bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism of potentiation of horseradish peroxidase-catalysed oxidation of NADPH by porphyrins
Authors: Van Steveninck J , Boegheim J P J , Dubbelman T M A R , Van der Zee J
Published: August 2015

Several porphyrins, including HpD (haematoporphyrin derivative), potentiate the oxidation of NADPH by horseradish peroxidase/H2O2. To elucidate the mechanism of potentiation, the following observations are relevant. During peroxidase-catalysed NADPH oxidation, O2-.(superoxide radical) is generated, as judged from superoxide dismutase-inhibitable cytochrome c reduction. This generation of O2-. is suppressed by HpD. Peroxidase-catalysed NADPH oxidation is stimulated by superoxide dismutase and by anaerobic conditions. Under anaerobic conditions HpD has no influence on peroxide-catalysed NADPH oxidation. Previous studies have shown that horseradish peroxidase is inhibited by O2-.. Thus the experimental results indicate that the potentiating effect of HpD can be explained by its ability to inhibit O2-. generation in the horseradish peroxidase/H2O2/NADPH system.
Probing the sequence-specific interaction of the cyclic AMP receptor protein with DNA by site-directed mutagenesis
Authors: Gent M E , Gronenborn A M , Davies R W , Clore G M
Published: August 2015

Mutants in the DNA-binding helix of the cyclic AMP receptor protein (CRP), as well as mutants in a synthetic DNA-binding site derived from the sequence in the lac regulatory region, have been constructed by oligonucleotide-directed mutagenesis, and used to study the effect of selected amino acid substitutions on CRP-mediated transcriptional activity and on sequence-specific DNA binding. It has been shown that mutation of Arg-180 to Lys or Leu abolishes both CRP-mediated expression of beta-galactosidase in vivo and CRP binding of DNA as measured by immunoprecipitation. In contrast, the mutation of Arg-185 to Leu or Lys and the mutation of Lys-188 to Leu does not appear to influence these two parameters significantly. On the DNA side, both substitutions studied, namely the exchange of the G . C base pair in position 2 of the consensus T1G2T3G4A5 motif into an A . T base pair and the exchange of the A . T base pair in position 5 for a G . C base pair, abolish specific binding. Implications of these findings with respect to the present models for specific CRP-DNA recognition are discussed.
Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases
Authors: Svensson B E , Domeij K , Lindvall S , Rydell G
Published: August 2015

Isolated neutrophils from healthy donors were used for the isolation of four highly purified forms of myeloperoxidase as determined by spectral (A430/A280 ratio 0.80-0.87) and enzyme-activity measurements. Although the myeloperoxidases exhibited different elution profiles on cation-exchange chromatography, gel filtration indicated similar relative molecular masses. When these forms were assayed for peroxidase and peroxidase-oxidase activities with several substrates, they all exhibited virtually the same specific activities. These results suggest that possible functional differences between the enzymes may be related to differences in their sites of action rather than to differences in enzyme activity. Myeloperoxidase from a patient with chronic myeloid leukaemia also revealed a similar heterogeneity on cation-exchange chromatography. However, this myeloperoxidase contained in addition one form with a lower and one form with a higher relative molecular mass, as indicated by gel-filtration chromatography.
Theoretical analysis of a translocation-like model with saturable kinetics
Authors: Maïsterrena B , Blum L J , Coulet P R
Published: August 2015

A theoretical analysis of the initial rates of product appearance in both compartments of a specifically designed diffusion cell separated by an asymmetrical enzyme membrane is presented. Variable substrate concentrations and different substrate diffusional limitations were considered. Our analysis shows that, under specific conditions, not only a product accumulation occurs in the compartment opposite to that in which the reaction takes place, but that substrate saturable kinetics can be obtained. These product translocation-like kinetics appear similar to those observed with translocation processes reported for biological situations. For such phenomena, a key role of the diffusion layer surrounding a bioactive surface is proposed.
Pseudomonas μtant strains that accumulate androstane and seco-androstane intermediates from bile acids
Authors: Leppik R A , Sinden D J
Published: August 2015

Transposon mutant strains which were affected in bile acid catabolism were isolated from four Pseudomonas spp. Two of the mutant groups isolated were found to accumulate 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione as the major product from deoxycholic acid. Strains in one of these two groups were able to grow on steroids such as chenodeoxycholic acid, which lacks a 12 alpha-hydroxy function, whereas the one member of the second group could not. With chenodeoxycholic acid, this latter strain accumulated a yellow muconic-like derivative, tentatively identified as 3,7-dihydroxy-5,9,17-trioxo-4(5),9(10)-disecoandrosta-1(10)2 -dien-4-oic acid. Members of two further mutant groups accumulated either 12 beta-hydroxyandrosta-1,4-diene-3,17-dione or 3,12 beta-dihydroxy-9(10)-secoandrosta-1,3,5(10)-triene-9,17-dione as the major product from deoxycholic acid. The relationship between the catabolism of m- and p-cresol, 3-ethylphenol and the bile acids was also examined.
The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides
Authors: Barrowcliffe T W , Havercroft S J , Kemball-Cook G , Lindahl U
Published: August 2015

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.
The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism
Authors: Murphy G J , Hruby V J , Trivedi D , Wakelam M J O , Houslay M D
Published: August 2015

Treatment of intact hepatocytes with glucagon, TH-glucagon [(1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.
Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities
Authors: Quistorff B , Grunnet N
Published: August 2015

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed ‘dual-digitonin-pulse perfusion’. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.
The rapid purification and partial characterization of human serum angiotensinogen
Authors: Campbell C J , Charlton P A , Grinham C J , Mooney C J , Pendlebury J E
Published: August 2015

Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.
Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth μscle cells in culture
Authors: Opsahl W P , Ehrhart L A
Published: August 2015

Rates of collagen and non-collagen protein synthesis in rabbit arterial smooth muscle cells (SMC) were determined by using the specific (radio)activity of [3H]proline in the extracellular, intracellular, and prolyl-tRNA pools. The intracellular free proline specific activity was only 25% of the extracellular value in cultures incubated for 12 h in 0.25 mM-proline. The specific activity of prolyl-tRNA was less than 10% of the extracellular specific activity. Increasing the extracellular proline concentration 10-fold (to 2.5 mM), while keeping the extracellular specific activity of proline constant, resulted in equilibration of the specific activities of intracellular and extracellular free proline, but the specific activity of prolyl-tRNA remained at less than 10% of the extracellular specific activity. Therefore, calculated rates of collagen and non-collagen protein synthesis were greatly underestimated using the intracellular or extracellular specific activity of proline. SMC were also incubated with 0.1 mM-[14C]ornithine in 0.25 nM or 2.5 mM non-labelled proline to examine synthesis de novo of proline and prolyl-tRNA from ornithine. In SMC cultures containing 0.25 mM unlabelled proline, the specific activity of intracellular ornithine was approx. 45% of the extracellular specific activity, due to the production of unlabelled ornithine. The specific activity of ornithine-derived intracellular free proline in SMC incubated with 2.5 mM-proline was significantly lower than in SMC incubated in 0.25 mM-proline, due to the influx of unlabelled proline. However, a corresponding difference in the specific activity of [14C]prolyl-tRNA between SMC in 0.25 mM- or 2.5 mM-proline was not observed. Ornithine-derived [14C]proline was incorporated into proteins in a manner different from that of exogenously added radiolabelled proline. A much higher proportion of the proline synthesized de novo was channelled into collagen synthesis relative to total protein synthesis. Together, these results show that intracellular proline pools are highly compartmentalized in arterial SMC. They also suggest that proline synthesized from ornithine may enter a prolyl-tRNA pool separate from that of proline entering from the extracellular medium.
The antioxidant action of human extracellular fluids. Effect of human serum and its protein components on the inactivation of α1-antiproteinase by hypochlorous acid and by hydrogen peroxide
Authors: Wasil M , Halliwell B , Hutchison D C S , Baum H
Published: August 2015

The elastase-inhibitory capacity of purified human alpha 1-antiproteinase is inactivated by low concentrations of the myeloperoxidase-derived oxidant hypochlorous acid, but much higher concentrations are required to inhibit the elastase-inhibitory capacity of serum samples. The protective effect of serum appears to be largely due to albumin. High concentrations of H2O2 also inactivate the elastase-inhibitory capacity of alpha 1-antiproteinase, by a mechanism not involving formation of hydroxyl radicals. Serum offers protection against H2O2 inactivation of alpha 1-antiproteinase. The relevance of these results to the tissue damage produced by activated phagocytes is discussed.
Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system
Authors: Fagan J M , Waxman L , Goldberg A L
Published: August 2015

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.
Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells
Authors: Jans D A , Resink T J , Hemmings B A
Published: August 2015

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The ‘receptorless’ mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.
Glycogenolytic and haemodynamic responses to heat-aggregated immunoglobulin G and prostaglandin E2 in the perfused rat liver
Authors: Buxton D B , Fisher R A , Briseno D L , Hanahan D J , Olson M S
Published: August 2015

Infusion of heat-aggregated immunoglobulin G (HAG) into perfused livers from fed rats caused transient increases in hepatic glycogenolysis and portal-vein pressure, accompanied by a transient increase in hepatic glycogen phosphorylase alpha content. The hepatic responses to HAG were inhibited by indomethacin (2 microM). In contrast, HAG was without effect on phosphorylase alpha content and glucose output in isolated hepatocytes. HAG infusion caused a transient decrease in hepatic cyclic AMP. Lowering the extracellular Ca2+ concentration to 6 or 50 microM attenuated markedly the glycogenolytic and haemodynamic responses to HAG; efflux of Ca2+ from the liver was not observed in response to HAG. Co-infusion of the specific platelet-activating-factor antagonist U-66985 (1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphoric acid 6′-trimethylammoniumhexyl ester) did not attenuate the glycogenolytic response to HAG. Infusion of prostaglandin E2 caused increases in glucose output, portal-vein pressure and the reduction state of the cytosolic NAD(H) redox couple similar to those seen with HAG. The present study suggests that the glycogenolytic activation after HAG infusion may be an indirect consequence of the haemodynamic response of the hepatic vasculature to stimulation of the reticuloendothelial cells of the liver.
Resolution and analysis of ‘native’ and ‘activated’ properdin
Authors: Farries T C , Finch J T , Lachmann P J , Harrison R A
Published: August 2015

A rapid and reproducible procedure for the resolution of ‘native’ and ‘activated’ forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only ‘native’ properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of ‘native’ into ‘activated’ properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the ‘native’ population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. ‘Activated’ properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin ‘activation’ is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the ‘native’ population.
Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase
Authors: Steenkamp D J
Published: August 2015

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.
Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location
Authors: Danson M J , Conroy K , McQuattie A , Stevenson K J
Published: August 2015

Dihydrolipoamide dehydrogenase has been discovered in the bloodstream form of the eukaryotic African parasite, Trypanosoma brucei. The enzyme catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+ and exhibited a hyperbolic dependence of catalytic activity on the concentrations of both dihydrolipoamide and NAD+. Chemical modification with the tervalent arsenical reagent p-aminophenyldichloroarsine indicates the involvement in catalysis of a reversibly reducible disulphide bond. Plasma-membrane sheets were purified from T. brucei, and it was shown that virtually all the dihydrolipoamide dehydrogenase remained closely associated with this membrane preparation. T. brucei apparently lacks the 2-oxoacid dehydrogenase multienzyme complexes of which dihydrolipoamide dehydrogenase is usually an integral component. In the context of this absence, the possible function of trypanosomal dihydrolipoamide dehydrogenase is discussed, with particular reference to its cellular location in the plasma membrane.
A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets
Authors: Nunn D L , Watson S P
Published: August 2015

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.
Sequence-selective binding of phleomycin to DNA
Authors: Fox K R , Grigg G W , Waring M J
Published: August 2015

The binding of phleomycin and bleomycin to DNA has been investigated by studying their effects on cleavage by DNAase I and micrococcal nuclease. In the presence of cobalt, cleavage of DNA by the antibiotics is suppressed, yet they still provide protection from nuclease attack in regions surrounding the drug cleavage sites. We conclude that cleavage by phleomycin occurs at bonds around which the antibiotic is already selectively bound.
An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver
Authors: Li F , Lim C K , Peters T J
Published: August 2015

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.
Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat
Authors: Del Boccio G , Di Ilio C , Alin P , Jörnvall H , Mannervik B
Published: August 2015

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.
Structural relationships between human erythrocyte sialoglycoproteins β and γ and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s)
Authors: Reid M E , Anstee D J , Tanner M J A , Ridgwell K , Nurse G T
Published: August 2015

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.
Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II
Authors: Job C , Dietrich J , Shire D , Teissere M , Job D
Published: August 2015

A kinetic study of the effect of elongating nucleotide concentration on the reactions of abortive elongation catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template suggests that the shift from abortive to productive elongation may involve the participation of at least two nucleotides, according to a mechanism very similar to that reported for Escherichia coli RNA polymerase. Experiments performed with non-complementary nucleotides with respect to the DNA template, and with substrate derivatives, allow an analysis of the substrate specificity during these reactions. Similar experiments performed with poly[d(A-A-T)].poly[d(T-T-A)] as template provide a starting point for a better understanding of the effect of DNA sequence on the rates of abortive and productive elongation catalysed by the plant enzyme.
Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes
Authors: Hegewald H , Müller E , Klinger R , Wetzker R , Frunder H
Published: August 2015

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.
Binding of NAD+ by cholera toxin
Authors: Galloway T S , van Heyningen S
Published: August 2015

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.
The interaction of spermine and pentamines with DNA
Authors: Basu H S , Marton L J
Published: August 2015

We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and ‘melting’ temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC). All pentamines caused aggregation of DNA at much lower concentrations than that of spermine. Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects. Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA. The biological role of pentamines in T. thermophilus appears to be related to their ability to promote DNA condensation at high temperature.
Chemistry of the collagen cross-links. Origin and partial characterization of a putative mature cross-link of collagen
Authors: Barnard K , Light N D , Sims T J , Bailey A J
Published: August 2015

The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen.
Bacterial killing by complement. C9-mediated killing in the absence of C5b-8
Authors: Dankert J R , Esser A F
Published: August 2015

The ability of serum complement to kill Gram-negative bacteria requires assembly of the membrane attack complex (MAC) on the cell surface. The molecular events that lead to cell killing after MAC assembly are unknown. We have investigated the effect of C9 on bacterial survival in the presence and absence of its receptor, the C5b-8 complex, on the outer membrane. A fluorescence assay of the membrane potential across the inner bacterial membrane revealed that addition of C9 to cells bearing the performed C5b-8 complex caused a rapid and complete dissipation of the membrane potential. No fluorescence change was observed in serum-resistant strains of Escherichia coli. Addition of trypsin, after C9 was bound to C5b-8, did not rescue the cells from the lethal effects of C9. Furthermore, assays of cell killing kinetics and C9 binding indicate that formation of tubular poly(C9) is not required for killing. When C9 was introduced into the periplasmic space in the absence of its receptor by means of an osmotic shock procedure, cell killing occurred. Other proteins, such as C8 or serum albumin, were not toxic, and C9 was ineffective against two resistant strains. The results presented here and previously [Dankert & Esser (1986) Biochemistry 25, 1094-1100], when considered together, indicate that the ‘lethal unit’ in complement killing of some Gram-negative bacteria is a C9-derived product that acts by dissipation of cellular energy.
Terminal-group oxidation of retinol by mouse epidermis. Inhibition in vitro and in vivo
Authors: Connor M J , Smit M H
Published: August 2015

Locally applied retinol is metabolized to retinoic acid in mouse epidermis in vivo. To characterize the oxidation system we investigated the ability of soluble extracts of hairless-mouse epidermis to convert retinol and retinal into retinoic acid. The extracts oxidized retinol to retinoic acid in two steps catalysed by two NAD+-dependent enzymes that were resolved on h.p.l.c. The first enzyme catalyses the reversible oxidation of retinol to retinal and is an alcohol dehydrogenase isoenzyme. The second enzyme oxidizes retinal to retinoic acid. Retinol oxidation by epidermal extracts was inhibited by the alcohol dehydrogenase inhibitor 4-methylpyrazole and by the polyene citral. The toxicity and relatively low potency at inhibiting the epidermal alcohol dehydrogenase isoenzyme curtailed the use of 4-methylpyrazole in vivo. However, citral significantly inhibited retinoic acid formation from retinol in the epidermis in vivo. The ability to inhibit the oxidation of retinol to retinoic acid in mouse epidermis provides a potential method to resolve the roles of retinol and retinoic acid in epithelial function.
Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces
Authors: Mejean C , Boyer M , Labbé J P , Marlier L , Benyamin Y , Roustan C
Published: August 2015

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.
Biphasic interaction of Triton detergents with the erythrocyte membrane
Authors: Trägner D , Csordas A
Published: August 2015

Octylphenoxy polyoxyethylene ethers (Triton detergents) interact with the erythrocyte membrane in a biphasic manner, i.e. they stabilize erythrocytes against hypo-osmotic haemolysis at low concentrations (0.0001-0.01%, v/v), but become haemolytic at higher concentrations. This biphasic behaviour was demonstrated with Triton X-114, Triton X-100 and Triton X-102. However, a critical chain length is a prerequisite for the haemolytic effect, because Triton X-45, which differs from the other Tritons only by the shorter chain of the polyoxyethylene residue, does not exhibit this biphasic behaviour, but goes on protecting against osmotic rupture up to saturating concentrations. Even a 1% solution of Triton X-45 does not cause haemolysis. This structural specificity of Triton X-45, namely the lack of haemolysis and efficient stabilization against osmolysis even at higher concentrations of the detergent, is exhibited at 0 degree and 37 degrees C as well as at room temperature. Three conclusions are reached: (i) a critical chain length of the octylphenoxy polyoxyethylene ethers is required for the haemolytic effect; (ii) the different structural requirements would suggest that different mechanisms are responsible for the haemolytic and the stabilizing effect of amphiphilic substances; (iii) the results suggest that haemolysis is not caused simply by dissolution of the membrane by the detergent but is a rather more specific process.
Immunochemical probing of the structure and cofactor of NADH dehydrogenase from Paracoccus denitrificans
Authors: George C L , Ferguson S J
Published: August 2015

Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates. The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes. The antibody immunoprecipitated NADH dehydrogenase from P. denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents. All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase. A polypeptide of Mr 46,000 in P. denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate. Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits. It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase. Flavoproteins were specifically radiolabelled by growth of P. denitrificans in the presence of [14C]riboflavin. Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions. Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000. Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization. Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I).
Heterogeneity of rat skin heparin chains with high affinity for antithrombin
Authors: Horner A A
Published: August 2015

Subfractions of 35S-labelled rat skin heparin proteoglycans with various degrees of high affinity for antithrombin were obtained by gradient elution from a column of antithrombin-agarose. Heparin chains released from the proteoglycan preparations by beta-elimination with alkali were re-fractionated on the same column. Proportions of chains with high affinity for antithrombin (HA-chains) ranged from 17% to 76%. These separations also revealed three overlapping subfractions of HA-chains. Their proportions varied in a manner consistent with a stepwise increase in the degree of affinity of HA-chains for antithrombin, this presumably being due to the biosynthesis of increasing numbers of antithrombin-binding sites per chain. The anticoagulant activity, with respect to thrombin neutralization, ranged from 32 units/mg to 287 units/mg. It is suggested that HA-chains may have from one to five or six antithrombin-binding sites. Thus the asymmetric distribution of these sites in rat skin heparin proteoglycans is much more marked than was realized from the earlier work of Horner & Young [(1982) J. Biol. Chem. 257, 8749-8754].
Changes in oleic acid oxidation and incorporation into lipids of differentiating L6 myoblasts cultured in normal or fatty acid-supplemented growth medium
Authors: Sauro V S , Strickland K P
Published: August 2015

L6 myoblasts accumulate large stores of neutral lipid (predominantly triacylglycerol) when cultured in fatty acid-supplemented growth medium. No accumulation of neutral lipid was evident in myotubes (differentiated myoblasts) when treated similarly. Triacylglycerol accumulation was rapid and dependent on exogenous fatty acid concentration. Triacylglycerol content in myoblasts cultured in fatty acid-supplemented growth medium was approx. 3-fold higher than that in myotubes treated similarly and 2-3-fold higher than that in myoblasts cultured in normal growth medium. Incorporation studies using [I-14C]oleic acid showed that myoblasts and myotubes take up exogenous fatty acid at similar rates. However, cells cultured in fatty acid-supplemented growth medium remove more exogenous fatty acid than do cells cultured in normal growth medium. Over 90% of the incorporated label was found in phospholipid and triacylglycerol fractions in all situations studied. Myoblasts incorporated a more significant proportion (P less than 0.001) of label into triacylglycerol compared with that of myotubes. No differences in fatty acid oxidation rates were detected when differentiating L6 cells cultured in normal growth medium were compared with those cultured in fatty acid-supplemented growth medium. However, fatty acid oxidation rates were observed to increase 3-5-fold upon myoblast differentiation. We conclude that there is a marked change in the pattern of lipid metabolism when myoblasts (primarily triacylglycerol-synthesizing cells) differentiate into myotubes (primarily phospholipid-synthesizing cells). Understanding these changes, which coincide with normal muscle development, may be important, since a defect in this natural switch could explain the observed accumulation of lipid in muscle characteristic of some of the muscular dystrophies and other lipid-storage myopathies.
The effect of concanavalin A on the rat electro-olfactogram. Differential inhibition of odorant response
Authors: Shirley S G , Polak E H , Mather R A , Dodd G H
Published: August 2015

When the rat olfactory mucosa is treated with concanavalin A, it subsequently shows diminished sensitivity towards 60% of the 112 odorants tested (as judged by the amplitude of the electro-olfactogram response). Odorants containing four to six carbon atoms tend to show the largest (absolute) diminutions, suggesting a receptor for this kind of odorant, although the structural specificity is weak. The receptor seems to be of particular importance in the detection of thiols, carboxylic acids and hydrocarbons of the above size, since these compounds loose the highest proportion of their original signal. The concanavalin A appears to be binding to the glycan of one or more cell-surface proteins. The binding may be at, or close to, at least one odorant receptor.