bluuebunny/crossref_metadata_2025 huggingface dataset:
A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases
Authors: Pâquet M R , Moscarello M A
Published: August 2015

UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase was partially purified from rat liver Golgi membranes and rat serum. The kinetic parameters of the two enzymes isolated by affinity chromatography were compared with each other and with those for commercial bovine milk galactosyltransferase. When N-acetyl-glucosamine was the acceptor the Km values for UDP-galactose were 65,52 and 43 microM for the rat liver Golgi, rat serum and bovine milk enzymes respectively. The Km values for N-acetylglucosamine were 0.33, 1.49 and 0.5 mM for the three enzymes respectively. The Km values for UDP-galactose, with glucose as acceptor in the presence of 1 mg of alpha-lactalbumin, were 23, 9.0 and 60 microM for the three enzymes respectively, and the Km values for glucose were 2.3, 1.8 and 2.0 mM respectively. The effects of alpha-lactalbumin in both the lactosamine synthetase and lactose synthetase reactions were similar. The activation energies were 94.0 kJ/mol (22.5 kcal/mol) and 96.0 kJ/mol (22.9 kcal/mol) for the Golgi and serum enzymes respectively. Although some differences in Km values were observed between the rat liver Golgi and serum enzymes, the values obtained suggest a high degree of similarity between the kinetic properties of the three galactosyltransferases.
d-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea
Authors: Keller U , Zocher R , Krengel U , Kleinkauf H
Published: August 2015

A D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea was purified about 145-fold. The enzyme was able to catalyse both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Both reactions were also catalysed to a decreased but significant extent with respect to dihydrolysergic acid. The molecular mass of the enzyme was estimated to lie between 135 and 140 kDa. The involvement of the enzyme in the biosynthesis of ergot peptide alkaloids is discussed.
The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA
Authors: Gianazza E , Emerson D L , Dykes D , Arnaud P
Published: August 2015

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a ‘pseudoligand’ for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.
Copper-phenanthroline-induced site-specific oxygen-radical damage to DNA. Detection of loosely bound trace copper in biological fluids
Authors: Gutteridge J M C
Published: August 2015

Copper(II) ions, in the presence of 1,10-phenanthroline, O2 and a reducing agent, degrade DNA with the release of thiobarbituric-acid-reactive material. This reaction, dependent on the formation of oxygen radicals, was made the basis of a sensitive and specific assay for loosely bound copper in body fluids. When applied to certain extracellular fluids, trace amounts of copper could be detected in the lower micromolar range.
Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate
Authors: Barankiewicz J , Cohen A
Published: August 2015

The catabolism of GTP and ATP in intact human thymocytes was studied, and the effect of intracellular Pi concentrations on various catabolic reactions was evaluated. Induction of nucleotide catabolism was performed with either NaN3 or deoxyglucose. NaN3 induced an increase in intracellular Pi, whereas deoxyglucose induced a decrease in Pi in human thymocytes. At elevated Pi concentrations, an intracellular accumulation of GMP, IMP and AMP was observed, and the entry of these nucleotides into the nucleoside cycles was diminished. In contrast, at lowered Pi concentrations, there was no accumulation of nucleoside monophosphates, but the production of purine bases and nucleosides was increased as a result of enhanced nucleotide entry into the nucleoside cycles. The dephosphorylation of nucleotides thus seems to be regulated by intracellular Pi concentrations. ATP catabolism proceeded mainly via adenylate deamination at both elevated and lowered Pi concentrations. Guanylate formed during GTP catabolism was mainly dephosphorylated, but significant amounts of GMP were also deaminated to yield IMP.
Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature
Authors: Mackenzie N E , Malthouse J P , Scott A I
Published: August 2015

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.
Regulation of arylsulphatase A and sulphogalactolipid turnover by cortisol in myelinogenic cultures of cells dissociated from embryonic mouse brain
Authors: Stephens J L , Pieringer R A
Published: August 2015

Myelinogenic cultures of cells dissociated from embryonic mouse brain were used to study the regulation of myelination-associated molecules by cortisol. Cortisol in physiological concentrations (0.03 microM) caused an increased accumulation of myelination-associated sulphogalactolipids. It also stimulated the myelin- and oligodendroglia-specific cyclic nucleotide phosphohydrolase. The increase in sulphogalactolipid content was caused by a cortisol-concentration-dependent inhibition in arylsulphatase A activity and not by an increase in either cerebroside sulphotransferase activity or an increase in availability of adenosine 3′-phosphate 5′-phosphosulphate. Of several steroid hormones tested only the glucocorticoid types brought about these changes. The relationship between net sulphogalactolipid accumulation and arylsulphatase A inhibition induced by cortisol was confirmed by sulphogalactolipid turnover studies. Depending on whether a single-phase or a two-phase decay calculation is used, the turnover of sulphogalactolipid with cortisol present was decreased at 22 days in culture by either 62% or 65% respectively of that without cortisol. This decrease in turnover can be attributed completely to the decrease of arylsulphatase activity by cortisol to 63% of the value for normal cells grown under the same conditions.
The inhibition of glucose-stimulated insulin secretion by primary amines. A role for transglutaminase in the secretory mechanism
Authors: Bungay P J , Potter J M , Griffin M
Published: August 2015

Rat pancreatic islets contain a Ca2+-activated and thiol-dependent transglutaminase (EC 2.3.2.13) comparable in activity with that found in rat liver, lung and spleen. The Ca2+-dependence of this enzyme is such that half-maximal velocity was obtained in the region of 40 microM. Preincubation of rat islets with primary-amine substrates of transglutaminase (monodansylcadaverine, methylamine, ethylamine, propylamine and cystamine) led to an inhibition of glucose-stimulated insulin release by these amines. Kinetic analysis of the competitive substrates methylamine, monodansylcadaverine, propylamine and ethylamine for their ability to inhibit islet transglutaminase activity indicated a potency that matched their ability to inhibit glucose-stimulated insulin release. When these amines were tested for their effects on glucose-stimulated protein synthesis and glucose utilization, the most potent inhibitor of insulin release, monodansylcadaverine, had no effect on either process at 100 microM. The amines cystamine, ethylamine, methylamine and propylamine had variable effects on these metabolic processes. For ethylamine, methylamine and propylamine, concentrations were found which inhibited glucose-stimulated insulin release in a manner which was found to be independent of their effects on either glucose oxidation or protein synthesis. Primary amines may therefore inhibit insulin release through their incorporation by islet transglutaminase into normal cross-linking sites. A role for protein cross-linking in the secretory mechanism is suggested.
Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain
Authors: Raina A , Hyvönen T , Eloranta T , Voutilainen M , Samejima K , Yamanoha B
Published: August 2015

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5′)-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5′)-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5′-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.
Measurement of the oxidation-reduction potentials for one-electron and two-electron reduction of electron-transfer flavoprotein from pig liver
Authors: Husain M , Stankovich M T , Fox B G
Published: August 2015

Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.
Simultaneous formation of 2- and 4-quinolones from quinolinium cations catalysed by aldehyde oxidase
Authors: Taylor S M , Stubley-Beedham C , Stell J G P
Published: August 2015

Quinolinium salts were incubated with partially purified aldehyde oxidase, and the products were separated by high-pressure liquid chromatography and fully characterized by u.v. spectroscopy, i.r. spectroscopy and mass spectrometry. Oxidation of N-methylquinolinium salts with either rabbit or guinea-pig liver aldehyde oxidase in vitro gave two isomeric products, N-methyl-4-quinolone and N-methyl-2-quinolone. Incubation of N-phenylquinolinium perchlorate similarly yielded two oxidation products, N-phenyl-4-quinolone and N-phenyl-2-quinolone. The ratio of 2- to 4-quinolone production was species-dependent, the proportion of 4-quinolone with the guinea-pig enzyme being greater than that obtained with the rabbit liver enzyme. Kinetic constants were determined spectrophotometrically for both the quinolinium salts and a number of related quaternary compounds. In general, quaternization facilitated oxidation of a substrate, but a number of exceptions were noted, e.g. N-methylisoquinolinium and N-methylphen-anthridinium. Km values varied with the nature of electron acceptor employed, and this difference was more marked for quaternary substrates than the unquaternized counterparts. The product ratio obtained from N-methylquinolinium salts was found to be constant under various conditions, including purification of the enzyme and the use of either induced or inhibited aldehyde oxidase, but a change in the ratio was found at high pH values and in the presence of a competing substrate, N-methylphenanthridinium. This may indicate that a quaternary substrate binds to aldehyde oxidase in two alternative positions.
Radioimmunoassay of soluble and insoluble collagenases in fibrotic liver
Authors: Takahashi S , Koda K
Published: August 2015

We have postulated that an insufficient active of collagenase relative to increased collagen synthesis may be the cause of the increased collagen accumulation in fibrotic tissues. In the present study, 125I-collagenase and rabbit anti-collagenase immunoglobulin G were used to develop a sensitive radioimmunoassay that detects 0.1 nM (3 ng) of collagenase protein in tissue samples. The assay also can detect collagenase protein that is associated with extracellular-matrix collagen fibrils. Good correlation with an assay of enzyme activity validates the radioimmunoassay for quantification of collagenase. The assay was used to measure amounts of collagenase in relation to fibrotic processes in livers of mice with schistosomiasis. Results indicate that the amounts of collagenase relative to synthesized collagens were significantly lower, and this may contribute to the progressive fibrosis. The occurrence of a maximum amount of collagenase at 7 weeks after infection with Schistosoma mansoni cercariae in concanavalin A-treated animals, as compared with 8 weeks in controls, could account for the large remission of fibrosis in mice so treated. The results emphasize the possible importance of collagenase in controlling or limiting fibrosis.
A mass-spectrometric investigation of the mechanism of the semisynthetic transformation of pig insulin into an ester of insulin of human sequence
Authors: Rose K , Gladstone J , Offord R E
Published: August 2015

In the trypsin-mediated semisynthetic transformation of pig insulin into an ester of insulin of human sequence, the B30 alanine residue of the pig hormone is replaced by an ester of threonine. The mechanism of this reaction was investigated by carrying out the transformation in a medium containing water enriched with 18O. Subsequent analysis by combined g.l.c.-mass spectrometry demonstrated that the oxygen isotope, is incorporated into the B29 carbonyl group of the insulin ester product. This result, together with those of appropriate controls, supports the conclusion that the transformation occurs, in the system studied, by a mechanism involving hydrolysis followed by coupling, and not by direct transpeptidation as has been previously found the case for another such system [Markussen & Schaumberg (1983) in Peptides 1982 (Bláha & Malon, eds.), pp. 387-394, W. de Gruyter, Berlin and New York].
Sequence comparison of pepsin-resistant segments of basement-membrane collagen α 1(IV) chains from bovine lens capsule and mouse tumour
Authors: Schuppan D , Glanville R W , Timpl R , Dixit S N , Kang A H
Published: August 2015

The C-terminal peptic fragment P1 (about 518 amino acid residues) of bovine lens-capsule collagen alpha 1(IV) chain was cleaved with CNBr and trypsin. The peptides were purified and characterized, allowing their ordering within the P1 fragment by comparison with a corresponding section of mouse collagen alpha 1(IV) chain [Schuppan, Glanville & Timpl (1982) Eur. J. Biochem. 123, 505-512]. About 67% of the sequence of bovine collagen fragment P1 was determined by Edman degradation. Comparison with the sequence of the corresponding mouse collagen fragment P1 showed 76% identity for positions Xaa and Yaa of the triplet structures Gly-Xaa-Yaa. Invariance was found for the positions of two non-triplet interruptions and of 3-hydroxyproline residues, pointing to the functional importance of these structures.
Formamide as a substrate of xanthine oxidase
Authors: Morpeth F F , George G N , Bray R C
Published: August 2015

Formamide is a substrate of xanthine oxidase. At pH 8.2 and 1.14 mM-O2, Vmax.(app.) is 3.1 s-1 and Km (app.) is 0.7 M. Mo(V) e.p.r. signals obtained by treating the enzyme with formamide were studied, and these provide new information about the ligation of molybdenum in the enzyme and about the enzymic mechanism. The substrate is the first compound that is not a nitrogen-containing heterocycle to give a Very Rapid signal. This supports the hypothesis that the Very Rapid signal, though it is not detectable with all substrates, represents an essential intermediate in turnover. Formamide also gives the Inhibited signal and is the first non-aldehyde substrate to do so. The Rapid type 1 signal obtained in the presence of formamide was examined in H2O enriched with 2H or with 17O. The single oxygen atom detectable in the signal is shown to be strongly and anisotropically coupled. This indicates that this atom remains as an oxo ligand of molybdenum in this signal-giving species. Other structural features of this species are discussed.
The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues
Authors: Wagenmakers A J M , Schepens J T G , Veldhuizen J A M , Veerkamp J H
Published: August 2015

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.
Effects of verapamil on lipolysis due to dibutyryl cyclic AMP
Authors: Goko H , Takashima S , Shimizu S , Kagawa S , Matsuoka A
Published: August 2015

The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.
Isolation and characterization of the two giant secretory proteins in salivary gland of Chironomus tentans
Authors: Rydlander L
Published: August 2015

The two giant secretory proteins, sp-Ia and sp-Ib, in salivary-gland cells of the larva of the fly Chironomus tentans, were isolated by preparative gel electrophoresis and characterized chemically. Their amino acid compositions are dominated by polar amino acids, with about 30% of basic amino acid residues. Crossed immunoelectrophoresis of sp-Ia and sp-Ib provided evidence that they share antigenic determinants. They also have major methionine-containing tryptic peptides in common. CNBr cleavage of sp-Ib gives a small number of low-Mr fragments, indicating that this protein has a repetitive structure.
Salt-induced inhibition of the precipitin reaction of concanavalin A with polysaccharides and glycoprotein
Authors: Salahuddin A , Begum R , Averill B K
Published: August 2015

The time course of the precipitin reactions of concanavalin A with glycogen, dextran and ovalbumin was investigated by a light-scattering method near 30 degrees C in 10 mM-Tris/HCl buffer, pH 7.4, containing neutral salts, i.e. NaCl, KCl, NaBr, KI and NaClO4. With 0.8 microM-lectin and 0.36 mg of glycogen/ml, the half-life, t 1/2, of the precipitin reaction was independent of salt concentration between 0.1 M and 1.5 M, and was the same (175s) in the presence of NaCl, KCl, NaBr and KI but was significantly (27%) higher in NaClO4. In contrast, the five salts caused significant to marked enhancement in t 1/2 for the reactions of concanavalin A with dextran and ovalbumin. Likewise, whereas the turbidity produced in 1 h as a result of lectin-glycogen precipitation remained unchanged, those measured for the binding of dextran and ovalbumin were decreased in the presence of three salts. The increase in t 1/2 and decrease in turbidity were found to be higher with NaClO4, followed by KI; NaBr produced moderate and NaCl (or KCl) small but generally significant inhibition of the precipitin reactions with dextran and ovalbumin. The results showed that the lectin-ligand precipitin reactions involve salt-sensitive polar interactions that are less pronounced with compactly folded ligands such as glycogen.
Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone
Authors: Davidson J M , Shibahara S , Schafer M P , Harrison M , Leach C , Tolstoshev P , Crystal R G
Published: August 2015

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.
Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain α-saturated polyprenyl monophosphate
Authors: Lang W C
Published: August 2015

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.
AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia
Authors: Doussière J , Vignais P V
Published: August 2015

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3′-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.
Specific inactivation of the phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system by diethyl pyrocarbonate
Authors: Arion W J , Burchell B , Burchell A
Published: August 2015

We have examined the interactions of the histidine-specific reagent diethyl pyrocarbonate (DEPC) with the components of the rat hepatic glucose-6-phosphatase system (EC 3.1.3.9). DEPC is the first known reagent that satisfies the criteria of an active-site-specific label for the phosphohydrolase component. (a) It inactivates through formation of a stable covalent bond. (b) It is effective at reasonably low concentrations (2-4 mM) under relatively mild conditions (e.g. 30 degrees C at neutral pH). (c) Inactivation is substantially blocked by glucose 6-phosphate, Pi and NaF, compounds which are known to interact quite specifically with the phosphohydrolase. (d) Under conditions where glucose 6-phosphate and NaF protect the enzyme, no protection is provided against DEPC-mediated inactivation of two other functional components of the membrane, the glucose 6-phosphate translocase and UDP-glucuronyltransferase. DEPC also shows potential for use at 0 degree C as a label for UDP-glucuronyltransferase.
Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase
Authors: Scott R , Yeoh G C T
Published: August 2015

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.
The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle
Authors: Challiss R A J , Arch J R S , Newsholme E A
Published: August 2015

Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.
Precursors of storage proteins in Lupinus angustifolius
Authors: Gayler K R , Boadle B G , Snook M , Johnson E D
Published: August 2015

The proteins that are synthesized during differentiation and development in the cotyledons of Lupinus angustifolius L. were characterized both in situ and after purification. The proteins present in situ were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and subjected to ‘Western’-blot analysis to identify immunologically related polypeptides. The major storage proteins of the lupin, conglutins alpha and beta, were both present in juvenile tissue only as higher Mr precursors. For conglutin beta, a family of at least three polypeptides of Mr 66 000-72 000 accumulated during the earliest phases of protein synthesis in the developing cotyledon (20-28 days after flowering). Later in development each of these polypeptides disappeared and there was the concurrent appearance in the cotyledon of the lower-Mr fragments characteristic of mature conglutin beta. For conglutin alpha, an equivalent family of precursor polypeptides of Mr 60 000-83 000 was detected. Multiple internal sites for proteolytic cleavage of all these precursors appeared to be present. However, processing of the precursors was sufficiently slow to allow them to accumulate to over 50% of total soluble protein in juvenile tissue. The precursors were purified by column chromatography under non-dissociating conditions and shown by ultracentrifugation to be multimeric proteins with Mr values in the range 150 000-200 000.
Binding of the asymmetric forms of acetylcholinesterase to heparin
Authors: Brandan E , Inestrosa N C
Published: August 2015

The interaction between acetylcholinesterase (EC 3.1.1.7) and heparin, a sulphated glycosaminoglycan, was studied by affinity chromatography. A specific binding of the asymmetric acetylcholinesterase to an agarose gel containing covalently bound heparin was demonstrated. This interaction required an intact collagenous tail, shown by the fact that the binding is abolished by pretreatment with collagenase. The globular forms did not bind to the column. Both total and intracellular asymmetric acetylcholinesterase forms isolated from the endplate region of the rat diaphragm muscle showed higher affinity for the heparin than did the enzyme from the non-endplate region. The binding to the resin was destabilized with 0.55 M-NaCl, and, among the various glycosaminoglycans tested, only heparin was able to displace the acetylcholinesterase bound to the column. Our results added further support to the concept that the asymmetric acetylcholinesterase forms are immobilized on the synaptic basal lamina via interactions with heparin-like molecules, probably related to heparan sulphate proteoglycans.
Flow cytometry is a new method for the characterization of intestinal plasma membrane
Authors: Gorvel J P , Mawas C , Maroux S , Mishal Z
Published: August 2015

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.
Swainsonine-induced oligosaccharide excretion in sheep. Time-dependent changes in the oligosaccharide profile
Authors: Daniel P F , Warren C D , James L F
Published: August 2015

Urinary oligosaccharides isolated from locoweed-intoxicated sheep were separated and quantified by reversed-phase high pressure liquid chromatography of the perbenzoylated alditols. Mannose-containing oligosaccharides were elevated as early as day 3 of feeding, but maximum levels (approx. 1 mumol/ml) were not attained until after 6 weeks of feeding. The relative abundance of individual oligosaccharides changed over the course of the feeding period. Man3GlcNAc2 reached a peak on day 3 and then rapidly declined. Two isomers were shown to be present in this fraction and the relative proportions altered with the duration of locoweed treatment. The major isomer present at early time points (less than 8 days) co-eluted with synthetic Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, was digested by endo-beta-N-acetyl-glucosaminidase D, and is probably derived from the trimannosyl core of complex glycoproteins synthesized prior to locoweed treatment. Man3GlcNAc2 isolated from day 53 urine was resistant to endo-beta-N-acetylglucosaminidase D digestion but was cleaved by endo-beta-N-acetylglucosaminidase H. This isomer has the probable structure Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, indicative of its origin from hybrid or high-mannose glycoproteins. Man5GlcNAc2 reached a peak on day 13 and then slowly declined, whereas Man4GlcNAc2 increased concomitantly. The rapid increase in Man5GlcNAc2 can probably be attributed to the breakdown of hybrid glycans produced as a result of swainsonine inhibition of Golgi alpha-D-mannosidase II. The onset of observable clinical signs on day 38 closely correlated with the time point at which the level of Man4GlcNAc2 exceeded Man5GlcNAc2. After locoweed feeding was discontinued, the amount of urinary oligosaccharides declined rapidly and reached baseline levels within 12 days.
Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits
Authors: Singh S V , Partridge C A , Awasthi Y C
Published: August 2015

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya′ is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa′) of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya′ subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively.
Identification of a rabbit liver cytosolic binding protein for human growth hormone
Authors: Ymer S I , Stevenson J L , Herington A C
Published: August 2015

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56×10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.
The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study
Authors: Clore G M , Gronenborn A M , Piper E A , McLaughlin L W , Graeser E , van Boom J H
Published: August 2015

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5′-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1′ ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3′-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a ‘melting’ temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3′-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5×10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.
The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo
Authors: Carrington M J , Bird T A , Levene C I
Published: August 2015

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.
Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization
Authors: DeSante D C , Little L , Peavy D E , Vinicor F
Published: August 2015

An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 × 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.
Purification and photoaffinity labelling of lipid methyltransferase from rat liver
Authors: Pajares M A , Alemany S , Varela I , Marin Cao D , Mato J M
Published: August 2015

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.
A computerized approach to the analysis of oligosaccharide structure by high-resolution proton n.m.r
Authors: Hounsell E F , Wright D J , Donald A S R , Feeney J
Published: August 2015

The 500 MHz proton-n.m.r. spectra of 21 oligosaccharides, predominantly of the lacto-N and lacto-N-neo series and their derivatives containing non-reducing terminal fucose, sialic acid or N-acetylgalactosamine and reduced-end hexitol or hexosaminitol, were examined with 2H2O as solvent. The chemical-shift data obtained from analysis of the spectra were collated with data from other laboratories who have used 250-500 MHz n.m.r. in the analysis of secreted and chemically synthesized oligosaccharides and of the O- and N-linked chains of glycoproteins. A referenced computer library was constructed that includes the chemical shifts of monosaccharides within oligosaccharide sequences that make up the majority of the carbohydrate structures found in mammalian glycoproteins. Examples are given of the computerized interrogation of this library for the assignment of possible structures of unknown oligosaccharides.
Pertussis toxin blocks the inhibitory effect of muscarinic cholinergic agonists on cyclic AMP accumulation and prolactin secretion in GH3 anterior-pituitary tumour cells
Authors: Brown B L , Wojcikiewicz R J H , Dobson P R M , Robinson A , Irons L I
Published: August 2015

The inhibition of prolactin secretion and cyclic AMP accumulation in GH3 cells by muscarinic agonists was blocked by preincubation of the cells with pertussis toxin (islet-activating protein). There was a lag of approx. 80 min in the onset of the effect on secretion. These results suggest that muscarinic agonists decrease prolactin secretion by inhibiting adenylate cyclase activity.