bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of indomethacin on the stimulation of protein synthesis by insulin in young post-absorptive rats
Authors: Reeds P J , Hay S M , Glennie R T , Mackie W S , Garlick P J
Published: August 2015

Groups of young rats (100 g body wt.) were starved from 23:00 to 11:00 h. The animals were then infused intravenously with diluent or insulin at three different doses to achieve plasma insulin concentrations of 20, 50 and 150 microunits/ml. Before the start of the infusion, animals received a single intravenous injection of indomethacin (250 micrograms) or diluent. After 20 min of infusion, the rats were injected with a large amount of labelled phenylalanine and were killed 10 min later. Insulin produced a dose-dependent decrease in plasma glucose and a dose-dependent rise in protein synthesis in cardiac, gastrocnemius, plantaris and soleus muscles. Protein synthesis in the liver was unaffected by insulin. Indomethacin had no effect on plasma glucose concentrations, but blocked the insulin-induced rise in protein synthesis in cardiac, gastrocnemius and plantaris, but not in soleus muscle. The hormone also increased the plasma concentration of prostaglandin E2 and of prostaglandins F2 alpha and E2 in gastrocnemius and plantaris muscle. The results show close similarities to previous observations with isolated rabbit muscles in vitro and suggest that the involvement of arachidonic acid metabolism in the action of insulin on protein synthesis is of physiological significance.
Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide
Authors: Bornheim L M , Kotake A N , Correia M A
Published: August 2015

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.
Ca2+-binding protein of the human placenta. Characterization, immunohistochemical localization and functional involvement in Ca2+ transport
Authors: Tuan R S
Published: August 2015

The Ca2+-binding protein (HCaBP) of the human placenta was studied with respect to its biochemical properties, tissue and cellular distribution, and possible involvement in placental Ca2+ transport. Optimal Ca2+ binding by the HCaBP occurs at pH 7-8 and in 100 mM-Na+ and 3 mM-Ca2+. The HCaBP possesses at least 10 Ca2+-binding sites with a Kd of 5 × 10(-6) M ([Ca2+]). Highly specific rabbit-derived anti-HCaBP antibodies were used for HCaBP immunoquantification and immunohistochemistry, which revealed that the HCaBP is localized in the chorionic villi and is primarily associated with the trophoblastic cells of the placenta. In addition, an ‘in vitro’ cell-free assay system for Ca2+ uptake was constructed with microsomal membranes isolated from term placental tissues. Ca2+ uptake by the placental microsomal fraction exhibited characteristics indicative of active Ca2+ transport such as temperature-dependence, saturability and energetic requirement. In this system, preincubation of microsomal membranes with anti-HCaBP antibodies inhibited Ca2+ uptake, suggesting that the HCaBP is functionally involved in placental membrane Ca2+ uptake.
H.p.l.c. analysis of di- and tri-carboxylic porphyrins in porphyric patients
Authors: Jackson A H , Rao K R N , Smith S G , Lash T D
Published: August 2015

New h.p.l.c. methods have been developed for the quantitative determination of di- and tri-carboxylic porphyrin methyl esters, and applied to the analysis of faecal extracts from patients with four different types of porphyria.
Immobilization of immunoglobulins on silica surfaces. Stability
Authors: Jönsson U , Malmqvist M , Rönnberg I
Published: August 2015

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.
Quantification of cytochrome P-450-dependent cyclohexane hydroxylase activity in normal and neoplastic reproductive tissues
Authors: Senler T I , Dean W L , Murray L F , Wittliff J L
Published: August 2015

It is well established that liver microsomal cytochrome P-450 participates in steroid metabolism and probably also in the metabolism of anti-oestrogens such as tamoxifen (Nolvadex). Thus it is possible that variations in cytochrome P-450 levels may influence the responsiveness of human breast and endometrial carcinomas to endocrine therapy. Therefore a simple sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylation activity in breast and uterine microsomes (microsomal fractions) has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumour microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. As previously described [Senler, Dean, Pierce & Wittliff (1985) Anal. Biochem. 144, 152-158], a direct method utilizing isotope-dilution/gas chromatography-mass spectrometry was also developed in order to confirm the results of the spectrophotometric assay. The average activity (cyclohexane-dependent NADPH oxidation) for 139 human breast-tumour microsome preparations was 1.34 nmol/min per mg, which is in the range of that found in untreated mammalian liver (1-3 nmol/min per mg). Also, high enzyme activity was demonstrated in human ovary, normal uterus as well as uterine leiomyomas. Endocrine status appeared to influence enzyme levels, in that mammary tissue from virgin rats contained significantly (P less than 0.025) higher amounts of activity than did tissues from either pregnant or lactating rats. Furthermore, carbon monoxide, as well as an antibody against rat liver cytochrome P-450, completely inhibited NADPH oxidation by breast-carcinoma microsomes. These results strengthen our hypothesis that tumours with high levels of cytochrome P-450 may have a reduced response to additive endocrine therapy.
Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase δ is composed of two distinct subunits (B1 and B2)
Authors: Stockman P K , Beckett G J , Hayes J D
Published: August 2015

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.
Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart
Authors: Cleeter M W J , Banister S H , Ragan C I
Published: August 2015

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.
Hemicellulosic complexes from the cell walls of runner bean (Phaseolus coccineus)
Authors: O'Neill M A , Selvendran R R
Published: August 2015

The 1 M-KOH extract from the depectinated cell walls of parenchymatous tissues of mature runner bean (Phaseolus coccineus) on neutralization, dialysis and concentration gave insoluble (hemicellulose A) and soluble (hemicellulose B) carbohydrate complexes in the weight ratio 2:1. Both fractions contained polysaccharide, protein and polyphenolic material. The structural features of the carbohydrates were examined by methylation analysis. Hemicellulose A contained mainly pectic arabinogalactan, with lesser amounts of arabinoxylan and glucan. Sequential fractionation of hemicellulose B by anion-exchange and hydroxyapatite chromatography gave a range of polysaccharide-protein-polyphenolic complexes. The main polysaccharides in these complexes were (acidic) arabinoxylans, galactans, arabinogalactans 1 and 2 and xyloglucans. The proteins contained small amounts of hydroxyproline, but were rich in aspartic acid and glutamic acid. Attempts to determine the nature of the polyphenolic material were unsuccessful. The structural features of the polysaccharide-protein-polyphenolic complexes are discussed in relation to the structure of the cell walls of parenchymatous tissues.
Characterization of a type VI collagen-related Mr-140 000 protein from cutis-laxa fibroblasts in culture
Authors: Crawford S W , Featherstone J A , Holbrook K , Yong S L , Bornstein P , Sage H
Published: August 2015

The precise biochemical defects in connective-tissue metabolism that are responsible for the laxity of skin seen in the syndrome of cutis laxa are largely unknown. We have studied fibroblasts cultured from skin explants of a 2-year-old male with the syndrome. Electron-microscopic examination of this skin revealed decreased amounts of amorphous elastin and an increase in elastin-associated microfibrils. Although the cultured fibroblasts were similar to control skin fibroblasts in morphology, growth rate and total protein synthesis, there was a 4-6-fold increase in accumulation of a collagenous protein of Mr 140 000 in both the culture medium and in the cell layer. This protein was structurally distinct from collagen types I, III, IV, V and VIII. It was found to be related to a cell-surface-associated glycoprotein, GP140, by both antigenic cross-reactivity and peptide mapping. Our data support observations that GP140 is a precursor of at least one form of pepsin-extracted type VI collagen.
Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei
Authors: Richardson L V , Richardson J P
Published: August 2015

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.
Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4
Authors: Willenbrock F , Brocklehurst K
Published: August 2015

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2′-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2′-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2′-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.
The mechanism of end-organ resistance to 1α,25-dihydroxycholecalciferol in the common marmoset
Authors: Takahashi N , Suda S , Shinki T , Horiuchi N , Shiina Y , Tanioka Y , Koizumi H , Suda T
Published: August 2015

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.
Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation
Authors: Sauro V S , Klamut H J , Lin C H , Strickland K P
Published: August 2015

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5′-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.
Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro
Authors: Douglas G C , King B F
Published: August 2015

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 × 10(-6) M, Kd,37 degrees C = 5.3 × 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 × 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.
Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene
Authors: Hyman M R , Wood P M
Published: August 2015

Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.
Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat
Authors: Van Veldhoven P , Mannaerts G P
Published: August 2015

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.
Studies of translatable mRNA for rabbit C-reactive protein
Authors: Samols D , MacIntyre S S , Kushner I
Published: August 2015

C-reactive protein (CRP) mRNA was assayed by cell-free translation of poly(A)-containing liver RNA isolated both from rabbits stimulated to undergo the acute-phase response and from unstimulated control rabbits. No CRP-related translation products were identified until the denaturant methylmercury hydroxide (CH3HgOH) was added to the RNA before cell-free translation. In the presence of the denaturant, a 24000-Da translation product was synthesized which was immunochemically identifiable as the CRP primary translation product. It is likely that rabbit CRP mRNA can form a stable intramolecular duplex which interferes with its translatability in vitro. The 24000-Da CH3HgOH-facilitated cell-free translation product was not detected in poly(A)-containing liver RNA from unstimulated animals, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response. On the basis of absorption experiments, the 24000-Da CRP primary translation product was immunochemically more closely related to denatured CRP than to native CRP.
Cytochrome b-245 from human neutrophils is a glycoprotein
Authors: Harper A M , Chaplin M F , Segal A W
Published: August 2015

Cytochrome b-245 is a glycoprotein. It runs as a broad band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its apparent Mr varies with the concentration of acrylamide. It stained positively with Schiff reagent and with silver stains after oxidation with periodic acid. It preferentially bound the lectin of Phaseolus vulgaris (type III), and cleavage of carbohydrate with endoglycosidase F resulted in a sharp band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 55000 G.l.c. analysis of carbohydrate showed this to account for about 15% of the Mr and N-acetylglucosamine and galactose to be the major sugars.
Kinetics of suicide substrates. Practical procedures for determining parameters
Authors: Waley S G
Published: August 2015

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.
[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate
Authors: Heath D F , Rose J G
Published: August 2015

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)
Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli
Authors: George G N , Bray R C , Morpeth F F , Boxer D H
Published: August 2015

The interconversion of nitrate reductase from Escherichia coli between low-pH and high-pH Mo(V) e.p.r. signal-giving species was re-investigated [cf. Vincent & Bray (1978) Biochem. J. 171, 639-647]. The process cannot be described by a single pK value, since the apparent pK for interconversion is raised by the presence of various anions. The low-pH form of the enzyme exists as a series of complexes with different anion ligands of molybdenum. Each complex has specific and slightly different e.p.r. parameters, but all show strong coupling of Mo(V) to a single proton, exchangeable with the solvent, having A(1H)av. 1.0 to 1.3 mT. Complexes with Cl-, F- [A(19F)av. 0.7 mT], NO3- and NO2- give particularly well-defined spectra. The high-pH form of the enzyme is now shown to bear a coupled proton. Like that in the low-pH species, this proton is exchangeable with the solvent, but the coupling is much weaker, with A(1H)av. 0.3 mT. Thus, contrary to earlier assumptions, the proton detectable by e.p.r. is probably not identical with the proton whose dissociation controls interconversion between the two species; the latter proton could be located in the protein rather than on a ligand of molybdenum. Treatment of the enzyme with trypsin [Morpeth & Boxer (1985) Biochemistry 24, 40-46] did not affect its Mo(V) e.p.r. signals.
Terminal deoxynucleotidyltransferase. Alignment of α- and β-subunits of the core enzyme along the primary translation product
Authors: Beach C M , Chan S K , Vanaman T C , Coleman M S
Published: August 2015

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.
Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes
Authors: Jordan P M , Gibbs P N
Published: August 2015

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid ‘side’ of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.
The elevation of sarcoplasmic reticulum Ca2+-ATPase levels by thyroid hormone in the L6 muscle cell line is potentiated by insulin-like growth factor-I
Authors: Muller A , van Hardeveld C , Simonides W S , van Rijn J
Published: August 2015

Net synthesis of the fast-type sarcoplasmic reticulum (SR) Ca2(+)-ATPase was studied in the muscle cell line L6AM using an immunochemical assay (e.l.i.s.a.). In addition, Ca2+ uptake by SR was monitored in muscle cell homogenates by a method employing the fluorescent Ca2+ indicator fura-2. Measurements were done both in differentiating myoblasts and in myotubes. Ca2(+)-ATPase levels were low (1 pmol/mg of protein) in undifferentiated myoblasts (controls) and only doubled over a period of 8 days in the absence of thyroid hormone (L-triiodothyronine; T3). This corresponded to a similar increase in Ca2+ uptake activity. Only half of the myoblasts fused under these conditions. Fusion was not increased in the presence of T3 (5 nM), but Ca2(+)-ATPase levels increased 4-fold and the Ca2+ uptake activity doubled compared with controls. In contrast, insulin-like growth factor-I (IGF-I) induced almost complete myotube formation (greater than 90% fusion), but only slightly stimulated (50%) net Ca2(+)-ATPase synthesis above control levels. However, the doubling of the Ca2+ uptake stimulation by IGF-I was comparable with that caused by T3. The effects of T3 plus IGF-I on Ca2(+)-ATPase levels and Ca2+ uptake activity were more than additive. Furthermore, the temporal relationship between the induction of Ca2(+)-ATPase net synthesis and Ca2+ uptake activity was identical with the two hormones. Qualitatively similar results were obtained when T3 and IGF-I were added to maximally fused cell cultures. The enhanced effect of T3 on Ca2(+)-ATPase net synthesis and Ca2+ uptake activity in the presence of IGF-I cannot therefore be explained by an increased myotube formation stimulated by the latter. In both differentiating myoblasts and myotubes the effect of T3 was more prominent on Ca2(+)-ATPase net synthesis than on Ca2+ uptake activity, whereas in myotubes the opposite was observed for IGF-I. This could imply complementary actions of the two agents in the development of a functional SR.
Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli
Authors: Ding V D H , Sheares B T , Bergstrom J D , Ponpipom M M , Perez L B , Poulter C D
Published: August 2015

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.
Distribution and characterization of dihydrodiol dehydrogenases in mammalian ocular tissues
Authors: Hara A , Nakayama T , Harada T , Kanazu T , Shinoda M , Deyashiki Y , Sawada H
Published: August 2015

The immunological relationship of two forms of dihydrodiol dehydrogenase (DD) in pig lens to pig muscle aldose reductase and kidney aldehyde reductase has been studied. Although the minor enzyme form, a monomer of Mr 35,000, was identical with aldose reductase, the major enzyme form, a dimer of Mr 65,000, was distinct from the two reductases. The two enzyme species, although their amounts were low, were distributed in the cornea, iris-ciliary body, retina and choroid of the pig eye. In other mammals, rabbit lens exhibited much higher DD activity than did lens of mice, rats, cats, hamsters, guinea pigs and monkeys, and contained large amounts of the Mr-65,000 enzyme form as well as the minor enzyme form of Mr 35,000. In contrast, only the Mr-35,000 form of the enzyme was found in the lens of other species, except that a small amount of the high-Mr enzyme was detected in mouse lens. The high-Mr enzyme, purified from rabbit lens, was functionally and immunologically similar to dimeric DD of pig lens. The low-Mr enzyme forms, isolated or partially purified from these animal lenses, showed several features in common with aldose reductases from mammalian tissues. The dimeric enzymes of pig and rabbit lenses were NADP(+)-specific, whereas the low-Mr enzymes exhibited dual cofactor specificity and their activities with NAD+ were more than 3-fold higher than those with NADP+.
Differential hepatic processing and biliary secretion of head-group and acyl chains of liposomal phosphatidylcholines
Authors: Verkade H J , Derksen J T , Gerding A , Scherphof G L , Vonk R J , Kuipers F
Published: August 2015

To investigate the contribution of plasma-derived phosphatidylcholine (PC) to bile PC, the hepatic processing and biliary secretion of liposome-associated PC was studied in rats. For this purpose, small unilamellar vesicles (SUV), containing trace amounts of [2-palmitoyl-9,10-3H]dipalmitoylphosphatidylcholine ([palmitoyl-3H]DPPC), [choline-14C]-dipalmitoylphosphatidylcholine ([choline-14C]DPPC), di[14C]palmitoylphosphatidylcholine ([14C]DPPC) or di[1-14C]-oleoylphosphatidylcholine ([14C]DOPC), were administered intravenously to unanaesthetized rats, equipped with permanent catheters in heart and bile duct. Biliary secretion of the 14C-head-group label of DPPC was very slow (0.3% of injected dose in 4 h), whereas the [3H]palmitoyl label was secreted at a much higher rate (16% in 4 h), but only after substantial catabolism of the acyl chain. To study the latter process in more detail, we compared hepatic metabolism and biliary secretion of [1-14C]acyl-labelled DPPC and DOPC. In rats with an 8-day bile drainage, degradation products of the oleoyl chain were utilized for synthesis of bile acids, which were subsequently secreted into the bile (2% in 6 h). A much smaller fraction (0.6% in 6 h) was secreted as PC and lyso-PC. When bile drainage was started immediately after SUV injection, i.e. a situation with a low hepatic bile acid synthesis rate and a high phospholipid secretion, the secretion of [14C]DOPC-derived radioactivity in the form of bile acids was decreased (0.2% in 6 h), and that as (lyso-)PC increased (1.5% in 6 h). Biliary secretion of DPPC palmitoyl chains in bile-diverted rats was much less than that of the oleoyl chains, and occurred predominantly as PC and lyso-PC (0.6%, compared with 0.4% as bile acids in 6 h). Breath analyses demonstrated that a considerable fraction of both acyl chains was oxidized to CO2 and expired: 25.1% of the administered label for oleoyl chains and 13.4% for palmitoyl chains respectively in a 4 h period. The results of this study indicate that liposomal PC is only minimally secreted into bile via a direct pathway; the bulk is extensively degraded in the liver. Resulting products are partly secreted into bile, as bile acid or as resynthesized PC. There appears to be a quantitative difference in the metabolism of oleoyl and palmitoyl acyl chains.
Infrared spectroscopy of heparins suggests that the region 750-950 cm-1 is sensitive to changes in iduronate residue ring conformation
Authors: Grant D , Long W F , Moffat C F , Williamson F B
Published: August 2015

By careful definition of polymer environment, heparin i.r. spectra were examined in a region (750-950 cm-1) in which sulphate half-ester absorptions occur. Changes seen in this region when metal ion-heparin complexes are converted into heparinic acid, when heparin is carboxy-group-reduced and when various concentrations of Li(+)-heparin are examined are tentatively interpreted in terms of changes in the ring conformation of iduronate residues.
Molecular cloning, sequencing and characterization of cDNA to rat liver rhodanese, a thiosulphate sulphurtransferase
Authors: Weiland K L , Dooley T P
Published: August 2015

Rhodanese (EC 2.8.1.1), a mitochondrial thiosulphate sulphurtransferase, is involved in the formation of iron-sulphur complexes and cyanide detoxification. By screening a rat liver cDNA library with oligonucleotide probes complementary to portions of the published bovine rhodanese peptide sequence, rat rhodanese cDNA clones were obtained and sequenced. Comparison of the rat rhodanese cDNA open reading frame with the bovine peptide sequence demonstrated in the rat open reading frame the presence of 27 amino acid substitutions, only five of which are highly non-conservative. Thus the rat enzyme is approx. 91% identical with bovine rhodanese, or about 98% similar when conservative substitutions are considered. In addition, the rat translation product contains a Gly-Lys-Ala C-terminal tripeptide that was not observed in the bovine peptide sequence. All cysteine and proline residues are invariant between the two mammalian proteins. Computer-generated structural modelling of rat rhodanese indicated that few amino acid substitutions were present within close proximity to the active site or within the hinge region (connecting loop) between the A and B domains. Furthermore, evidence is presented showing that rhodanese is highly conserved at the DNA level among rodents, primates and a variety of other vertebrates.
Quantitative analysis of metabolic regulation. A graph-theoretic approach using spanning trees
Authors: Sen A K
Published: August 2015

A graph-theoretic technique using spanning trees is described for the evaluation of Flux Control Coefficients of metabolic pathways. The technique is illustrated by investigating a linear pathway (a) in the absence of feedback and feedforward regulation. (b) with its first enzyme inhibited by the end product and (c) with multiple feedback loops. It is shown that the Flux Control Coefficients of a linear pathway with one or more feedback loops can be derived in a systematic manner by superimposing the effect of the feedback loop(s) on the expressions pertaining to the Flux Control Coefficients of the unregulated pathway.
Identification of complexes between the tyrosine-O-sulphate-binding protein and tyrosine-sulphated proteins in bovine liver membrane lysates
Authors: Liu M C , Lu R L , Han J R , Tang X B , Suiko M , Liu C C
Published: August 2015

Rabbit antiserum against electrophoretically purified bovine liver tyrosine-O-sulphate (TyrS)-binding protein was prepared. Affinity-purified antibodies from the antiserum were found to be capable of immunoprecipitating the TyrS-binding protein from the sodium choleate extract of a bovine liver microsomal membrane fraction. Using purified specific antibodies as the probe, Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates was performed. It was found that the TyrS-binding protein co-precipitated with three tyrosine-sulphated proteins (fibronectin, fibrinogen and complement C4) immunoprecipitated by their respective antibodies. In contrast, for the two non-tyrosine-sulphated proteins (haptoglobin and transferrin) tested, co-precipitation of the TyrS-binding protein was not observed. On employing an affinity gel fractionation technique, it was shown that partially purified TyrS-binding protein exhibited binding affinity towards Sepharose gels covalently bonded to fibronectin or fibrinogen, but not towards Sepharose gels bonded to albumin or transferrin. These results indicate that the TyrS-binding protein formed complexes with tyrosine-sulphated proteins both in vivo and in vitro, and thus provide support for the putative role of the former being the receptor of the latter.