P-chloromercuribenzoate Research Articles

Purification and Characterization of Isoamyl Alcohol Oxidase ("Mureka"-Forming Enzyme).

Isoamyl alcohol oxidase (IAAOD) was purified to apparent homogeneity on SDS-PAGE from ultrafiltration (UF) concentrated sake. IAAOD was a glycoprotein, a monomeric protein with an apparent molecular mass of 73 and 87 kDa, by SDS-PAGE and gel filtration on HPLC, respectively. IAAOD showed high substrate specificity toward C5 branched-chain alkyl alcohol (isoamyl alcohol), and no activity toward shorter (C1-C4) or longer (C7-C10) alkyl alcohols tested. IAAOD was stable between pH 3.0-6.0 at 25°C. The optimum pH was 4.5 at 35°C. Heavy metal ions, p-chloromercuribenzoate (PCMB), hydrazine, and hydroxylamine strongly inhibited the enzyme activity, and an anti-oxidant like L-ascorbate did also. Isovaleraldehyde was produced markedly in pasteurized sake by adding purified IAAOD, therefore, we concluded that it was the enzyme that causes formation of mureka, an off-flavor of sake, the main component of which is isovaleraldehyde.

OCCURRENCE OF TRANSGLUTAMINASE IN GREY MULLET (MUGIL CEPHALUS) MUSCLE AND IT'S EFFECT ON MINCED FISH PRODUCT

The gel strength of the minced fish product made from the unwashed mullet mince was found higher than that of the washed product. Transglutaminase (TGase) was found in the water soluble fraction of mullet muscle. It's optimal temperature was at 30C to 35C and the optimal pH was 7.5 to 7.7, Increased setting time at 30C, increased the gel strength of the minced fish product produced from the unwashed mullet mince. p-Chloromercuribenzoate (PCMB), an inhibitor to TGase was added to the mullet mince up to 2.0 mM, the mince was set at 35C and then heated to 85C. The gel strength of the product was inversely correlated to the PCMB concentration, Y 1 = - 0.45X 1 + 1.66 (r = 0.95). Studies of setting of the mullet mince at temperatures ranging 20 to 60C indicated that maximal gel strength was obtained at 35C, coinciding with the optimal temperature of TGase. Meat from 21.5 month-old grey mullet produced a minced product of higher gel strength than those produced from younger fish, although the muscle TGase activities were not significantly different.

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.

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca 2+ affinity of regulatory Ca 2+ sites in single channel recordings and [ 3H]ryanodine binding

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid ( p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20–100 μM) increased the mean open probability ( P o) of the calcium channel at subactivating (20 nM), maximally activating (20–100 μM) and inhibitory (1–4 mM) Ca 2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 μM 4-CMPS caused a transient increase in P o, followed by a low activity state within 1 min. At inhibitory Ca 2+ concentrations P o was increased to values observed with maximally activating Ca 2+ or lower, inhibitory Ca 2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [ 3H]Ryanodine binding was increased up to four-fold with 3–15 μM 4-CMPS/ p-CMB (Hill coefficient 1.7–2.0) at 4 μM Ca 2+ and reduced at high concentrations (50–200 μM). The increase in [ 3H]ryanodine binding by 10 μM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [ 3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca 2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 μM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.

Chemical inactivation of bacterial GABA aminotransferase.

The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme. The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner. Inhibition kinetics were consistent with a simple bimolecular reaction. The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1. In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site. p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme. 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA). The inhibitor constant (Ki) was 0.83 +/- 0.44 mM. We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.

Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body ( Bombyx mori)

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a procedure including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195 kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375 and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which K m values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 μM, respectively. Azaserine, 6-diazo-5-oxo-norleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These results show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes.

Isolation and properties of myoglobins from rat ( Rattus norvegicus) skeletal muscles

One major (Mb I) and two minor (Mbs II and III) myoglobins, were isolated by a heat-denaturation–gel-filtration–chromatofocusing procedure from aqueous extract of rat skeletal muscles. The primary structure of the major component, as determined by an automatic Edman degradation method, revealed amino acid replacements at nine positions compared with murine myoglobin. As in murine myoglobin, a cysteine residue, highly reactive to p-chloromercuribenzoate (PMB), was present at position 66. The two minor components, with higher anodic mobilities and with SH group unreactive to PMB, were found to be derived from Mb I through mixed disulfide formation with cysteine (Mb II) or glutathione (Mb III) at the Cys66 residue. In view of the hydropathy profile, secondary structures of rat and mouse myoglobins showed few differences from each other. The three rat myoglobins displayed essentially identical oxygen equilibrium properties with neither homotropic nor heterotropic allosteric interactions, which agreed very well with those computed from the kinetic measurements.

Purification and characterization of an acid phosphatase from cell membrane fraction of Lactococcus lactis ssp lactis 303

An acid phosphatase was purified from the cell membrane fraction of Lactococcus lactis ssp. lactis 303 by anion exchange chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl Sepharose and gel filtration on Sephacryl S-200 HR and Sepharose 4B-200. Gel filtration on Sepharose 4B-200 showed that activity was confined to a single peak eluted at the void volume of the column. The molecular mass of the enzyme was estimated to be 430 kDa by gel filtration equilibrated with 20 mM Tris-HCl buffer, pH 7.2, containing SDS and β-mercaptoethanol. Activity was optimal at pH 5.2 and 50°C. The enzyme was relatively heat-stable; 23% of activity remained after heating at 70°C for 30 min. The enzyme was stable in 6 M urea; after standing for 18 h at 4°C, 83% of the original activity remained when assayed in the present of 3 M urea. The enzyme was inhibited by F −, Al 3+, phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid and iodoacetic acid and was activated by dithiothreitol and β-mercaptoethanol. Pyrophosphate behaved as a competitive inhibitor for the hydrolysis of p-nitrophenylphosphate, with a K i of 6.4 mM. Kinetic measurements at pH 5.2 gave a K m for p-nitrophenylphosphate of 0.1 mM. The enzyme released inorganic phosphate from a mixture of casein phosphopeptides, mainly αs1-casein (fragment 59–79) and β-casein (fragment 1–25), at a rate of 0.0071 mmol min −1 mg −1 but did not dephosphorylate sodium caseinate.

Lysosomal sulphate transport is dependent upon sulphydryl groups.

Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 microM versus a control value of 126 microM and a modest decrease in Vmax to 99 pmolperunit beta-hexosaminidase per 30 s versus a control value of 129 pmolperunit beta-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 microM and a Vmax of 77 pmolperunit beta-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.

Extracellular alpha-amylase from Thermus filiformis Ork A2: purification and biochemical characterization.

An extracellular alpha-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5-6.0 and 95 degrees C, and the enzyme was stable in the pH range of 4.0-8.0. Calcium enhanced thermostability at temperatures above 80 degrees C, increasing the half-life of activity to more than 8 h at 85 degrees C, 80 min at 90 degrees C, and 19 min at 95 degrees C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The alpha-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and beta-mercaptoethanol activated the enzyme. The alpha-amylase exhibited Michaelis-Menten kinetics for starch, with a K(m) of 5.0 mg.ml-1 and kcat/K(m) of 5.2 x 10(5) ml.mg-1s-1. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an alpha-amylase from a strain of the genus Thermus.

Possible involvement of cysteine and histidine residues in the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Amphibacillus xylanus.

Effect of various inhibitors on the (NH4+ + Na+)-activated ATPase of an anaerobic alkaliphile, Ep01(a strain of Amphibacillus xylanus), was examined. Among the chemicals tested, the enzyme was drastically inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate. The ATPase activity of the enzyme, which was inactivated by p-chloromercuribenzoic acid and diethyl pyrocarbonate, was remarkably restored by beta-mercaptoethanol and hydroxylamine, respectively, suggesting the involvement of cysteine and histidine residues in the enzyme activity. Analysis of the inhibition kinetics by diethyl pyrocarbonate indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme.

Purification and Some Properties of p-Nitrophenyl-β-D-glucoside-hydrolyzing Enzymes in Culture Filtrate of Bacillus circulans KA-304 Grown on Cell-wall Preparation of Schizophyllum commune.

Hydrolyzing activities toward p-nitrophenyl (p-NP)-β-D-glucoside and laminarin in a culture filtrate of Bacillus circulans KA-304, which has been observed to form protoplasts from Schizophyllum commune mycelia, increased when the bacterium was grown on a cell-wall preparation (CWP) of S. commune or laminarin as a carbon source. An analysis of the filtrate with the CWP suggested occurrence of two major p-NP-β-D-glucoside-hydrolyzing enzymes (β-D-glucosidases I and II) and a laminarin-hydrolyzing enzyme. After separation by DEAE-cellulose column chromatography, β-D-glucosidases I and II were isolated (β-D-glucosidase I: 13-fold purification with 34% yield; β-D-glucosidase II: 26-fold with 8%). The enzymes resembled each other in their properties except for their molecular weight, subunit structure (β-D-glucosidase I: 200,000, tetramer; II: 100,000, dimer), and susceptibility to such substances as p-chloromercuribenzoic acid and Ag(+) ion. β-D-Glucosidases I and II hydrolyzed gentiobiose (β-1,6 glucosidic linkage; Km=3.6 mM, β-D-glucosidase I; 4.6 mM, β-D-glucosidase II) and laminaribiose (β-1,3 glucosidic linkage; Km=6.1 mM, β-D-glucosidase I; 6.7 mD β-D-glucosidase II), and showed a certain reactivity toward laminarin as well.

Purification and some properties of a hepatic NADPH-dependent reductase that specifically acts on 1,5-anhydro-D-fructose.

Glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is then reduced to 1,5-anhydro-D-glucitol (AG) in animal livers. An enzyme that catalyzes NADPH-dependent reduction of AF to AG was isolated and purified to homogeneity from porcine liver. Its apparent molecular mass was about 38 kDa on the basis of SDS-PAGE, and its monomeric dispersion in aqueous solution was indicated by gel filtration on a Superose 12 column. Amino acid sequences were determined for four peptides obtained from the purified enzyme. The resulting sequences covered about 50% of the whole sequence and indicated a remarkable similarity between the enzyme and aldose reductase. The purified enzyme showed molecular activity of 8.7 s(-1) on the basis of a molecular mass of 38 kDa, and a Km value of 0.44 mM for AF at the optimum pH of 7.0. It reduced pyridine-3-aldehyde and 2,3-butanedione effectively, acetaldehyde, glucosone, and glucuronic acid poorly and showed no detectable action on glucose, mannose and fructose. It was inactivated by p-chloromercuribenzoic acid to a considerable extent, and the inactivation was partially reversed by 2-mercaptoethanol treatment. It was also sparingly inhibited by relatively high concentrations of glucose, glucose-1(6)-phosphate and 1,5-anhydroglucitol. The reverse reaction, i.e., NADP+-dependent AG oxidation, was not observed. The observed catalytic properties and partial amino acid sequences rule out the possibility that the isolated protein is identical with any known reductase.

X-ray diffraction studies of bacteriorhodopsin. Determination of the positions of mercury label at several engineered cysteine residues.

The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p-chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X-ray diffraction of purple membrane films, with better than 1 A accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol. 259, 393-421, 1996). Given that the distance between the mercury and the C alpha atom of the cysteine in the xy plane must be shorter than 4.5 A and that the mercury atom is located at the delta position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.

Functions of Characteristic Cys-Gly-His-Cys (CGHC) and Gln-Glu-Asp-Leu (QEDL) Motifs of Microsomal ER-60 Protease

The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.

Control of cytosolic pH in two-cell mouse embryos: roles of H(+)-lactate cotransport and Na+/H+ exchange.

In this study we used imaging techniques with the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to investigate the control of cytosolic pH (pHi) in two-cell mouse embryos in nominally HCO3(-)-free conditions. We found that the resting pHi of two-cell embryos (40-50 h after human chorionic gonadotropin) in HCO3(-)-free M2 was 7.31 +/- 0.01 (n = 172 embryos), which is significantly above the level predicted if H+ is at electrochemical equilibrium. We showed that two-cell embryos contain a H(+)-monocarboxylate cotransport system with apparent Michaelis constants for D-lactate, L-lactate, and pyruvate of 11.5, 3.7, and 3.5 mM, respectively. It is inhibited by p-chloromercuribenzoic acid (300 microM), p-chloromercuriphenylsulfonic acid (300 microM), and alpha-cyano-4-hydroxycinnamate (1 mM) and is insensitive to 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (500 microM). We also showed that the pHi response to the acid load produced by an NH4Cl pulse has two components, one due to H(+)-monocarboxylate cotransport and the other due to Na+/H+ exchange. We found no evidence that a H+ conductance was responsible in these cells for the recovery in pHi after an acid load.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Effects of sulfhydryl modification reagents on the kinase activity of the epidermal growth factor receptor.

Earlier reports have indicated that epidermal growth factor (EGF) receptor autophosphorylation, thought to be a key step in receptor transmembrane signaling, can be inactivated with the relatively sulfhydryl-specific reagent N-ethylmaleimide (NEM); however, no Cys residue has been implicated in the catalytic mechanism of the kinase. In an effort to address the mechanism of inhibition by NEM, we have investigated effects of several sulfhydryl-modifying reagents on EGF receptor autophosphorylation and on the kinase activity of the receptor toward an exogenous peptide substrate. Kinase activity is relatively insensitive to iodoacetic acid (IAAcid) and iodoacetamide (IAAmide), though IAAmide-treated receptor displays a higher Km(app) with respect to ATP, relative to untreated receptor. In contrast, even low concentrations of the very specific sulfhydryl reagent p-chloromercuribenzoic acid (PCMB) inactivate the receptor kinase. Pretreatment of the receptor with IAAmide, but not IAAcid, provides substantial protection of the kinase from subsequent treatment with NEM and a degree of protection from subsequent treatment with PCMB. Further, inactivation by NEM, and to a lesser extent PCMB, is inhibited by coincubation of the receptor with the hydrolysis-resistant ATP analog AMP-PNP. The protective effect of IAAmide from inactivation by NEM is also lost when AMP-PNP is present during the IAAmide treatment. Pretreatment of receptor with IAAcid has no effect on subsequent modification by IAAmide. These results, taken together, suggest that NEM, PCMB, and IAAmide, but not IAAcid, modify a Cys residue of the EGF receptor kinase that is inaccessible when nucleotide is bound. Modification of this residue by a bulky reagent (NEM, PCMB) inactivates the kinase by a steric mechanism, while modification with the smaller reagent (IAAmide) results in an active enzyme with altered affinity for ATP. Further, PCMB appears to react with an additional Cys residue (or residues), also resulting in steric inactivation.

Lead transport in IEC-6 intestinal epithelial cells.

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 microM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature significantly reduced the total cellular Pb content of IEC-6 cells. Simultaneous exposed of cells to zinc (Zn) and Pb resulted in decreased total cellular Pb contents compared to total cellular Pb contents of cells exposed to Pb only. IEC-6 cells treated with ouabain (1 mM) or sodium azide (1 mM) and 5 microM Pb accumulated more Pb than cells exposed to Pb only. Cells treated with p-chloromercuribenzensulfonic acid (50 microM), p-chloromercuribenzoic acid (50 microM), or iodoacetimide (50 microM) accumulated less Pb than cells treated with Pb only. We conclude that Pb uptake by IEC-6 cells depends on the extracellular Pb concentration. Our data suggest that the mechanism of Pb uptake by IECs is complex, and that Pb transport in IEC-6 cells is time- and temperature-dependent, involves sulfhydryl groups, and is decreased by the presence of Zn. Extrusion of Pb is at least partially dependent on metabolic energy.

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.