Presence Of N-ethylmaleimide Research Articles

Strong binding of myosin modulates length-dependent Ca2+ activation of rat ventricular myocytes.

Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca2+ sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca2+ sensitivity of tension (ie, pCa50) were measured in the absence and presence of N-ethylmaleimide-modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non-tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of approximately 1.90 microm, application of NEM-S1 markedly increased submaximal Ca2+-activated tensions and thereby increased Ca2+ sensitivity; ie, pCa50 increased from 5.40+/-0.02 to 5.52+/-0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca2+ sensitivity of tension, in that the DeltapCa50 between short and long lengths was 0. 02+/-0.01 pCa units in the presence of NEM-S1 compared with a DeltapCa50 of 0.10+/-0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca2+ sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.

Resistance of γA/γ′ Fibrin Clots to Fibrinolysis

Elevated plasma fibrinogen levels are a major risk factor for thrombosis. This report shows two mechanisms by which fibrinogen can affect the fibrinolysis rate in vitro and thus may lead to thrombosis. First, the lysis rate of fibrin decreases as the initial concentration of fibrinogen increases. Second, a minor variant form of fibrinogen decreases the rate of fibrinolysis. This variant, gammaA/gamma' fibrinogen, has one altered gamma chain and is known to bind to factor XIII zymogen. In a fibrinolysis assay containing purified thrombin, fibrinogen, tissue-type plasminogen activator, and plasminogen, clots from gammaA/gammaA and gammaA/gamma' fibrinogen lysed at similar rates. However, when factor XIII was added, slower lysis was seen in gammaA/gamma' fibrin clots when compared with gammaA/gammaA fibrin clots. A D-dimer agglutination assay showed that the gammaA/gamma' clots were more highly cross-linked than the gammaA/gammaA clots. The lysis rates of gammaA/gamma' clots were similar to gammaA/gammaA clots in the presence of N-ethylmaleimide, a specific inhibitor of factor XIIIa. The gammaA/gamma' fibrin clots made in the presence of factor XIII showed increased proteolytic resistance to both plasmin and trypsin. Clots made from afibrinogenemic plasma reconstituted with gammaA/gamma' fibrinogen also showed significant resistance to lysis compared with gammaA/gammaA fibrinogen. These data demonstrate gammaA/gamma' fibrin is resistant to fibrinolysis, possibly as a result of concentrating factor XIII on the clot. The total fibrinogen concentration and the amount of gammaA/gamma' fibrinogen increase clot stability in vitro and thus may contribute independently to the risk of thrombosis in humans.

Efficient Autoproteolytic Processing of the MHV-A59 3C-like Proteinase from the Flanking Hydrophobic Domains Requires Membranes

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates.In vitroexpression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence ofN-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance thetransprocessing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed fromEscherichia coli.Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pellets. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.

Plasmalemmal vesicles function as transcytotic carriers for small proteins in the continuous endothelium.

We investigated the location and the structural identity of the small pore system, postulated by the pore theory of capillary permeability, using a murine heart perfusion system and small protein molecules as preferential probes for the small pores. Dinitrophenylated proteins were perfused in situ in the absence and in the presence of N-ethylmaleimide (NEM), a reagent known to interfere with membrane fusion of vesicular carriers with their target membranes. The exit pathways of the tracers from vascular lumina to the interstitia were followed by immunoelectron microscopy and by tissue fractionation biochemistry to quantitate their transport and to estimate the extent of transport inhibition by NEM. After 5 min of perfusion, all tracers used were found essentially restricted to plasmalemmal vesicles (PVs) within the endothelium and NEM inhibited their transport by 80-85%. The transport of [14C]inulin and [14C]sucrose, assumed to follow the paracellular pathway, was marginally affected by NEM. These findings indicate that PVs function as structural equivalents of the small pore system for molecules >2 nm in diameter.

Modification of the single unpaired sulfhydryl group of β-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation [Single SH of β-lactoglobulin and pressure denaturation

Chemical modification reactions of the unpaired sulfhydryl group of β-lactoglobulin (LG) under high pressure and the role of this group in the pressure-induced denaturation were investigated. When LG was incubated at 400 MPa (pH 6.8) for 1 h, dimerization through intermolecular reaction of SH was observed. The generation of the covalently linked dimers were prevented by the presence of N-ethylmaleimide (NEM), an agent for SH-specific modification. The reactivity of the SH group of LG, which is buried inside in its native state, was increased by high pressure, as a result of its exposure to the protein surface accompanied by the pressure denaturation. The effect of NEM was also observed in the fluorescence change caused by high pressure, in both the intrinsic fluorescence of LG and the retinol fluorescence of the LG-retinol complex. The control showed an irreversible change at neutral pH, but it became mostly reversible in the presence of NEM. Compatible results were obtained by CD spectroscopy. Inter- and intramolecular reactions of the SH group are suggested to be main causes for the pressure-induced irreversible denaturation of LG

Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.

Role of an N-ethylmaleimide-sensitive factor in the selective cellular uptake of low-density lipoprotein free cholesterol.

Low-density lipoprotein (LDL) was the major contributor to an influx of free sterol from plasma which balances high-density lipoprotein (HDL)-mediated efflux from cultured skin fibroblasts. When HDL was absent, the uptake of LDL free cholesterol was associated with an increase in total cell cholesterol, due in part to accumulation of esterified cholesterol. This influx was mediated by a high-capacity, low-affinity pathway whose magnitude was similar in normal and LDL receptor-deficient cells. In the presence of HDL, some of the interiorized labeled LDL free cholesterol became available for HDL-mediated efflux and some was interiorized, as a result of a transport mechanism which was sensitive to N-ethylmaleimide (NEM) and nitrate ion but resistant to progesterone, azide, or vanadate. We suggest that normal free cholesterol homeostasis in these cells includes the initial binding of LDL followed by the selective transfer of free cholesterol to a compartment from which it is either returned to the membrane for efflux or internalized for storage or further metabolism within the cell. In the presence of NEM, LDL-derived free cholesterol remained mostly accessible for efflux from the cell surface. This free cholesterol pathway may function physiologically to stabilize plasma membrane cholesterol levels against the effect of varying concentrations of HDL and LDL.

Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells.

The role of sulfhydryl groups (SH) and disulfide bonds as well as disulfide oxidoreductases in regulation of the catalytic activity of the membrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatment of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N-ethylmaleimide (NEM) (10 to 50 microM) or with the intramolecular disulfide-forming agent diamide (20 to 100 microM) resulted in the reduction of NO synthase activity in a dose-dependent fashion. Similar loss of enzyme activity was observed when purified NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity. In contrast, when intact PAEC or isolated total membranes derived from PAEC were treated with increasing concentrations (1 to 5 mM) of the disulfide-reducing agent dithiothreitol (DTT), but not oxidized DTT, NO synthase activity was increased by 20 to 85%. DTT reduction of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/thioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide-treated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fold more efficient than the DTT-induced reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate and Product Binding Sites of Yeast Fatty Acid Synthase. Stoichiometry and Binding Kinetics of Wild-Type and in vitro Mutated Enzymes

The four known substrate binding sites of yeast fatty acid synthase (FAS), Ser819 (acetyltransferase, OHAc) and Ser5421 (malonyl/palmitoyl transferase, OHMal) of subunit β and Ser180 (pantetheine binding site, SHc) and Cysl305 (3-oxoacyl synthase, SHp) of subunit α were replaced, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins together with two pairs of double mutants (OHAc/OHMal and SHc/SHp) were episomally expressed in appropriate Δfas1 or Δfas2 deletion strains. The purified enzymes isolated from these trans-formants were used for comparative acyl binding studies with the substrates [1-14 C]acetyl-CoA and [2-14 C]malonyl-CoA. Malonate was found to be transacylated to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydroxyl group of malonyltransferase (OHMal) while acetate could use both the acetyl (Ser819) and the malonyl (Ser5421) transferase active sites on its way to the SHc and SHp binding sites. Acylation of SHc with either substrate was unaffected by the absence of the ‘peripheral’ SH group (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regarding the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a maximum of 2–3 mol rather than the expected 12 mol of malonate and of 6–7 mol rather than 24 mol of acetate bound/mol hexameric yeast FAS. Only 20–30% of the malonyl-enzyme and 35–50% of the acetyl enzyme represented per-formic-acid-labile thioester bonds. The binding characteristics of both substrates, exhibiting Hill coefficients distinctly lower than 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subunits. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of the complex appears unlikely since the coenzyme-A:FAS transacylation equilibrium may be shifted towards the fully acetylated enzyme in the presence of N-ethylmaleimide. In contrast to the limited acylation capacity of the ‘resting’ enzyme, complete acylation of yeast FAS at all of its 12 SHc and SHp sites is observed under steady-state conditions of fatty acid biosynthesis. Under these conditions, the enzyme exhibits full-site reactivity at its SHp, SHc and OHAc sites, but a concomitant 18-fold increase in Km of the coenzyme-A:OHAc transacylation reaction keeps the acyl-O-ester content of the acylated enzyme at less than 5% of the total. Thus, the differential acyl binding characteristics of yeast fatty acid synthase with its strictly limited capacity for acetate and malonate obviously ensure that essentially the full complement of binding sites is reserved for the intermediates and products of the reaction process.

Substrate and Product Binding Sites of Yeast Fatty Acid Synthase. Stoichiometry and Binding Kinetics of Wild-Type and in vitro Mutated Enzymes

The four known substrate binding sites of yeast fatty acid synthase (FAS), Ser819 (acetyltransferase, OHAC) and Ser5421 (malonyl/palmitoyl transferase, OHMa1) of subunit beta and Ser180 (pantetheine binding site, SHc) and Cys1305 (3-oxoacyl synthase, SHp) of subunit alpha were replaced, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins together with two pairs of double mutants (OHAc/OHMa1 and SHc/SHp) were episomally expressed in appropriate delta fas1 or delta fas2 deletion strains. The purified enzymes isolated from these transformants were used for comparative acyl binding studies with the substrates [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Malonate was found to be transacylated to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydroxyl group of malonyltransferase (OHMa1) while acetate could use both the acetyl (Ser819) and the malonyl (Ser5421) transferase active sites on its way to the SHc and SHp binding sites. Acylation of SHc with either substrate was unaffected by the absence of the 'peripheral' SH group (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regarding the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a maximum of 2-3 mol rather than the expected 12 mol of malonate and of 6-7 mol rather than 24 mol of acetate bound/mol hexameric yeast FAS. Only 20-30% of the malonyl-enzyme and 35-50% of the acetyl enzyme represented performic-acid-labile thioester bonds. The binding characteristics of both substrates, exhibiting Hill coefficients distinctly lower than 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subunits. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of the complex appears unlikely since the coenzyme-A:FAS transacylation equilibrium may be shifted towards the fully acetylated enzyme in the presence of N-ethylmaleimide. In contrast to the limited acylation capacity of the 'resting' enzyme, complete acylation of yeast FAS at all of its 12 SHc and SHp sites is observed under steady-state conditions of fatty acid biosynthesis. Under these conditions, the enzyme exhibits full-site reactivity at its SHp, SHc and OHAc sites, but a concomitant 18-fold increase in Km of the coenzyme-A:OHAc transacylation reaction keeps the acyl-O-ester content of the acylated enzyme at less than 5% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate and Product Binding Sites of Yeast Fatty Acid Synthase. Stoichiometry and Binding Kinetics of Wild-Type and in vitro Mutated Enzymes

The four known substrate binding sites of yeast fatty acid synthase (FAS), Ser819 (acetyltransferase, OHAC) and Ser5421 (malonyl/palmitoyl transferase, OHMa1) of subunit beta and Ser180 (pantetheine binding site, SHc) and Cys1305 (3-oxoacyl synthase, SHp) of subunit alpha were replaced, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins together with two pairs of double mutants (OHAc/OHMa1 and SHc/SHp) were episomally expressed in appropriate delta fas1 or delta fas2 deletion strains. The purified enzymes isolated from these transformants were used for comparative acyl binding studies with the substrates [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Malonate was found to be transacylated to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydroxyl group of malonyltransferase (OHMa1) while acetate could use both the acetyl (Ser819) and the malonyl (Ser5421) transferase active sites on its way to the SHc and SHp binding sites. Acylation of SHc with either substrate was unaffected by the absence of the 'peripheral' SH group (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regarding the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a maximum of 2-3 mol rather than the expected 12 mol of malonate and of 6-7 mol rather than 24 mol of acetate bound/mol hexameric yeast FAS. Only 20-30% of the malonyl-enzyme and 35-50% of the acetyl enzyme represented performic-acid-labile thioester bonds. The binding characteristics of both substrates, exhibiting Hill coefficients distinctly lower than 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subunits. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of the complex appears unlikely since the coenzyme-A:FAS transacylation equilibrium may be shifted towards the fully acetylated enzyme in the presence of N-ethylmaleimide. In contrast to the limited acylation capacity of the 'resting' enzyme, complete acylation of yeast FAS at all of its 12 SHc and SHp sites is observed under steady-state conditions of fatty acid biosynthesis. Under these conditions, the enzyme exhibits full-site reactivity at its SHp, SHc and OHAc sites, but a concomitant 18-fold increase in Km of the coenzyme-A:OHAc transacylation reaction keeps the acyl-O-ester content of the acylated enzyme at less than 5% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

コイ・ミオシン加熱時のＳＳ結合によるタンパク質の高分子化機構

The polymer of protein through SS bonding on the top of 5% SDS-PAGE gel was found to be made up of only a myosin heavy chain from the analysis of two-step SDS-PAGE, and from the results it was a dimer, trimer, tetramer, and above-decamer with 1.8% SDS-PAGE gel. The polymerization was suppressed in the presence of N-ethylmaleimide, dithiothreitol, or EDTA. The polymerization of myosin light chains as well as myosin heavy chain was promoted in the presence of transition ion (Cu+, Cu2+), while Ca2+ and Mg2+ did not affect the polymerization. From the above results, it was made clear that the polymerization of protein through SS bonding during the heating of myosin is the reaction that the SH groups of myosin heavy chain are oxidized in the presence of catalysts such as copper ion, and contribute to the smooth and swift formation of the polymer larger than decamer.

Effect of binding of fibrinogen to each bacterium on coaggregation between Porphymmonas gingivalis and Streptococcus oralis

Fibrinogen inhibits the coaggregation between Porphyromonas gingivalis and Streptococcus oralis. In this study, we determined which bacterium interacts with fibrinogen in this inhibitory process. Although preincubation of each bacterium with fibrinogen did not inhibit coaggregation, its activity was completely eliminated by the addition of protease inhibitors such as N-ethylmaleimide (NEM), p-chloromercuriphenyl sulfonate and N alpha-p-tosyl-L-lysine chloromethyl ketone to the preincubation mixture with fibrinogen and P. gingivalis. However, the inhibition of coaggregation was not found after preincubation of S. oralis with fibrinogen in the presence or absence of the protease inhibitors. Labelled materials were recovered from the extract of P. gingivalis cells incubated with radioiodinated fibrinogen in the presence of NEM but not in the absence of NEM. In the binding experiment, P. gingivalis showed a much higher binding activity to fibrinogen than S. oralis. These findings suggest that fibrinogen and its fragment(s) may mask directly or indirectly the aggregation site with S. oralis on the P. gingivalis cells in its inhibitory process of coaggregation.

Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates.

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10(-9) to 10(-10) M and their formation was prevented in the presence of N-ethylmaleimide.

Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide.

In a murine heart perfusion system, we were able to "turn off" the transport of derivatized albumin [dinitrophenylated albumin (DNP-albumin)] from the perfusate to the tissue, by preperfusing the system with 1 mM N-ethylmaleimide (NEM) for 5 min at 37 degrees C, followed by a 5-min perfusion of DNP-albumin in the presence of NEM. Using a postembedding immunocytochemical procedure, we showed that (i) a 30-sec to 1-min treatment of heart vasculature with 1 mM NEM reduces the transendothelial transport of DNP-albumin and nearly stops it after 5 min, and (ii) DNP-albumin is detected exclusively in plasmalemmal vesicles (PVs) while in transit across endothelial cells. Perfusion with 10 mM dithiothreitol for 1 min before NEM prevents the inhibition of vesicular transport. To quantitate the NEM effect on vesicular transport inhibition, we developed an ELISA and a dot-blot assay for measuring DNP-albumin in supernatants of perfused whole-heart homogenates. The results obtained indicate that the treatment of the heart vasculature with 1 mM NEM decreases the vesicular transport of DNP-albumin by 78-80%. Since NEM is known to inhibit the fusion of different types of vesicular carriers with their target membranes in other cell types and in in vitro reconstituted cellular systems, by alkylating a NEM-sensitive factor, we assume that the same mechanism applies in our in situ system. The decrease of vesicular transport can be explained by NEM preventing the fusion of recycling vesicles with their targets--i.e., the abluminal and luminal domains of the plasmalemma. The results open to question previous interpretations from other laboratories according to which plasmalemmal vesicles are sessile, immobile structures.

Involvement of Ca(2+)-stimulated adenosine 5'-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei.

The regulatory role of Ca(2+)-stimulated adenosine 5'-triphosphatase (Ca(2+)-ATPase) in Ca2+ transport system of rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca(2+)-ATPase activity was calculated by subtracting Mg(2+)-ATPase activity from (Ca(2+)-Mg2+)-ATPase activity. The release of Ca2+ from the Ca(2+)-loaded nuclei was evoked progressively after Ca2+ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca2+ from the Ca(2+)-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca2+ uptake and induced a promotion of Ca2+ release from the Ca(2+)-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca(2+)-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca2+ uptake and release. Meanwhile, verapamil and diltiazem (10 microM), a blocker of Ca2+ channels, did not prevent the NAD+ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 microM) and arachidonic acid (10 microM)-induced increase in nuclear Ca2+ release, suggesting that Ca2+ channels do not involve on Ca2+ release from the nuclei. These results indicates that an inhibition of nuclear Ca(2+)-ATPase activity causes the decrease in nuclear Ca2+ uptake and the release of Ca2+ from the Ca(2+)-loaded nuclei. The present finding suggests that Ca(2+)-ATPase plays a critical role in the regulatory mechanism of Ca2+ uptake and release in rat liver nuclei.

Generation of hydrogen peroxide in resting and activated platelets.

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells

Incubation of big endothelin-1 (big ET-1, 1–39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reversephase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1–21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied pig ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.

Characterization of cell-bound and cell-free hemolytic activity of proteus strains

The relationship between growth condition and production of cell-bound hemolysin by Proteus mirabilis S1959 strain was investigated. Hemolytic activity was not dependent on type of medium, oxygen accessibility and was inhibited in presence of N-ethylmaleimide, different sera or trypsine. Cell-free hemolysin was released to the medium during stationary-death phases of growth of fluid Proteus mirabilis and vulgaris cultures.

Roles of sulfhydryl compounds in the gastric mucosal protection of the herb drugs composing oren-gedoku-to (a traditional herbal medicine).

We investigated the involvement of sulfhydryl compounds in the cytoprotective effect of each component herb drug composing Oren-gedoku-to (OGT) against ethanol-induced gastric lesions and potential difference (PD) reduction in comparison with that of OGT in rats. Pretreatment with N-ethylmaleimide (NEM) significantly blocked the cytoprotective effects of OGT, Coptidis rhizoma and Phellodendri cortex, but did not block the cytoprotective effects of Gardeniae fructus and Scutellariae radix. The inhibitory effects of OGT, Coptidis rhizoma and Phellodendri cortex against the PD reduction disappeared in the presence of NEM or diethyldithiocarbamate (DDC), whereas NEM or DDC had little or no effect with Gardeniae fructus and Scutellariae radix. These results suggest that the gastric mucosal protection of Coptidis rhizoma and Phellodendri cortex may be ascribed to the reinforcement of mucosal barrier resistance through endogenous sulfhydryl compounds and DDC-sensitive compounds, but those of Gardeniae fructus and Scutellariae radix may be independent of NEM- or DDC-sensitive compounds.