bluuebunny/crossref_metadata_2025 huggingface dataset:
The structural basis for neutrophil inactivation of C1̅ inhibitor
Authors: Pemberton P A , Harrison R A , Lachmann P J , Carrell R W
Published: August 2015

Limited proteolysis of C1 inhibitor (C1-INH) by neutrophil elastase, Pseudomonas elastase and snake venoms resulted in initial cleavage within the molecule's N-terminus followed by further cleavage within the molecule's C-terminally placed reactive centre. N-Terminal proteolysis occurred at peptide bonds 14-15, 36-37 and 40-41. This had no effect on either the inhibitory activity or the heat-stability of C1-INH. Proteolysis within the reactive centre occurred at peptide bonds 439-440, 440-441, 441-442 and 442-443. Cleavage at any one of these sites inactivated C1-INH and conferred enhanced heat-stability upon a previously heat-labile molecule. Released neutrophil proteinases also cleaved and inactivated C1-INH, suggesting that they may physiologically regulate C1-INH during inflammatory episodes.
Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids
Authors: Honoré B , Pedersen A O
Published: August 2015

pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9.
Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures
Authors: Sinclair P , Frezza J , Sinclair J , Bement W , Haugen S , Healey J , Bonkovsky H
Published: August 2015

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.
The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides
Authors: Rhodes C J , Zumbrunn A , Bailyes E M , Shaw E , Hutton J C
Published: August 2015

Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.
Differentiation of rapid and slower-acting effects of insulin on mitochondrial processes in brown adipose tissue from streptozotocin-diabetic rats
Authors: Gualberto A , Saggerson E D
Published: August 2015

Insulin treatment of streptozotocin-diabetic rats restores the depressed palmitoyl-group oxidation observed in brown-adipose-tissue mitochondria from diabetic rats. A relatively rapid effect of insulin (5 h) to increase carnitine-dependent oxidation of palmitoyl-CoA and to increase overt carnitine palmitoyltransferase activity is differentiated from a slower effect of the hormone (1 day) to increase palmitoylcarnitine oxidation.
Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro
Authors: Holm I , Persson L , Stjernborg L , Thorsson L , Heby O
Published: August 2015

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.
Analysis of progress curves for enzyme-catalysed reactions. Automatic construction of computer programs for fitting integrated rate equations
Authors: Duggleby R G , Wood C
Published: August 2015

The computer analysis of progress curves for enzyme-catalysed reactions involves a series of mathematical and computational tasks. The three most daunting of these are the derivation of an integrated rate equation, solving this equation so that the amount of product formed by the reaction at any time can be calculated, and incorporating this solution into a non-linear-regression computer program. This paper describes the basis of a computer program that greatly simplifies the problem. The proposed mechanism is specified in the familiar kinetic constant form, which is automatically translated into a program capable of fitting this mechanism to a series of experimental progress curves. The approach is illustrated for a reversible reaction with one substrate and one product, and tested with some data obtained for the fumarase reaction. A copy of the program has been deposited as Supplementary Publication SUP 50148 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.
A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system
Authors: Azrolan N I , Coleman P S
Published: August 2015

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.
pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase
Authors: Ascenzi P , Brunori M , Coletta M , Desideri A
Published: August 2015

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.
Phosphorylation-dependent and -independent pathways of platelet aggregation
Authors: Watson S P , Hambleton S
Published: August 2015

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.
Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae
Authors: Gemoets J P , Bravo M , McKenna C E , Leigh G J , Smith B E
Published: August 2015

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.
Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane
Authors: Shioi J , Naito S , Ueda T
Published: August 2015

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5′-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.
Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase
Authors: Salam W H , Wilcox H G , Cagen L M , Heimberg M
Published: August 2015

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.
Developmental regulation of murine mammary-gland 90 kDa heat-shock proteins
Authors: Catelli M G , Ramachandran C , Gauthier Y , Legagneux V , Quelard C , Baulieu E E , Shyamala G
Published: August 2015

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.
Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization
Authors: Grant D , Long W F , Williamson F B
Published: August 2015

Of a range of glycosaminoglycans, heparin and heparan sulphate were the most effective inhibitors in vitro of CaCO3 (calcite) crystallization as assayed by conductimetric measurements. The possible role of such glycosaminoglycans in modulating calcium-salt crystallizations in vivo is discussed.
Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers
Authors: Sharma R J , Woods N M , Cobbold P H , Grant D A W
Published: August 2015

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.
Ba2+ stimulation of luteinizing-hormone release demonstrates two mechanisms of Ca2+ entry in gonadotrope cells
Authors: Smith C E , Davidson J S , Millar R P
Published: August 2015

Kinetic studies on gonadotropin-releasing-hormone (gonadoliberin, GnRH)-stimulated luteinizing-hormone (lutropin, LH) release in the cultured rat gonadotrope demonstrated a biphasic pattern of LH release. The first rapid phase of release was unaffected by the voltage-gated Ca2+-channel blockers methoxyverapamil (D600) and nifedipine [a dihydropyridine (DHP)], whereas the later second phase was partially inhibited by both drugs. These results suggested that the initial phase of LH release is independent of Ca2+ entry through dihydropyridine (DHP)-sensitive Ca2+ channels and might depend on entry of extracellular Ca2+ by another mechanism. These mechanisms were further studied by utilizing Ba2+ as a Ca2+ substitute. Ba2+, which freely permeates DHP-sensitive Ca2+ channels in the absence of GnRH, induced LH release which was sensitive to blockade by D600 and nifedipine. However, in the presence of the channel blockers, Ba2+-induced LH release could be elicited when GnRH was added to the system. This indicates that GnRH stimulates LH release by initially activating a DHP-insensitive Ca2+-entry mechanism and then a DHP-sensitive mechanism. The DHP-sensitive mechanism freely allows Ba2+ entry in the absence of GnRH-receptor occupancy, whereas the DHP-insensitive mechanism requires GnRH-receptor activation for Ba2+ entry.
Reaction of 3-dehydroecdysone with certain n.m.r. solvents
Authors: Howarth O W , Thompson M J , Rees H H
Published: August 2015

Synthetically prepared 3-dehydroecdysone shows by n.m.r. spectroscopy a mixture of two and three components in 2H2O and [2H4]methanol respectively; only 3-dehydroecdysone is indicated in [2H5]pyridine. Although 3-dehydroecdysone is the sole component in [2H5]pyridine, it represents only 62% and 55% in 2H2O and [2H4]methanol respectively. Evidence indicates that the other component in 2H2O is a 3-[2H2]hydrate of 3-dehydroecdysone, and that in [2H4]methanol the other two components are isomeric [2H3]hemiacetals of 3-dehydroecdysone.
Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein
Authors: Tatham A S , Drake A F , Shewry P R
Published: August 2015

C hordein, a storage protein from barley grains, has an Mr of about 53,000, and consists predominantly of repeated octapeptides with a consensus sequence of Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. Previously reported hydrodynamic and c.d. studies indicate the presence of beta-turns, the repetitive nature of which may lead to the formation of a loose spiral. In order to study these turns we have compared the structures of a synthetic peptide corresponding to the consensus repeat motif and total C hordein by using c.d. and Fourier-transform i.r. spectroscopy. The synthetic peptide exhibited spectra typical of beta I/III reverse turns when dissolved in trifluoroethanol at 22 degrees C and in water at 70 degrees C, but ‘random-coil’-like spectra in water at 22 degrees C. The whole protein also showed increases in beta I/III reverse turns when dissolved in increasing concentrations of trifluoroethanol (50-100%, v/v) or heated in ethanol/water (7:3, v/v). Two cryogenic solvent systems were used to determine the c.d. spectra of the peptide and protein at temperatures down to -100 degrees C. Methanol/glycerol (9:1, v/v) and ethanediol/water (2:1, v/v) were selected as analogues of trifluoroethanol/water and water respectively. The peptide exhibited beta I/III-reverse-turn and ‘random-coil’-like spectra in methanol/glycerol and ethanediol/water respectively at 22 degrees C, but a spectrum similar to that of a poly-L-proline II helix in both solvents at -100 degrees C. Similarly the proportion of this spectral type also increased when the whole protein was cooled in both solvents. These results indicate that a poly-L-proline II conformation at low temperatures is in equilibrium with a beta I/III-turn-rich conformation at higher temperatures. The latter conformation is also favoured in solvents of low dielectric constant such as trifluoroethanol. The ‘random-coil‘-like spectra exhibited by the protein and peptide in high-dielectric-constant solvents at room temperature may result from a mixture of the two conformations rather than from the random-coil state.
Iodothyronine 5′-deiodinase activity as an early event of prenatal brown-fat differentiation in bovine development
Authors: Giralt M , Casteilla L , Viñas O , Mampel T , Iglesias R , Robelin J , Villarroya F
Published: August 2015

Iodothyronine 5'-deiodinase activity appears to be a type I enzyme in bovine brown adipose tissue, on the basis of its high Km for 3,3',5'-tri-iodothyronine (‘reverse T3’) (in the micromolar range) and sensitivity to propylthiouracil inhibition. This enzyme activity is already detectable in perirenal adipose tissue of bovine fetuses in the second month of gestation, reaches peak values around the seventh month of fetal life, declines before birth, becomes lower after parturition and finally undetectable in the adult cow. Iodothyronine 5'-deiodinase activity is present in the pericardic, peritoneal and intermuscular adipose depots of the neonatal calf, but it is always undetectable in the subcutaneous adipose tissue. It is concluded that iodothyronine 5'-deiodinase is a specific feature of brown fat in the bovine species that is not shared by white adipose tissue. white adipose tissue. Peak values of 5'-deiodinating activity appear as an early event in the prenatal differentiation programme of bovine brown-fat cells as they occur when uncoupling-protein-gene expression first starts.
A gated 31P-n.m.r. study of bioenergetic recovery in rat skeletal muscle after tetanic contraction
Authors: Challiss R A , Blackledge M J , Shoubridge E A , Radda G K
Published: August 2015

1. Gated 31P-n.m.r. spectra were obtained from the ankle flexor muscles of the rat at various times after 3 s isometric tetanic contraction. This allowed the time course of changes in phosphocreatine (PCr), Pi and free ADP concentrations and intracellular pH to be monitored in skeletal muscle in vivo with 1 s time resolution. 2. ATP concentration did not change significantly, either during the recovery from a 3 s tetanus or during the overall protocol. 3. The calculated rate of recovery of ADP towards pre-stimulation levels was very rapid (t1/2 less than 5 s). The rate of Pi disappearance (t1/2 = 14 s) was more rapid than the rate of PCr synthesis (t1/2 = 24 s), resulting in a significant transient decrease in n.m.r.-visible PCr + Pi between 25 and 45 s after tetanic contraction. 4. The rates of PCr, Pi and ADP recovery are higher than those previously reported for recovery from steady-state exercise in humans or twitch isometric contraction in animals.
Lactoperoxidase from human colostrum
Authors: Langbakk B , Flatmark T
Published: August 2015

The present study has confirmed that human colostrum contains a lactoperoxidase (EC 1.11.1.7) [Langbakk & Flatmark (1984) FEBS Lett. 174, 300-303], which represents about 0.004% of the total protein in crude colostrum. An apparent 32-fold purification of the enzyme was obtained by a multistep procedure, as modified from that of the bovine enzyme, with a recovery of about 7%. By use of chromatography on an immunoaffinity column (directed against bovine lactoperoxidase B), an apparent 1450-fold purification was obtained in a single step, with a recovery of 21%. The enzyme behaved as a glycoprotein (binding to concanavalin A-Sepharose), and revealed spectral properties (Soret peak at 412 nm) and an Mr (80,000) similar to those of the bovine enzyme.
Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium
Authors: Taylor C W , Berridge M J , Cooke A M , Potter B V L
Published: August 2015

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.
Transfection of insulin-producing cells with a transforming c-Ha-ras oncogene stimulates phospholipase C activity
Authors: Berggren P O , Hallberg A , Welsh N , Arkahammar P , Nilsson T , Welsh M
Published: August 2015

Pancreatic islet beta-cells and insulin-producing RINm5F cells were electroporated in the presence of the c-Ha-ras oncogene, to assess the possible involvement of the encoded product in coupling extracellular receptors to phospholipase C. After two days the c-Ha-ras-transfected cells increased their expression of c-Ha-ras mRNA. These cells were also found to contain more [3H]InsP3, suggesting an increased basal (non-ligand-activated) phospholipase C activity. In addition, the transfected cells were unable to respond to ligand (bombesin) activation of phospholipase C. The ras-transfected insulin-producing cells showed enhanced phosphorylation of a 200 kDa substrate crossreacting with an antibody to an 80 kDa protein kinase C substrate. The phorbol ester 12-O-tetradecanoyl 13-acetate and bombesin also induced phosphorylation of the 200 kDa substrate. All of these changes occurred without changes in the rates of [3H]thymidine incorporation. The results suggest that the mutated c-Ha-ras oncogene directly or indirectly stimulates the basal phospholipase C activity of these cells.
The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step
Authors: Rigney E , Mantle T J , Dickinson F M
Published: August 2015

When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a ‘burst’ was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the ‘burst’. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state ‘burst’ reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.
N.m.r. assignments and temperature-dependent conformational transitions of a mutant trp operator-promoter in solution
Authors: Lane A N
Published: August 2015

A total of 145 protons in the mutant trp operator-promoter sequence CGTACTGATTAATCAGTACG were assigned by one-dimensional and two-dimensional n.m.r. methods. Except at the sites of mutation (underlined), the chemical shifts and other n.m.r. parameters are very similar to those observed in the symmetrized wild-type sequence [Lefèvre, Lane & Jardetzky (1987) Biochemistry 26, 5076-5090]. Spin-spin-relaxation rate constants of the resolved base protons and intra- and inter-nucleotide nuclear-Overhauser-enhancement intensities argue for a sequence-dependent structure similar to that of the wild-type, except at and close to the sites of the mutation. The overall tumbling time as a function of temperature was determined from cross-relaxation rate constants for the H-6-H-5 vectors of the four cytosine residues. The values are consistent with the oligonucleotide maintaining a double-helical conformation over the entire temperature range 5-45 degrees C, and that internal motions of the bases are of small amplitude on the subnanosecond time scale. The temperature-dependence of chemical shifts, spin-spin-relaxation rate constants and cross-relaxation rate constants show the occurrence of two conformational transitions localized to the TTAA sequence in the centre of the molecule. The thermodynamics of the transition at the lower temperature (tm = 16 degrees C) were analysed according to a two-state process. The mid-point temperature is about 6 degrees C higher than in the wild-type sequence. The conformational transition does not lead to rupture of the Watson-Crick hydrogen bonds, but probably involves changes in the propellor twists of T.A-9 and T.A-10. The second transition occurs at about 40 degrees C, but cannot be fully characterized. This conformational variability seems to be a property of the sequence TTAA, and may have functional significance in bacterial promoters.
The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration
Authors: Zawalich W S , Zawalich K C , Rasmussen H
Published: August 2015

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.
Identification and characterization of parathyroid hormone receptors on dog kidney, human kidney, chick bone and human dermal fibroblast. A comparative study of functional and structural properties
Authors: Pun K K , Ho P W M
Published: August 2015

To evaluate the functional and structural characteristics of the parathyroid hormone (PTH) receptors on different tissues and the possible heterogeneity in structure and function, PTH receptors on dog kidney membrane, human kidney membrane, chick bone cell membrane and human dermal fibroblast membrane were evaluated. The results showed that human kidney plasma membrane, canine kidney plasma membrane and chick bone cell membrane possess one single class of PTH receptor with a Kd (dissociation constant) of 1-5 nM and an IC50 also of 1-5 nM. The number of binding sites was 800 fmol per mg of protein for chick bone cell particulate membrane, 1-5 pmol per mg of protein for human kidney plasma membrane and 2.2 pmol per mg of protein for dog kidney plasma membrane. Photoaffinity labelling identified a major binding component with a molecular mass of 70 kDa in all three types of membrane. The plasma membrane fraction from human dermal fibroblast contained two different binding sites for PTH with high (Kd = 2 nM) and low (Kd = 580 nM) affinities respectively. The IC50 for the adenylate cyclase is about 2 nM, which is similar to the Kd of the high-affinity site. Photoaffinity labelling also demonstrated a major binding component with a molecular weight of 70 kDa. We conclude that structural and functional similarity exists among the PTH receptors present on chick bone cell membrane, dog kidney membrane and human kidney membrane. The human dermal fibroblast possesses two different binding sites, one of which is coupled to adenylate cyclase.
Activation and phosphorylation of the ‘dense-vesicle’ high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase
Authors: Kilgour E , Anderson N G , Houslay M D
Published: August 2015

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity ‘dense-vesicle’ cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the ‘dense-vesicle’ phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the ‘dense-vesicle’ phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the ‘dense-vesicle’ phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.
Interaction of chicken cystatin with inactivated papains
Authors: Björk I , Ylinenjärvi K
Published: August 2015

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido)butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.
Phosphoenolpyruvate carboxykinase from the moderate halophile Vibrio costicola. Purification, physicochemical properties and the effect of univalent-cation salts
Authors: Salvarrey M S , Cannata J J B , Cazzulo J J
Published: August 2015

Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophilic bacterium Vibrio costicola. The enzyme is monomeric, with an Mr of 62,000, as determined by the Svedberg equation, by using values of s0(20,w) 4.4 x 10(-13) s, D20,w 6.13 x 10(-7) cm2.s-1 and v 0.719 cm3.g-1. Compared with other, non-halophilic, PEPCKs, the enzyme from V. costicola had a significantly lower total content of hydrophobic amino acids. The contents of glycine and serine were higher in the V. costicola enzyme (16.7 and 10.22% respectively) than in the non-halophilic PEPCKs (6.8-9.6% and 4.67-6.28% respectively). These results resemble those obtained by De Médicis & Rossignol [(1979) Experientia 35, 1546-1547] with the pyruvate kinase from V. costicola, and agree with the proposal by Lanyi [(1974) Bacteriol. Rev. 38, 272-290] of partial replacement of hydrophobic amino acids by glycine and serine to maintain the balance between hydrophobic and hydrophilic forces in halophilic enzymes. In agreement with this ‘halophilic’ characteristic, the PEPCK was somewhat stabilized by 1 M-KCl or -NaCl and by 20% (v/v) glycerol, and its oxaloacetate-decarboxylation and 14CO2-oxaloacetate-exchange reactions were activated by KCl and NaCl up to 1 M, whereas the fixation of CO2 on PEP had a maximum at 0.025-0.05 M salt. These facts suggest that the salts, at concentrations probably physiological for the bacterium, increase the formation of the complex of oxaloacetate and ATP with the enzyme, and the liberation of the products, PEP and ADP, thus favouring PEP synthesis.
Structural and immunological studies of keratan sulphates from mature bovine articular cartilage
Authors: Thornton D J , Morris H G , Cockin G H , Huckerby T N , Nieduszynski I A , Carlstedt I , Hardingham T E , Ratcliffe A
Published: August 2015

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.
Synthesis of 2-aminoethylarsonic acid. A new synthesis of primary
Authors: Geoghegan K F , Dixon H B
Published: August 2015

2-Aminoethylarsonic acid was prepared from 2-choloethylarsonic acid. The route constitutes a new procedure for making primary amines from haloalkanes; chloride was displaced by treatment with 2-aminoethanol at 70 degrees C, and the product was converted into the required primary amine by treatment with periodate.