2015

Pyrazole and 4-methylpyrazole, which are inhibitors of alcohol dehydrogenase, were also found to be effective inhibitors of the oxidation of ethanol by liver microsomes (microsomal fractions) in vitro. Ethanol oxidation by microsomes from rats previously treated for 2 or 3 days with either pyrazole or 4-methylpyrazole appeared to be especially sensitive to inhibition in vitro by pyrazole or 4-methylpyrazole. The kinetics of inhibition by pyrazole or 4-methylpyrazole in all microsomal preparations were mixed, as the Km for ethanol was elevated while Vmax was lowered. However, Ki values for pyrazole (about 0.35 mM) and especially 4-methylpyrazole (about 0.03-0.10 mM) were much lower than those found with the saline controls (about 0.7-1.1 mM). In contrast, Ki values for dimethyl sulphoxide as an inhibitor of microsomal ethanol oxidation were similar in all microsomal preparations. Pyrazole and 4-methylpyrazole reacted with microsomes to produce type II spectral changes whose magnitude increased after treatment with either pyrazole or 4-methylpyrazole. Thus the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole appears to be associated with increased interactions with the cytochrome P-450 isoenzyme(s) induced by these compounds. These isoenzymes have properties similar to those of the isoenzyme induced by chronic ethanol treatment. Therefore, caution is needed in the use of pyrazole or 4-methylpyrazole to assess pathways of ethanol metabolism, especially after chronic ethanol treatment, since these agents, besides inhibiting alcohol dehydrogenase, are also effective inhibitors of microsomal ethanol oxidation.
Mechanism of enzymic isomerization and epimerization of d-erythrose 4-phosphate
Authors: Hosomi S , Nakai N , Kogita J , Terada T , Mizoguchi T
Published: August 2015

The mechanism of the enzymic isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) by an enzyme preparation from bovine liver was investigated with the use of 2H2O. The incorporation of 2H was quantitatively determined by a procedure using gas chromatography-mass spectrometry. About one atom of 2H was incorporated per molecule of the enzymic epimerization reaction product of Ery4P (D-threose 4-phosphate) or that of D-ribulose 5-phosphate. Computer simulation of the Ery4P isomerization reaction indicated that the 2H of 2H2O was not directly incorporated into the enzymic reaction product (D-erythrulose 4-phosphate). Instead, intramolecular transfer of hydrogen atoms had occurred.
Determination of the molecular mass of apolipoprotein B-100 A chemical approach
Authors: Yang C Y , Lee F S , Chan L , Sparrow D A , Sparrow J T , Gotto A M
Published: August 2015

Apolipoprotein B-100 (apo B-100) is the protein ligand in low-density lipoproteins that binds to a specific cell-surface receptor. Its molecular mass has been a subject of controversy. We have determined the molecular mass of the protein by a chemical approach. After complete CNBr cleavage, the C-terminal fragment of apo B-100 was purified by reverse-phase h.p.l.c. Amino acid N- and C-terminal analyses confirm that this peptide represents the C-terminal peptide as deduced from the DNA sequence of a human apo B-100 cDNA clone. A chemically synthesized peptide was used to determine the recovery of the peptide (74.72%). On the basis of these data, the molecular mass of apo B-100 was determined to be 496.82 +/- 24.84 kDa.
Site recognition by protein-primed T cells shows a non-specific peptide size requirement beyond the essential residues of the site Demonstration by defining an immunodominant T site in myoglobin
Authors: Bixler G S , Bean M , Atassi M Z
Published: August 2015

In previous studies, six T sites within myoglobin (Mb) were localized. To define precisely the boundaries of the T sites, a new approach is introduced and applied here to the T site residing within residues 107-120 of Mb. Two sets of peptides were synthesized. One set represents a stepwise elongation by one-residue increments of the Mb sequence. The other set represents an identical stepwise addition of one-residue increments of the Mb sequence, but which were extended by additional unrelated (nonsense) residues to a uniform size of 14 residues. The longer peptides (nonsense-extended) usually gave higher proliferative responses than did their shorter counterparts having the same Mb region. Thus a minimum peptide size is required for optimal T-cell stimulation. The T site subtends, in three high-responder mouse strains, residues 109-119 or 110-120, depending on strain, and, in three low-responder strains, maps to residues 108-120. Thus, in this case, the T site coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.
Chemical modification of the haem propionate of cytochrome c A re-evaluation of the reaction of cytochrome c with a water-soluble carbodi-imide
Authors: Mathews A J , Brittain T
Published: August 2015

Horse heart and tuna heart cytochromes c were treated with the water-soluble carbodi-imide 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide. When the reaction is followed spectroscopically two kinetic phases are apparent. Alteration of the reactivity of the proteins with such ligands as CO, however, occurs in a single phase identical with the faster phase detected spectroscopically. The modified proteins both show spectroscopic and redox properties identical with those described for the tuna heart cytochrome c derivative by Timkovich [Biochem. J. (1980) 185, 47-57]. The use of radiolabelled carbodi-imide identifies two or three sites of reactivity. However, the addition of glycine methyl ester to the reaction mixture leads to the addition of nine glycine moieties in the case of the horse protein and seven in the case of the tuna protein, indicating a larger number of reactive sites than previously reported. A further set of reaction sites was identified by peptide mapping of the modified proteins, and these sites take part in intramolecular reactions leading to internal cross-linking and the formation of an enzymically indigestible ‘core particle’. The haem group was identified as a site of reaction with the carbodi-imide, and is as a consequence covalently linked to the peptide by a bond in addition to the thioether bonds normally present. In the light of these findings, the alterations in the properties of the tuna protein, subsequent to reaction with the carbodi-imide, which have been previously explained in structural terms, must be re-evaluated. This study also highlights the importance of internal cross-link formation, which can occur by intramolecular nucleophilic attack, a process that has often been overlooked by investigators employing carbodi-imide modification of carboxylate groups in proteins.
Structure and stability of Ricinus communis haemagglutinin
Authors: Frénoy J P , Tran A T , Bourrillon R
Published: August 2015

The molecular properties of the haemagglutinin of Ricinus communis (RCA I or RCA 120) were evaluated by analytical ultracentrifugation, light-scattering, c.d. and fluorescence. The native molecule had a fairly expanded structure (f/f0 = 1.43) and dissociated into two subunits of equal size in 6 M-guanidinium chloride. This native structure was stable in alkali (up to pH 11) and resistant to thermal denaturation at neutrality. A pH-triggered change in the haemagglutinin conformation was observed and characterized by analytical ultracentrifugation, c.d. and fluorescence between pH 7 and 4.5, the range in which its affinity for galactosides decreased [Yamasaki, Absar & Funatsu (1985) Biochim, Biophys. Acta 828, 155-161]. These results are discussed in relation to those reported in the literature for other lectins and more especially ricin, for which a pH-dependent conformation transition has been observed in the same range of low pH.
Muscarinic-agonist and guanine nucleotide activation of polyphosphoinositide phosphodiesterase in isolated islet-cell membranes
Authors: Dunlop M E , Larkins R G
Published: August 2015

Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5′-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.