Sulfhydryl Group Reagents Research Articles

Insight into the inhibitory effects of Zanthoxylum nitidum against Helicobacter pylori urease and jack bean urease: Kinetics and mechanism.

Zanthoxylum nitidum (Roxb.) DC. is a traditional Chinese medicine characterised by anti-inflammatory and anti-Helicobacter pylori, which is widely used to treat H. pylori-induced gastric disease in China. However, the underlying mechanism related to its anti-H. pylori activity remains unclear. Urease plays a crucial role in the colonisation and survival of H. pylori. The root aqueous extract of Z. nitidum against H. pylori urease (HPU) and jack bean urease (JBU) was investigated to illuminate the inhibitory potency, kinetics and potential mechanism. Z. nitidum components were determined by UPLC. The enzyme inhibitory effects of Z. nitidum were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. Urease inhibition kinetics were determined by Lineweaver-Burk plots. Sulfhydryl group reagents and Ni2+-binding inhibitors were used in the mechanism study. Moreover, the molecular docking technique was used to investigate the binding conformations of the main compounds of Z. nitidum on Urease. According to UPLC results, the major components of Z. nitidum were magnoflorine, sanguinarine, nitidine chloride, chelerythrine, skimmianine and L-Sesamin. Z. nitidum has higher enzyme inhibitory activity on HPU (IC50=1.29±0.10mg/mL) than on JBU (IC50=2.04±0.27mg/mL). Enzyme inhibitory kinetic analysis revealed that the type of Z. nitidum inhibition against HPU was a slow-binding and mixed-type, whereas a slow-binding and non-competitive type inhibited JBU. Further mechanism study indicated that the active site of sulfhydryl group might be the target of inhibition by Z. nitidum. The molecular docking study indicated that the above six main components of Z. nitidum exhibited stronger affinity to HPU than to JBU through interacting with the key amino acid residues located on the mobile flap or interacting with the active site Ni2+. Results indicated that these components are potential active ingredients directed against urease. Z. nitidum inactivated urease in a concentration-dependent manner through slow-binding inhibition and binding to the urease active site sulfhydryl group. Our investigation might provide experimental evidence for the traditional application of Z. nitidum in the treatment of H. pylori-associated gastric disorders.

Direct Evidence that Scorpion α-Toxins (Site-3) Modulate Sodium Channel Inactivation by Hindrance of Voltage-Sensor Movements

The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 + trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).

Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

GPEET procyclin is a major glycosylphosphatidylinositol-anchored protein of procyclic (insect stage) trypanosomes in culture and is heavily phosphorylated in the GPEET pentapeptide repeat. The phosphorylation reaction is a late event and occurs during maturation and transport of GPEET or on the parasite surface by an ecto-protein kinase. Initial biochemical characterization of the GPEET kinase activity now shows that it depends on bivalent cations for maximal activity, is stimulated by sulfhydryl group reagents, and is specific for ATP as phosphoryl donor. No kinase activity is detected in bloodstream form trypanosomes in culture, whereas strong phosphorylation is observed in early procyclic forms. In addition, the GPEET kinase activity is absent from procyclic trypanosomes that have repressed GPEET synthesis but can be induced in these same stocks by conditions, which also induce GPEET expression. However, the presence of an active kinase does not depend on the presence of (functional) GPEET because it can be detected in parasites expressing a non-phosphorylatable GPEET mutant protein and in procyclin null mutant trypanosomes. Interestingly, the presence of the glycosylphosphatidylinositol lipid moiety seems necessary for GPEET to become phosphorylated. Together, the results demonstrate that GPEET and its kinase are expressed during the same life cycle stages and that factors that induce the expression of GPEET in vitro also induce the expression of the GPEET kinase.

Purification and characterization of an arginine aminopeptidase from Lactobacillus sakei.

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37 degrees C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K(m) values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 microM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

Purification and characterisation of a glutaminase from Debaryomyces spp.

A glutaminase was purified from the cell-free extract of Debaryomyces spp. CECT 11815 by protamine sulphate treatment and several chromatographic procedures including anion exchange chromatography and gel filtration. The purified enzyme consisted of two subunits, with molecular masses of 65 and 50 kDa, respectively. Activity was optimal at 40 °C and pH 8.5, and the K m value for l-glutamine was 4.5 mM. The glutaminase exhibited activity against l-γ-Glu-methyl ester, l-γ-Glu-hydrazide, and l-albiziin, while l-asparagine, CBZ- l-Gln, CBZ- l-Gln-Gly, glutathione, l-γ-Glu-pNA and l-γ-Glu-AMC were not hydrolysed. The enzyme was not affected by PMSF, DTT and EDTA. However, the enzyme was inhibited by sulfhydryl group reagents, DON, l-albizziin, l-asparagine and high concentrations of l-glutamine and ammonium, while l-aspartate did not affect the activity. Phosphate and acetate did not produce any significant effect on the glutaminase activity, but it was slightly stimulated by lactate and borate.

Valine Transport and Biodiversity of Leuconostoc Wild Strains from French Raw Milk Cheeses

The rate of L-valine transport in whole cells of Leuconostoc was at the maximum at 30 degrees C, pH 6.0 in the presence of an energy source. Transport was inhibited by 40-55%, in the presence of the ionophores (valinomycin, nigericin or monensin), and uncouplers (carbonyl cyanide-m-chloro-phenylhydrazone or 2,4-dinitrophenol) confirming the previously described delta p-driven branched-chain amino acid transport system described in cytoplasmic membranes (Winters et al., 1991, Appl. Environ. Microbiol., 57, 3350-3354). Sulfhydryl group reagents (p-chloro-mercuribenzoate, iodoacetate and N-ethyl maleimide) all inhibited valine transport by 60-70%, indicating that valine is actively transported at high valine concentration. Three kinetically distinguishable transport systems were identified for each strain using whole cells, confirming results obtained with membranes. L-valine transport Kt and Vmax could be an additional tool to estimate the biodiversity of 18 Leuconostoc strains belonging to the dominant flora of French raw milk cheeses. Kt values varied from 20 to 510 nmol/l for the very high affinity system, from 26 to 427 pmol/l for the high affinity system and from 0.65 to 4.40 mmol/l for the low affinity system. No correlation existed between valine transport rates and a particular strain's ability to acidify milk or complex media, suggesting that valine transport is not a growth-limiting function in species of the genus Leuconostoc.

Purification and characterization of an intracellular esterase from Propionibacterium freudenreichii ssp. freudenreichii ITG 14

An intracellular esterase from Propionibacterium freudenreichiissp. freudenreichii ITG 14 was purified by anion exchange and gel filtration chromatography. The enzyme had a mole- cular weight of 47 400 g.mol -1 as determined by gel filtration chromatography, with an optimum acti- vity on a-naphthyl-acetate at pH 6.0 and at 65 °C, with KM = 1.2 mmol.L -1 . The esterase hydroly- zed synthetic substrates of low molecular weight (C2-C4), and among triglycerides only triacetin. Sulfhydryl group reagents and metal chelators had limited or no effect on enzyme activity; highest inhibition was observed with phenylmethylsulfonylfluoride (PMSF).

Hydroxyhydroquinone reductase, the initial enzyme involved in the degradation of hydroxyhydroquinone (1,2,4-trihydroxybenzene) by Desulfovibrio inopinatus.

The recently isolated sulfate reducer Desulfovibrio inopinatus oxidizes hydroxyhydroquinone (1,2,4trihydroxybenzene; HHQ) to 2 mol acetate and 2 mol CO2 (mol substrate)-1, with stoichiometric reduction of sulfate to sulfide. None of the key enzymes of fermentative HHQ degradation, i.e. HHQ-1,2,3,5-tetrahydroxybenzene transhydroxylase or phloroglucinol reductase, were detected in cell-free extracts of D. inopinatus, indicating that this bacterium uses a different pathway for anaerobic HHQ degradation. HHQ was reduced with NADH in cell-free extracts to a nonaromatic compound, which was identified as dihydrohydroxyhydroquinone by its retention time in HPLC separation and by HPLC-mass spectrometry. The compound was identical with the product of chemical reduction of HHQ with sodium borohydride. Dihydrohydroxyhydroquinone was converted stoichiometrically to acetate and to an unknown coproduct. HHQ reduction was an enzymatic activity which was present in the cell-free extract at 0.25-0.30 U (mg protein)-1, with a pH optimum at 7.5. The enzyme was sensitive to sodium chloride, potassium chloride, EDTA, and o-phenanthroline, and exhibited little sensitivity towards sulfhydryl group reagents, such as copper chloride or p-chloromercuribenzoate.

The monomeric polypeptide comprises the functional flavanone 3β-hydroxylase from Petunia hybrida

Flavanone 3β-hydroxylase catalyzes the FeII/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins. The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size. More recently, the Petunia 3β-hydroxylase was cloned and shown to be encoded in a 41 655 Da polypeptide. In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity. Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3β-hydroxylase exists in functional monomeric and oligomeric forms. Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution. Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39.2±12 kDa for the recombinant flavanone 3β-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3β-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.

Differential potency of two crosslinking plant lectins to induce formation of haptenic-sugar-resistant aggregates of rat thymocytes by post-binding signaling

To evaluate the significance of post-binding events for stable aggregate formation, the aggregation/dissociation of rat thymocytes initiated by two crosslinking plant lectins, namely concanavalin A (Con A) and Solanum tuberosum agglutinin (STA), were comparatively studied. Despite intimate cell contacts in the aggregates only Con A led to establishment of haptenic-sugar-resistant (HSR) complexes. The presence of inhibitor II of diacylglycerol kinase, a dual calmodulin antagonist/protein kinase C inhibitor (trifluoperazine), and a sulfhydryl group reagent ( N-ethylmaleimide) impaired this process. The obtained results indicate that the formation of HSR cellular contacts is not an automatic response to lectin-dependent cell association. In contrast to STA, Con A binding elicits this reaction with involvement of diacylglycerol kinase, protein kinase C and/or calmodulin as well as thiol level perturbation, as inferred by the application of target-selective inhibitors.

Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O 2 •−) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O 2 •−. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O 2 •− production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.

Purification and characterization of apyrase from the tick, Ornithodoros savignyi

An apyrase, ATP-diphosphohydrolase (EC 3.6.1.5) was purified from the soft tick, Ornithodoros savignyi. SDS–PAGE and native PAGE analysis, showed that tick apyrase has a molecular mass of 67 kDa and a basic iso-electric point. The purified enzyme conformed to properties associated with apyrases, including low substrate specificity, a dependence on bivalent metal ions and an insensitivity to normal ATPase inhibitors and sulfhydryl group reagents. It was, however, inhibited by chelating agents, mercuric chloride, dithiothreitol and fluoride. Mg 2+ had a stabilizing effect with respect to inactivation by DTT, suggesting that the enzyme is a metalloprotein. Compared to other apyrases, tick apyrase had higher kinetic rate constants (mM range). The enzyme also inhibited ADP- and collagen-, but not thrombin-induced platelet aggregation and disaggregated platelets that were aggregated by ADP. These properties indicate that apyrase fulfils an important function during tick feeding.

Purification and characterisation of an intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4

An intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4, isolated from traditional Greek yoghurt, was purified by anion exchange and gel filtration chromatography. A single band of molecular weight of about 80 000 appeared in SDS–PAGE; by gel filtration it was shown that the native enzyme was dimeric. The peptidase showed optimum activity on glycyl-prolyl 4-nitroanilide at pH 7.0 and at 50°C, with K m=3.1 mM and V max=3500 U mg −1; over 50°C the enzyme activity declined rapidly. It was inactivated by PMSF; sulfhydryl group reagents and metal chelators had little effect on enzyme activity.

Long-Lifetime Ru(II) Complexes as Labeling Reagents for Sulfhydryl Groups

We report the synthesis and spectral properties of two long-lifetime highly luminescent Ru(II) complexes containing either a sulfhydryl reactive iodoacetamido group or a less reactive chloroacetamido group, [Ru(bpy)2(5-iodoacetamido-1,10-phenanthroline)](PF6)2and [Ru(bpy)2(5-chloroacetamido-1,10-phenanthroline)](PF6)2, respectively, where bpy is 2,2′-bipyridine. Ru(bpy)2(phen-IA)](PF6)2was covalently linked to human serum albumin (HSA) and human immunoglobulin G (IgG). The photoluminescence lifetime of protein-bound probes approaches 1 μs under ambient conditions. In the absence of rotational motions, this probe displayed an anisotropy of 0.18 for excitation at 472 nm. Anisotropy decay data were used to determine the overall rotational correlation times of HSA and IgG. These long-lifetime sulfhydryl-reactive probes can be used to recover microsecond rotational motions and/or domain motions of proteins and/or macromolecular complexes.

Purification and Characterization of a Dipeptidase from Lactobacillus casei ssp. casei IFPL 731 Isolated from Goat Cheese Made from Raw Milk

A dipeptidase was purified to homogeneity from the cell-free extract of Lactobacillus casei ssp. casei IFPL 731 by a combination of heat treatment, hydrophobic interaction chromatography, anion-exchange chromatography, and gel filtration. A purification factor of 395-fold was obtained, and yield was 20%. The dipeptidase was shown to be a metaldependent enzyme; optimal activity was at pH 7.5 and 60 to 75°C, and the enzyme had a high degree of thermal stability. Molecular mass was estimated by SDS-PAGE and gel filtration to be 46 kDa, which suggested that the enzyme existed as a monomer. Enzyme activity was most effectively inhibited by metal-chelating agents, reducing agents, or sulfhydryl group reagents. After inhibition with phenanthroline, activity was partially restored by Co2+ and Mn2+. The kinetics of Phe-Ala and Leu-Leu did not follow Michaelis-Menten saturation kinetics but exhibited a mixture of positive and negative cooperativity for the successive binding of molecules of the same substrate.

Purification and Characterization of an Aminopeptidase fromLactobacillus sake

An aminopeptidase was purified from the cell-free extract of Lactobacillus sake IATA115 by ammonium sulfate fractionation and several chromatographic procedures including hydrophobic interaction, gel filtration, and anion exchange chromatography. The purified enzyme was a 35-36 kDa monomer. Activity was optimal at 37 °C and pH 7.5, and the K m values estimated for Leu and Met-AMC (7-amido-4-methylcoumarin) were 0.091 and 0.174 mM, respectively. The aminopeptidase exhibited maximal activity against Leu- and Ala-AMC, while Lys- and Arg-AMC were not hydrolyzed. Among peptides, highest activity was observed against Ala-Ala, Ala-Leu, and LeuAla, while dipeptides containing basic amino acids at the N terminus were not hydrolyzed. Serin and aspartic proteinase inhibitors had no effect on the activity. However, the enzyme was inhibited by puromycin, amastatin, bestatin, arphamenine B, and sulfhydryl group reagents but activated by reducing reagents. The presence of Hg 2+ , Cu 2+ , Cd 2+ , and Ni 2+ as well as high concentrations of chelator agents caused inhibition, while other divalent cations such as Ca 2+ , Sn 2+ , Mg 2+ , Ba 2+ , and Mn 2+ stimulated the activity.

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K m values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH 2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with K i values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC 50 value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.

Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.

Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.

Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing–thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A 2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24°C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze–thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.