P-chloromercuriphenylsulfonic Acid Research Articles

High-performance liquid chromatographic–colorimetric assay for glycine carboxypeptidase activity

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4′-sulfonyl-Gly- l-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4′-sulfonyl-Gly- l-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4′-sulfonyl-Gly- l-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The K m and V max values were respectively 21.1 μmol and 3.73 pmol/μg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag +, Cu 2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Pulsatile stretch stimulates superoxide production and activates nuclear factor-kappa B in human coronary smooth muscle.

There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and p-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor-alpha activated nuclear factor-kappa B (NF-kappa B) with a peak at approximately 3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-kappa B was abolished by cotreatment with N-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-kappa B inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-kappa B in cultured human coronary artery smooth muscle cells.

Transport of exogenous 5-hydroxytryptamine across the outer plasma membrane of the syncytial tegument ofHymenolepis diminutais by simple diffusion

The uptake of 5-hydroxytryptamine (5HT) from a 10 μM solution of exogenous [3H]5HT into the tegument of Hymenolepis diminuta was linear for the first 20 min of incubation. The rate of transport was 0.04 ± 0.01 pmol∙mg wet mass−1∙min−1, and there were no significant differences in the rate of uptake by the anterior, middle, and posterior regions of the body. The initial uptake was not Na+-dependent, was not saturable at up to 100 μM, was not highly temperature-dependent (Q10~ 1.2), and displayed activation energy of 11.8 kJ∙mol−1. Furthermore, uptake was not inhibited by p-chloromercuriphenyl sulphonic acid, imipramine, amiloride, or 5HT analogues, which collectively support a non-carrier-mediated uptake mechanism. Washing of the tissues with 10 mM 5HT after incubation in 10 μM [3H]5HT displaced less than 10% of the remaining [3H]5HT associated with the tissues, and little radioactivity was extracted by washing in acetone or chloroform. The uptake of [3H]5HT, however, was pH-dependent, the rate of uptake being closely correlated with the proportion of unprotonated 5HT. Only a small portion of the transported [3H]5HT was metabolized to a product associated with 5-hydroxyindoleacetic acid, and metabolism was significantly inhibited by the monoamine oxidase inhibitors iproniazid phosphate, deprenyl, and clorgyline. The present study showed that small amounts of [3H]5HT were taken up by H. diminuta by simple diffusion, little of the [3H]5HT was adsorbed to the surface of the worms or dissolved in the lipid phase of the plasma membrane, and some of the [3H]5HT taken up was metabolized by a monoamine oxidase-like enzyme.

Novel phosphoserine phosphatase inhibitors

Phosphoserine phosphatase (EC 3.1.1.3) catalyzes the final step in the major pathway of l-serine biosynthesis in brain. This enzyme may also regulate the levels of glycine and d-serine, the known and putative co-agonists for the glycine site of the N-methyl- d-aspartate receptor in caudal and rostral brain regions, respectively. Using l-phosphoserine as substrate, the rank order potency for inhibition of phosphoserine phosphatase was p-chloromercuriphenylsulfonic acid (CMPSA)>glycerophosphorylcholine≫hexadecylphosphocholine≥phosphorylcholine> N-ethylmaleimide≥ l-serine>fluoride> d-2-amino-3-phosphonopropionic acid ( d-AP3). Glycerylphosphorylcholine (IC 50 18 μM) was found to be an uncompetitive inhibitor of phosphoserine phosphatase. Glycerylphosphorylcholine probably binds a novel site on the enzyme since the known allosteric inhibitor l-serine is highly selective for its feedback regulatory site, indicated by the inactivity of 25 l-serine analogs. Fluoride ion (IC 50 770 μM) may bind the active site as has been shown for other Mg 2+-dependent enzymes. The sulfhydryl reagent CMPSA is a potent, noncompetitive inhibitor of the enzyme using l-phosphoserine as substrate (IC 50 9 μM) but is >300-fold less potent using d-phosphoserine as substrate. Substrate-dependent differences are also observed with the sulfhydryl alkylator N-ethylmaleimide, which inhibits l-phosphoserine, but stimulates d-phosphoserine hydrolysis. These sulfhydryl reagents may dissociate multimeric forms of the enzyme to form monomers; the multimeric forms and monomers may preferentially cleave l- and d-phosphoserine, respectively. Phosphorylcholine esters and sulfhydryl reagents may prove useful in determining the contribution of phosphoserine phosphatase to the biosynthesis of glycine and d-serine in neuronal tissue in vitro.

N-ARACHIDONYLETHANOLAMINE (ANANDAMIDE) FORMATION FROM N-ARACHIDONYLPHOSPHATIDYLETHANOLAMINE IN RAT BRAIN MEMBRANES

Labeled L-N-arachidonylphosphatidylethanolamine (L-N-arachidonyl PE), a likely precursor of N-arachidonylethanolamine (anandamide), as well as its D-isomer, were synthesized using [ 14C]arachidonic acid. Anandamide was formed by incubating L-N-arachidonyl PE and rat brain membrane with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of anandamide amidohydrolase. Formation of anandamide from L-N-arachidonyl PE was inhibited by p-chloromercuriphenylsulfonic acid (p-CMPS), sulfhydryl reagent, and heat inactivete pre-treatment. D-N-Arachidonyl PE, an unnatural analog for N-arachidonyl PE, did not form anandamide.

Cochlear outer hair cell bending in an external electric field

We have used a high-resolution motion analysis system to reinvestigate shape changes in isolated guinea pig cochlear outer hair cells (OHCs) evoked by low-frequency (2-3 Hz) external electric stimulation. This phenomenon of electromotility is presumed to result from voltage-dependent structural changes in the lateral plasma membrane of the OHC. In addition to well-known longitudinal movements, OHCs were found to display bending movements when the alternating external electric field gradients were oriented perpendicular to the cylindrical cell body. The peak-to-peak amplitude of the bending movement was found to be as large as 0.7 microm. The specific sulfhydryl reagents, p-chloromercuriphenylsulfonic acid and p-hydroxymercuriphenylsulfonic acid, that suppress electrically evoked longitudinal OHCs movements, also inhibit the bending movements, indicating that these two movements share the same underlying mechanism. The OHC bending is likely to result from an electrical charge separation that produces depolarization of the lateral plasma membrane on one side of the cell and hyperpolarization on the other side. In the cochlea, OHC bending could produce radial distortions in the sensory epithelium and influence the micromechanics of the organ of Corti.

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.

Stable expression of recombinant AMPA receptor subunits: binding affinities and effects of allosteric modulators.

Homomeric AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously reported. [3H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN-). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K(D) values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of higher-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [3H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC50 = 7 microM) was higher than that for receptors made of flop subunits (49-130 microM). In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 microM) than for any of the flop isoforms (18-40 microM). These results predict that the action of centrally active AMPA-receptor modulators varies across brain regions depending on their flip/flop composition.

Increased Production of Extracellular Glutamate by the Mitochondrial Glutaminase following Neuronal Death

Elevated extracellular concentrations of the excitatory transmitter glutamate are an important cause of neuronal death in a variety of disorders of the nervous system. The concentrations and rates of clearance and production of extracellular glutamate were measured in the medium of primary cultures from mouse neocortex containing neurons, astrocytes, or both cell types. Measurements were performed in the presence and absence of 2 mM glutamine with or without neuronal injury caused by 5-h exposure to hypoxia or 500 microM N-methyl-D-aspartate or a freeze-thaw cycle. High rates of glutamate generation (0.5-0.8 microM/min in the 0.4-ml culture well) occurred if neurons were both damaged and exposed to glutamine. Intact neurons or glia exposed to glutamine generated only small amounts of glutamate (0.03 microM/min). Glutamate generation by damaged neurons was dependent on the presence of glutamine, activated by phosphate, and inhibited by 6-diazo-5-oxo-L-norleucine and p-chloromercuriphenylsulfonic acid (pCMPS), strongly implicating the mitochondrial glutaminase. Following 5-h exposure to 500 microM N-methyl-D-aspartate, the glutaminase was localized to fragments of damaged neurons and was accessible to inhibition by the membrane-impermeant pCMPS. The glutaminase activity from damaged neurons is sufficient to account for the neurotoxic concentrations of glutamate in hypoxic mixed neuronal-glial cultures exposed to 2 mM glutamine. Finally, pCMPS is neuroprotective and also prevents the increased rate of generation of glutamate observed in neuronal cultures after prolonged exposure to glutamine. The cumulative data indicate the following: 1) excitotoxic neuronal death activates the hydrolysis of extracellular glutamine by the mitochondrial glutaminase, and 2) the glutaminase in damaged neurons is sufficient to cause neuronal death in in vitro models of neuronal injury.

Cleavage of CCK 33 by Recombinant PC2in Vitro

The specificity and kinetic properties of CCK 33 cleavage by recombinant prohormone convertase 2 (PC2) was investigated using a combination of Sephadex G-50 chromatography, HLPC and RIA methods. It is shown that CCK 33 can be cleaved effectively by PC2 to form CCK 8, the reaction of which has a Kmof 104.8 μM. No CCK 22 or other products were detected and the reaction is completely inhibited by the PC2 inhibitor, p-CMS (p-chloromercuriphenylsulfonic acid), suggesting that the cleavage is PC2 specific. The time course of this reaction shows an initial lag phase followed by a rapid increase in velocity consistent with a previously reported spontaneous transformation from a 71 kDa relatively inactive form into a more active form of 62 kDa (1). PC2 did not cleave recombinant pro CCK or CCK 1-21. These results demonstrate that PC2, an enzyme usually associated with dibasic cleavages, can cleave easily at single basic residues.

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.

Properties of a Gram-Posi tive Bacteriolytic Activity from an Oral Clinical Isolate

The aim of this study was to characterise an oral bacterial isolate possessing extracellular bacteriolytic activity and to determine the basic properties of this activity. The lytic strain L1 was a gram-positive pleomorphic rod that grew only under anaerobic conditions. Glucose and raffinose were fermented whereas catalase and urease were not produced. The activity spectrum of a crude lytic fraction was restricted to strains of Actinomyces naeslundii and Actinomyces viscosus . On the basis of its molecular weight (>30 kDa) as well as sensitivity to heat and trypsin, the factor(s) has a proteinaceous component. The activity was highly sensitive to EDTA suggesting that cations may be essential for optimal activity. Sulfhydryl groups are also of major importance in the lytic process as activity was highly stimulated by dithiothreitol and inhibited by p-chloromercuriphenylsulfonic acid. Strain L1 was found to grow on a solid basal medium containing heat-killed A. naeslundii cells, and suggests a physiological role for the bacteriolytic activity. The bacteriolytic activity demonstrated in the present study may constitute a selective advantage for establishment of the bacteria in a complex ecosystem like the oral cavity. Keywords: bacteriolytic activity, enzyme, oral bacteria, oral ecology, Actinomyces .

Modulation of frog skeletal muscle Ca2+ release channel gating by anion channel blockers.

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.

Inhibition and metal ion activation of pig kidney aminopeptidase P: Dependence on nature of substrate

Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to homogeneity after its solubilisation from brush border membranes by phosphatidylinositol-specific phospholipase C. The effects of various activators and inhibitors of AP-P activity have been examined with a number of different substrates for the enzyme. The hydrolysis of bradykinin and ArgProPro is inhibited at Mn 2+ concentrations above 10 −5 M, whereas the hydrolysis of other substrates (GlyProHyp, β-casomorphin, substance P) is substantially activated, with 4–10 mM Mn 2+ being optimal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of bradykinin. A number of inhibitors of angiotensin converting enzyme (ACE; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyProHyp, have no effect on the hydrolysis of bradykinin except in the presence of Mn 2+. Differences were also observed in the degree of inhibition of GlyProHyp and bradykinin hydrolysis by EDTA and their reactivation by divalent cations. The hydrolysis of GlyProHyp follows Michaelis-Menten kinetics with a K m value of 2.7 mM. Bradykinin inhibits GlyProHyp hydrolysis with an I 50 of 1.4 μM. The hydrolysis of bradykinin by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this substrate. These results indicate that substrates for AP-P can be divided into 2 groups based on their responses to inhibitors and cation activators.

Inactivation of Cholesteryl Ester Transfer Protein by Cysteine Modification

The present studies examine the effects of various cysteine-modifying reagents on human recombinant cholesteryl ester transfer protein (CETP) activity. Dithiothreitol or other reducing agents had no effect on CETP transfer activity. Alkylating agents, including iodoacetamide and N-ethyl maleimide, also did not affect transfer activity. However, incubation of CETP with hydrophobic thiol-modifying reagents such as p-chloromercuriphenylsulfonic acid (IC50= 0.02 μM), 4,4′-dithiodipyridine (IC50= 0.5 μM), or 4,4′-dithiobis (phenyl azide) (IC50= 0.5 μM) resulted in complete, time-dependent inactivation of both the cholesteryl ester and triglyceride transfer activities. Inactivation could be prevented by including dithiothreitol in the incubation. Long chain fatty acyl coenzyme A compounds were also found to be effective CETP inhibitors. The extent of inhibition was time-dependent, and proportional to the chain length of the fatty acyl portion of the molecule. These results suggest that CETP contains an essential free cysteine that resides in a hydrophobic environment within the protein.

Simultaneous but Independent Activation of Adenylate Cyclase and Glycosylphosphatidylinositol-Phospholipase C under Stress Conditions in Trypanosoma brucei

Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.

Neuroligand-evoked calcium-dependent release of excitatory amino acids from cultured astrocytes.

The release of excitatory amino acids (EAAs) from neuron-free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose-dependent receptor-mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin-induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium-dependent release of EAAs. Inhibitors of the glutamate transporters p-chloromercuriphenylsulfonic acid, L-trans-pyrrolidine-2,4-dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. alpha-Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium-containing saline. In calcium-depleted saline, alpha-latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium-induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long-term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.

Purification and characterization of beta-glucosidase from Bacteroides JY-6, a human intestinal bacterium.

A beta-glucosidase (EC 3.2.1.21.) was purified 2500-fold from Bacteroides JY-6, an intestinal anaerobic bacterium of human. The specific activity of the homogeneously purified enzyme was 210 mumol/min/mg protein. The enzyme (M(r) 75kDa) was an monomer whose pI and optimal pH values were 4.6 and 5.5-6, respectively. The best substrates were p-nitrophenyl beta-D-glucopyranoside and natural beta-bound glucosides, such as prunin and poncirenin. Puerarin, which is a C-glycoside, was weakly effective. However, cellobiose, alpha-bound glycosides and rhamnoglucosides were not effective. The apparent Kms for prunin and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.08 and 0.19 mM, respectively. The enzyme was strongly inhibited by p-chloromercuriphenylsulfonic acid and reaction products such as p-nitrophenol and glucose.

Properties of a Bacteriolytic Activity from an Oral Gram-Positive Clinical Isolate

The aim of this study was to characterise an oral bacterial isolate possessing extracellular bacteriolytic activity and to determine the basic properties of this activity. The lytic strain L1 was a gram-positive pleomorphic rod that grew only under anaerobic conditions. Glucose and raffinose were fermented whereas catalase and urease were not produced. The activity spectrum of a crude lytic fraction was restricted to strains of Actinomyces naeslundii and Actinomyces viscosus. On the basis of its molecular weight (>30 kDa) as well as sensitivity to heat and trypsin, the factor(s) has a proteinaceous component. The activity was highly sensitive to EDTA suggesting that cations may be essential for optimal activity. Sulfhydryl groups are also of major importance in the lytic process as activity was highly stimulated by dithiothreitol and inhibited by p-chloromercuriphenylsulfonic acid. Strain L1 was found to grow on a solid basal medium containing heat-killed A. naeslundii cells, and suggests a physiological ...

Peptidase inhibitors improve recovery of substance P and calcitonin gene-related peptide release from rat spinal cord slices.

The purpose of present study was to determine whether peptidase activity affects the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from spinal cord slices. When slices were exposed to various inhibitors of endopeptidase 24.11, the resting and capsaicin-stimulated release of SP were less than 0.04% and 0.20% total content per minute, respectively. Resting CGRP release was approximately 0.10% and stimulated release was 0.40%. The combination of 20 microM bacitracin, 100 microM phenylalanylalanine (Phe-Ala), and 50 microM p-chloromercuriphenylsulfonic acid (PCMS) significantly increased both resting and stimulated release of SP and CGRP at least two- or threefold. Doubling the concentration of PCMS and Phe-Ala did not further improve peptide release. These results demonstrate that recovery of peptides released from spinal cord slices is dependent in part on activity of multiple peptidases in the tissues.