bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane transport in relation to net uptake of glucose in the perfused rat hindlimb. Stimulatory effect of insulin, hypoxia and contractile activity
Authors: Idström J P , Rennie M J , Scherstén T , Bylund-Fellenius A C
Published: August 2015

The paired-tracer dilution method applied to the perfused rat hindlimb model was used to study glucose transport in relation to net glucose uptake in skeletal muscle tissue. 2-deoxyglucose was used as an analogue for glucose, since this eliminates the problem with release of labelled metabolites. The affinity of 2-deoxyglucose for the glucose carrier was shown to be indistinguishable from that of glucose. An insulin dose-response study showed maximal stimulation of glucose uptake and transport at 0.1 unit/l, and 75% of maximal stimulation at 0.01 unit of insulin/l. Hypoxia and contractile activity stimulated the 2-deoxyglucose transport rate similarly, and the stimuli were not additive, suggesting a common mechanism. The presence of insulin did not increase the effect of hypoxia or contractile activity, indicating no permissive effect of insulin. The 2-deoxyglucose transport rate was closely correlated with and always higher than that of glucose uptake, demonstrating that the transport is never rate-limiting for the net glucose uptake and that both processes are regulated together. Significant correlations between the 2-deoxyglucose transport rate and the intramuscular concentration of phosphocreatine suggest regulation of the glucose utilization by the energy state of the skeletal muscle tissue.
The γ-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum
Authors: Kirkness E F , Turner A J
Published: August 2015

The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.
Trans-stimulation and trans-inhibition of uridine efflux from human erythrocytes by permeant nucleosides
Authors: Jarvis S M
Published: August 2015

The ability of nucleoside permeants to accelerate the efflux of uridine from human erythrocytes has been compared. In contrast to uridine, 2-chloroadenosine acted as a trans-inhibitor of uridine efflux from fresh human erythrocytes, and adenosine had little effect. These results are consistent with the lower maximum velocity for influx of 2-chloroadenosine and adenosine as compared with uridine and demonstrate that trans acceleration experiments do not discriminate between transported and non-transported permeants for the human erythrocyte nucleoside carrier.
Proteins of the human placental microvillar cytoskeleton. α-Actinin
Authors: Vanderpuye O A , Edwards H C , Booth A G
Published: August 2015

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.
Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q
Authors: Tenner A J , Volkin D B
Published: August 2015

An enzyme-linked immunosorbent assay (e.l.i.s.a.) that is capable of quantifying C1q concentrations as low as 2 ng/ml and a sensitive haemolytic assay were used to study the appearance of material that cross-reacts with human serum C1q as well as C1q haemolytic activity in human monocyte culture media. This material was detected in the medium after 10-14 days and continued to be secreted through to day 28 of culture, at which time the cultures were terminated. Material specifically immunoabsorbed with Sepharose-anti-C1q antibody from a culture medium of cells that was metabolically labelled with [3H] proline or [35S] methionine demonstrated a polypeptide pattern identical with that of serum C1q on SDS/polyacrylamide-gel electrophoresis. Under non-reducing conditions two protein bands were detected migrating with the same Rf values as the serum C1q A-B and C-C dimers. On reduction three bands were evident, which migrated identically with the A, B and C chains of serum C1q. The amount of radioactivity in these bands increased with time in culture, consistent with the e.l.i.s.a. and haemolytic C1q assays. These bands were reactive with monospecific anti-C1q antibody after transfer to nitrocellulose.
Purification and characterization of ascamycin-hydrolysing aminopeptidase from Xanthomonas citri
Authors: Osada H , Isono K
Published: August 2015

A nucleoside antibiotic, ascamycin (9-beta-[5′-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.
Simultaneous induction of Pb-metallothionein-like protein and Zn-thionein in the liver of rats given lead acetate
Authors: Ikebuchi H , Teshima R , Suzuki K , Terao T , Yamane Y
Published: August 2015

Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-metallothionein (Pb-MT)-like protein (Pb-BP) and Zn-thionein (Zn-BP) in the liver. The Pb-BP had an apparent molecule mass of 6900 Da and seemed to bind preferentially to lead in the liver cytosol. The Zn-BP was identified by comparison of the Mr, elution profiles from Sephadex G-75 and DEAE-Sephadex A-25 columns, and polyacrylamide-gel-electrophoretic mobility, with those of rat liver Zn-MT-II. The Pb-BP accumulated in the liver to a maximum 6 h after the intraperitoneal injection of lead acetate and accounted for about 60% of the lead in the liver cytosol at this stage. However, after that, it gradually decreased in the liver, until it was close to the basal amount 24 h after the induction. In contrast, the amount of Zn-MT increased gradually, reached a maximum 12 h after the administration of lead acetate and maintained a constant value until at least 24 h after the induction. Amino acid analysis of the Pb-BP indicated that it contained about 28% half-cysteine. These results strongly suggest that lead acetate induces the synthesis of Pb-MT as well as Zn-MT in rat liver.
Kinetic studies on the nitrite reductase of Wolinella succinogenes
Authors: Blackmore R , Brittain T
Published: August 2015

The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 × 106 M-1 × S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.
Stimulation of mouse liver glutathione S-transferase activity in propylthiouracil-treated mice in vivo by tri-iodothyronine
Authors: Williams M T , Carrington H , Herrera A
Published: August 2015

Female C57Bl/6J mice were given drinking water containing 0.05% propylthiouracil to induce a hypothyroid condition. Mitochondrial glycerol-3-phosphate dehydrogenase activity, used as an index of hypothyroidism, was 57.1 +/- 4.5 and 29.4 +/- 3.8 nmol/min per mg of protein for control and propylthiouracil-treated animals respectively. Administration of tri-iodothyronine resulted in an approx. 4.5-fold increase in dehydrogenase activity in propylthiouracil-treated animals. A dose-dependent increase in hepatic GSH S-transferase activity in propylthiouracil-treated animals was observed at tri-iodothyronine concentrations ranging from 2 to 200 micrograms/100 g body wt. This increase in transferase activity was seen only when 1,2-epoxy-3-(p-nitrophenoxy)propane was used as substrate for the transferase. Transferase activity with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrate was decreased by tri-iodothyronine. Administration of actinomycin D (75 micrograms/100 g body wt.) inhibited the tri-iodothyronine induction of transferase activity. Results of these studies strongly suggest that tri-iodothyronine administration markedly affected the activities of GSH S-transferase by inducing a specific isoenzyme of GSH S-transferase and suppressing other isoenzymic activities.
Nature and properties of human platelet vasopressin receptors
Authors: Vittet D , Rondot A , Cantau B , Launay J M , Chevillard C
Published: August 2015

The binding of 3H-labelled [8-arginine]vasopressin to human platelets or crude platelet membranes was examined. Both preparations specifically bound [8-arginine]vasopressin. The binding increased linearly with protein concentration, it was temperature- and time-dependent, saturable and could be reversed to a large extent by EDTA (10 mM). In this latter case, addition of an excess of MgCl2 (20 mM) restored the initial level of binding. Intact platelets and membranes derived from these platelets presented a single population of binding sites with a dissociation constant (Kd) of 1.3 +/- 0.2 and 1.8 +/- 0.3 nM and a maximal binding capacity of 142 +/- 48 and 270 +/- 17 fmol/mg of protein, respectively. The Kd values of various analogues correlated well with those determined on rat liver membrane V1 vasopressin receptors but not with those determined on rat kidney membrane V2 receptors.
Purification and kinetic properties of ox brain histamine N-methyltransferase
Authors: Gitomer W L , Tipton K F
Published: August 2015

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.
Inhibition of pyruvate dehydrogenase complex by moniliformin
Authors: Gathercole P S , Thiel P G , Hofmeyr J H S
Published: August 2015

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.
Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat
Authors: Hayes J D , Mantle T J
Published: August 2015

The glutathione S-transferases are dimeric enzymes whose subunits can be defined by their mobility during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as Yf (Mr 24,500), Yk (Mr 25,000), Ya (Mr 25,500), Yn (Mr 26,500), Yb1 (Mr 27,000), Yb2 (Mr 27,000) and Yc (Mr 28,500) [Hayes (1986) Biochem. J. 233, 789-798]. Antisera were raised against each of these subunits and their specificities assessed by immuno-blotting. The transferases in extrahepatic tissues were purified by using, sequentially, S-hexylglutathione and glutathione affinity chromatography. Immune-blotting was employed to identify individual transferase polypeptides in the enzyme pools from various organs. The immuno-blots showed marked tissue-specific expression of transferase subunits. In contrast with other subunits, the Yk subunit showed poor affinity for S-hexylglutathione-Sepharose 6B in all tissues examined, and subsequent use of glutathione and glutathione affinity chromatography. Immuno-blotting was employed to identify a new cytosolic polypeptide, or polypeptides, immunochemically related to the Yk subunit but with an electrophoretic mobility similar to that of the Yc subunit; high concentrations of the new polypeptide(s) are present in colon, an organ that lacks Yc.
An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase
Authors: Sutherland J , Greenwood C , Peterson J , Thomson A J
Published: August 2015

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.
Changes in permeability to protons and other cations at high proton motive force in rat liver mitochondria
Authors: Brown G C , Brand M D
Published: August 2015

We have confirmed that the respiration rate of rat liver mitochondria can be substantially inhibited with only a small drop in proton motive force. We have directly measured the passive proton permeability as a function of delta psi by using K+ diffusion potentials and have shown that there is a large increase in proton permeability at high delta psi. This can quantitatively account for the inhibitor titrations of respiration. delta psi and delta pH were shown to have roughly equal effects on the relatively high respiration rate in static head. The permeabilities to K+, tetramethylammonium+ and choline+ were shown to increase greatly at high delta psi, in a similar way to proton permeability, indicating a similar mechanism of entry.
Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese
Authors: Gutteridge J M C , Bannister J V
Published: August 2015

When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2.- is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid-reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.- -dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+-EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex.
The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum
Authors: Jona I , Martonosi A
Published: August 2015

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.
Effects of monensin on insulin interactions with isolated hepatocytes. Evidence for inhibition of receptor recycling and insulin degradation
Authors: Whittaker J , Hammond V A , Taylor R , Alberti K G M M
Published: August 2015

Recent evidence suggests that, during endocytosis, receptors for many polypeptide ligands are spared degradation and are recycled to the plasma membrane for re-utilization. The univalent ionophore monensin was shown to inhibit membrane recycling. We therefore examined its effects on insulin interactions with isolated rat hepatocytes to characterize further receptor endocytosis and recycling in these cells. At 10 degrees C, in the absence of endocytosis, no change in insulin binding was observed. However, at 37 degrees C a concentration-dependent decrease in 125I-insulin binding was seen in the presence of insulin; this reached a maximum of 60% at 1 nM-insulin. Competitive binding studies showed this to be due to a 50-60% decrease in cell-surface insulin-receptor concentration, although the total cellular receptor concentration remained unchanged, suggesting that monensin causes the intracellular sequestration of receptors. Time-course studies of the processing of 2.5 nM-insulin showed that monensin produced a 50-60% decrease in surface binding, accompanied by a similar decrease in internalization and total inhibition of insulin degradation. When hepatocytes with 125I-insulin prebound to their surface receptors at 10 degrees C were warmed to 37 degrees C, monensin had no effect on internalization, but caused marked impairment of intracellular insulin degradation. It is concluded that monensin inhibits receptor recycling and cellular insulin degradation.
Removal of N-terminal methionine from haemoglobin nascent peptides by a membrane-bound rat liver methionine aminopeptidase
Authors: Termignoni C , Freitas J O , Guimarães J A
Published: August 2015

A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates.
Regulation of low-density-lipoprotein metabolism in the rat
Authors: Bhattacharya S , Balasubramaniam S , Simons L A
Published: August 2015

The mechanism of regulation of plasma low-density-lipoprotein (LDL) metabolism in the rat was studied under a number of experimental conditions. LDL clearance and uptake in the liver was measured after intravenous pulse injection of [14C]sucrose-labelled LDL alone or in combination with reductively methylated [3H]sucrose-labelled LDL. Hyperthyroid rats showed a significant increase in fractional catabolic rate (FCR) and the proportion of LDL degraded in the liver, whereas the synthetic rate of LDL increased by 50%. Receptor-mediated clearance increased 2-fold. Hypothyroid rats showed a significant increase in LDL concentration. The FCR and proportion of LDL degraded in the liver were decreased significantly. Receptor-mediated clearance was also reduced. Cholesterol feeding increased chylomicron, very-low-density-and intermediate-density-lipoprotein cholesterol concentrations, but there was no change in the LDL concentration, FCR or the synthetic rate of LDL. Cholestyramine feeding did not induce changes in the kinetic parameters. These results indicate that, in the rat, the hepatic LDL-receptor pathway is under hormonal control, whereas cholesterol and cholestyramine feeding have no demonstrated effect on LDL metabolism.
Transient kinetics of subunit-III-depleted cytochrome c oxidase
Authors: Malatesta F , Antonini G , Sarti P , Brunori M
Published: August 2015

Cytochrome c oxidase from ox heart was depleted of subunit III and its transient kinetic properties studied by stopped-flow and flash photolysis. It was found that the overall mechanism of electron transfer is very similar for subunit-III-depleted and native oxidase, although significant differences in some kinetic parameters have been detected. These include the second-order rate constant for cytochrome c oxidation and the rate-limiting step of the overall process. Moreover, at low cytochrome c/oxidase ratios (where the number of reducing equivalents is insufficient), the rate of reoxidation of cytochrome a was found to be very slow, even in air, and in fact for the subunit-III-depleted enzyme is even slower than for the native oxidase. The stability of reduced cytochrome a excludes the likelihood that removal of subunit III leads to a new O2-binding site, and the result may be relevant to the lowered vectorial H+/e- stoichiometry. The subunit-III-depleted oxidase can be pulsed under appropriate conditions and its combination with CO is unchanged, as shown by kinetic experiments and difference spectroscopy.
Chemical cross-linking analyses of ox neurofilaments
Authors: Carden M J , Eagles P A M
Published: August 2015

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among ‘rod-domain’ fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.
A probable new pathway for the biosynthesis of putrescine in Escherichia coli
Authors: Cataldi A A , Algranati I D
Published: August 2015

Some cultures of Escherichia coli BGA8, a mutant unable to synthesize putrescine, showed a change of behaviour and could grow almost equally well in either the absence or the presence of polyamines after repeated periods of polyamine starvation. Experiments in vivo with radioactive precursors showed that the bacteria which evaded the polyamine requirement had recovered their ability to synthesize putrescine from glucose or glutamic acid, but not from ornithine or arginine. These results are in agreement with the fact that the polyamine-independent cells were still deficient in the enzymes ornithine decarboxylase and agmatinase. Our findings seem to indicate the existence of a new pathway synthesize putrescine which does not involve ornithine or arginine as intermediates.
Identification and partial characterization of the human erythrocyte membrane component(s) that express the antigens of the LW blood-group system
Authors: Mallinson G , Martin P G , Anstee D J , Tanner M J A , Merry A H , Tills D , Sonneborn H H
Published: August 2015

Rhnull human erythrocytes lack the antigens of the Rhesus blood-group system, have an abnormal shape, have an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Rhnull erythrocytes also lack all antigens of the LW blood-group system, but the functional significance of this deficiency is unknown. We have identified, by immunoblotting with two mouse monoclonal antibodies (BS46 and BS56), the LW-active component(s) in normal human erythrocytes as a broad band of Mr 37 000-47 000 on SDS/polyacrylamide-gel electrophoresis. Treatment of intact human erythrocytes with endoglycosidase F preparation destroyed the epitopes recognized by antibodies BS46 and BS56, suggesting that one or more N-glycosidically linked oligosaccharides are required for the formation of the LW antigens. Estimation of the number of LW antigen sites per erythrocyte by using radioiodinated purified antibody BS46 gave average values of 4400 molecules/cell for Rh(D)-positive adult erythrocytes and 2835 molecules/cell for Rh(D)-negative adult erythrocytes. Like the Rh(D) polypeptide, the LW polypeptide(s) is (are) associated with the cytoskeleton of normal erythrocytes. These results suggest the possibility that the absence of the LW polypeptide may also contribute to the functional and/or morphological abnormalities of Rhnull erythrocytes.
Trypsin-catalysed formation of pig des-(23-63)-proinsulin from desoctapeptide-(B23-30)-insulin
Authors: Kubiak T , Cowburn D
Published: August 2015

Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, previously unreported, proinsulin analogue was determined on the basis of amino acid analysis, dansylation and digestion with Staphylococcus aureus V8 proteinase. Receptor-binding ability of des-(23-63)-proinsulin was 20% of that of pig desoctapeptide-(B23-30)-insulin and 0.02% of that of pig insulin.
Analysis of Hill slopes predicted by the four-ligand exponential model for a regulatory enzyme
Authors: Ainsworth S
Published: August 2015

The four-ligand exponential model for a regulatory enzyme is described as it is applied to kinetic studies of yeast pyruvate kinase in which the concentrations of four ligands are systematically varied. The Hill slopes predicted by this model are calculated for the two situations in which the fourth ligand is either a substrate or an effector. It is shown that the individual terms that make up the expression for the Hill slope assist the interpretation of the observed behaviour in terms of the constants employed by the model.
Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown
Authors: Guillon G , Gallo-Payet N
Published: August 2015

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.
Nucleotide sequence of the putative regulatory region of mouse lactate dehydrogenase-A gene
Authors: Fukasawa K M , Li S S L
Published: August 2015

The nucleotide sequence of approx. 3 kilobases including the regulatory region, a non-coding exon and the first protein-coding exon from mouse lactate dehydrogenase-A (LDH-A) gene has been determined. The putative initiation sites of transcription and translation were deduced by comparing the nucleotide sequence of mouse LDH-A gene with those of a mouse LDH-A processed pseudogene and the LDH-A full-length cDNAs from rat and human. The tentative TATA and CAAT boxes, and the hexanucleotides CCGCCC have been identified. The sequence of AAATCTTGCTCAA of mouse LDH-A gene has also been found to show striking homology to the cyclic AMP-responsive sequences of eukaryotic genes regulated by cyclic AMP. It has been reported previously that the protein-coding sequence of mouse LDH-A gene is interrupted by six introns and the 3′ untranslated sequence of 485 nucleotides is not interrupted [Li, Tiano, Fukasawa, Yagi, Shimiza, Sharief, Nakashima & Pan (1985) Eur. J. Biochem. 149, 215-225]. An additional intron of 1653 base-pairs was found in the 5′ untranslated sequence of 101 nucleotides at 24 nucleotides upstream to the translation start site. Thus, mouse LDH-A gene containing seven introns spans approx. 11 kilobases and its length of mature mRNA is 1582 nucleotides, excluding the poly(A) tail.
Target size of the adenosine Ri receptor
Authors: Frame L T , Yeung S M , Venter J C , Cooper D M F
Published: August 2015

The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes.
Conformational changes induced by polyanions in haemoglobin from Camelus dromedarius. Studies on the ferric derivatives
Authors: Santucci R , Ascoli F , Amiconi G , Brunori M
Published: August 2015

The effect of inositol hexakisphosphate on the redox equilibria and on the c.d. spectra of ferric derivatives of haemoglobin from Camelus dromedarius has shown that: two distinct functionally relevant binding sites for polyanions are present on the protein; conformational changes promoted by inositol hexakisphosphate are largely dependent on spin state of the iron; tertiary and quaternary changes are not necessarily linked; structures induced by polyanions can be mixed forms that are neither T-state nor R-state.
Metabolism of the diacetyl derivatives of stereoisomeric monoacyl-sn-glycerols by rat adipocytes in vitro
Authors: Christie W W , Hunter M L
Published: August 2015

Diacetyl long-chain 1(3)- and 2-acyl-sn-glycerols containing either [9,10-3H]oleic acid or [1-14C]palmitic acid were synthesized by partial hydrolysis of the corresponding labelled triacylglycerols and acetylation. They were obtained in a high degree of stereochemical purity by preparative h.p.l.c. on a column containing a diol bonded phase. Each compound was rapidly metabolized by adipocyte preparations in vitro, and a high proportion of the label was recovered in the unesterified fatty acid and triacylglycerol fractions. Negligible amounts of intermediate products of hydrolysis were detected. Triacylglycerols were formed from [9,10-3H]oleic acid and from diacetyl-1(3)-[9,10-3H]oleoyl glycerol precursors at about the same rate, but the 2-isomer was metabolized rather more slowly. The results were consistent with the hypothesis that essentially complete hydrolysis occurred in the medium or at the plasma membrane, through the actions of lipoprotein lipase and monoacylglycerol lipase, and that subsequent esterification took place within the cell. To confirm that no putative intermediate monoacylglycerols were utilized for triacylglycerol biosynthesis via the monacylglycerol pathway, the positional distributions of fatty acids in triacylglycerols from each substrate were determined. No positional selectivity was observed. It was concluded that monoacylglycerols, of an origin exogenous to the tissue, e.g. those derived from plasma triacylglycerols, were not utilized to a significant degree for triacylglycerol biosynthesis in adipose tissue. The diacetyl derivatives of monoacylglycerols may serve as useful stereochemical probes in studies of triacylglycerol biosynthesis via the monoacylglycerol pathway in other tissues.
Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen
Authors: Pollock W K , Rink T J , Irvine R F
Published: August 2015

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.
The human erythrocyte anion-transport protein. Further amino acid sequence from the integral membrane domain homologous with the murine protein
Authors: Brock C J , Tanner M J A
Published: August 2015

Sequences from the human erythrocyte anion-transport protein homologous with residues 417-449 and 794-813 of the murine erythrocyte anion-transport protein have been determined. The former sequence includes the putative transmembrane helix closest to the N-terminus of the protein. The latter sequence traverses almost all of the lipid bilayer and is located towards the C-terminus of the protein. Sites have been identified by alignment with the murine sequence in the integral membrane domain that are accessible to proteolytic enzymes. Sequences from the integral membrane domain of the erythrocyte anion-transport protein are highly conserved.
Processing and secretion in the neurohypophysis. Stability of isolated secretory vesicles and role of internal pH
Authors: Saermark T , Andersen N M , Atke A , Jones P M , Vilhardt H
Published: August 2015

A possible role of low pH in secretory vesicles for processing and secretion in the neurohypophysis was investigated. Subcellular fractionation of guinea-pig neural lobes revealed that a proton present in the membranes from this tissue could not be ascribed to secretory vesicles. However, a proton pump was found in coated microvesicles. Secretory vesicles isolated from rats and guinea pigs were stable under conditions known to lyse secretory vesicles from the adrenal medulla owing to the generation of a proton gradient. These results suggest that the internal pH of secretory vesicles from the neurohypophysis is closer to neutral than is the pH in chromaffin secretory vesicles. Processing of a neurophysin-glycopeptide intermediate from the biosynthesis of vasopressin in intact secretory vesicles incubated in vitro was activated by the addition of NH4Cl, known to increase the intravesicular pH. This activation of neurohormone processing was also apparent in isolated nerve endings incubated in the presence of NH4Cl, suggesting that NH4Cl can also be used to increase the intravesicular pH in intact nerve endings. However, NH4Cl did not affect the secretion of neurohormones, indicating that a low intravesicular pH is not important for exocytosis in the neurohypophysis. Our results indicate that a low pH generated during processing by mechanisms other than ATP-dependent proton transport may inhibit the processing enzymes, thereby preventing extensive breakdown of neurohormone precursors.
The effect of purified aminoaldehydes produced by polyamine oxidation on the development in vitro of Plasmodium falciparum in normal and glucose-6-phosphate-dehydrogenase-deficient erythrocytes
Authors: Morgan D M L , Bachrach U , Assaraf Y G , Harari E , Golenser J
Published: August 2015

Purified aminoaldehydes produced by polyamine oxidation were toxic to the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes. There was a profound effect on young ring forms, and, during maturation, parasites became more sensitive to the aldehydes. Oxidation of the aldehydes abolished the lethal effect. The plasmodia within glucose-6-phosphate-dehydrogenase (G6PD)-deficient erythrocytes were more sensitive to mono- and di-aldehydes than were parasites in normal erythrocytes. G6PD-deficient erythrocytes were also more sensitive to pretreatment with the dialdehyde produced by the oxidation of spermine. Pretreatment prevented further invasion by the parasites.
The effect of urea synthesis on extracellular pH in isolated perfused rat liver
Authors: Häussinger D , Gerok W , Sies H
Published: August 2015

In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.
Conformational changes in the bilirubin-human serum albumin complex at extreme alkaline pH
Authors: Honoré B , Frandsen P C
Published: August 2015

Light-absorption, c.d. and fluorescence of the bilirubin-albumin complex were investigated at extreme alkaline pH. Above pH 11.1 albumin binds the bilirubin molecule, twisted oppositely to the configuration at more neutral pH. On the basis of light-absorption it is shown that two alkaline transitions occur. The first alkaline transition takes place at pH between 11.3 and 11.8, co-operatively dissociating at least six protons. The second alkaline transition takes place at pH between 11.8 and 12.0. It probably implies a reversible unfolding of the albumin molecule, increasing the distance between tryptophan-214 and bilirubin, and partly exposing the liganded bilirubin to the solvent.
Detection of radical species in haematin-catalysed retinoic acid 5,6-epoxidation by using h.p.l.c.-e.p.r. spectrometry
Authors: Iwahashi H , Ikeda A , Negoro Y , Kido R
Published: August 2015

E.p.r. signals were detected in an all-trans-retinoic acid/haematin incubation mixture by using an e.p.r. spin-trapping technique. The spin adducts are presumably attributable to some intermediates in haematin-catalysed retinoic acid 5,6-epoxidation, since addition of nitrosobenzene to the reaction mixture dose-dependently inhibited the epoxidation. Analysing the reaction mixture by h.p.l.c.-e.p.r. spectrometry resulted in the detection of three peaks (III–1, III–2, IV) ascribable to the radical species. Two (peaks III–1 and −2) of the three peaks, which appeared 10 min after the reaction had started, seem to be attributable to the radical species directly participating in the epoxidation. The radicals trapped by nitrosobenzene do not appear to be derived from active oxygen, since none of these peaks were detected in a similar h.p.l.c. analysis of O2- and OH.-generating systems. They are presumably derived from retinoic acid. This view is also supported by the following results: none of these peaks were detected in the h.p.l.c. elution profile of the reaction mixture when retinoic acid was absent; peaks III-1 and 2 were detected even under anaerobic conditions, and their peak heights were unchanged under aerobic conditions.