P-chloromercuribenzoate Research Articles

Purification and characterization of β-galactosidase from rat epididymal fluid

Beta-galactosidase from rat epididymal fluid was purified by a combination of chromatographic techniques and precipitation with ammonium sulphate. Specific activity of the enzyme in the final precipitate was 18 times greater than in the original fluid, and it was practically free of N-acetyl-beta-D-glucosaminidase. A single major band was seen when the precipitate was analysed by sodium dodecylsulphate polyacrylamide gelectrophoresis (SDS-PAGE). The activity of the purified enzyme has an optimum at pH 4.5, and the temperature optimum is around 45 degrees C. The activity was inhibited by p-chloromercuribenzoic acid and ions such as Cd(II), Co(II), Cu(II) and Ag(I). Lactose does not appear to be a substrate for this enzyme.

FACTORS AFFECTING THE (PRODUCTION OF EXTRACELLULAR NEUTRAL PROTEASE) BY STREPTOMYCES MICROFLAVUS

In order to produce neutral protease from Streptomyces microflavus, it was cultivated in basal medium containing soluble starch, potassium nitrate, dipotassium hydrogen phosphate, magnesium sulphate, sodium chloride, calcium carbonate and ferrous sulphate. Protease production increased with decreasing the ratio of culture broth to vessel volume 1:5 and shaking at 150 rpm. Protease production was low when pH was 9. The productivity of protease decreased sharply when the incubation temperature increased from 30 to 450C. The maximum yield of protease was obtained at the third day of incubation. Soluble starch and yeast extract were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease production. In addition, the dipotassium hydrogen phosphate was the best phosphorous source. Para chloromercuribenzoic acid and phenyl methylsulfonylfloride had significant inhibitory effect on protease production.

Purification and Characterization of L-Phenylalanine Aminopeptidase from Chick-Pea Cotyledons (Cicer arietinum L.)

Chick-pea (Cicer arietinum L.) cotyledons are unique source of aminopeptidase - 8-9 U/g cotyledons was observed using L-leucine-p-nitroanilide as substrate. The aminopeptidase was purified (65 kDa, pI 4.8 ) reaching a specific activity of 220 U/mg at pH 7.0-7.2 and 35-40 degrees C. The determined constant of specificity k(cat)/K(m) during hydrolysis of N-unsubstituted amino acid-p-nitroanilides showed a decrease order: Phe>Leu>Pro>Ile>Val>Ala. The enzyme was strongly inhibited by p-chloromercuribenzoic acid as well as in a competitive rate by the antihypertensive peptides Ile-Pro-Pro and Val-Pro-Pro.

Characterization of the brain-specific non-AT 1, non-AT 2 angiotensin binding site in the mouse

In the present study the existence of a non-AT 1, non-AT 2 angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT 1, non-AT 2 angiotensin binding site was similar to the rat brain: Sar 1-Ile 8-Ang II > Ang III ≥ Ang II > Ang I> p-aminophenylalanine 6 Ang II> CGP42112 >> Ang IV > Ang 1–7 ≅ shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K i values > 10 µM, while substance P and VIP had K i values of ~ 2 µM. The non-AT 1, non-AT 2 angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [ 125I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT 1a, AT 1b, AT 2, Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT 1, AT 2, AT 4 and Ang 1–7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study.

Further characterization of the novel, non‐AT1, non‐AT2 brain‐specific angiotensin binding site in the mouse brain

To further characterize the non‐AT1, non‐AT2 binding site in mouse brain, differential centrifugation was used to assess its subcellular distribution. The fraction enriched in plasma membranes (P2) from both cerebral cortex (CTX) and hypothalamus (HT) showed a higher density of 125I‐Ang II binding relative to the nuclear/mitochondrial/debris (P1) fraction: CTX P1 Bmax= 21.9±3.6 fmol/mg protein, CTX P2 Bmax = 63.1±11.1 fmol/mg protein; HT P1 Bmax = 24.5±7.6 fmol/mg protein, HT P2 Bmax = 38.3±9.8 fmol/mg protein. The enriched binding in CTX and HT plasma membranes (71 and 66% of total binding, respectively) suggests that the non‐AT1, non‐AT2 angiotensin binding site is primarily localized to plasma membrane. Since p‐chloromercuribenzoate (PCMB), the agent that unmasks this binding site, may simulate pathophysiological conditions in the brain associated with an altered redox state, we examined the effect of reduced glutathione (GSH) on the ability of PCMB to unmask the binding site in P2 membranes. GSH at concentrations ranging from 0.3 to 10 mM inhibited 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site from 78 to 87% in a concentration‐related manner. A small residual amount of 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site in the absence of PCMB was also inhibited by 0.3 to 10 mM GSH by 51 to 70%. Functionality of the non‐AT1, non‐AT2 binding site may thus depend on redox state of the brain.

Characterization of an Aminopeptidase and a Proline Iminopeptidase from Cabbage Leaves

Aminopeptidase, preferring phenylalanine-p-nitroanilide as substrate, and proline iminopeptidase, highly-specific for proline-p-nitroanilide, were isolated from cabbage leaves (Brassica oleraceae var. capitata). As pH optima, 7.2-7.5 for aminopeptidase activity and 8.0-8.5 for proline iminopeptidase were determined. Both peptidases were strongly inhibited by p-chloromercuribenzoic acid, heavy metal ions and urea. The molecular weights were determined by gel filtration to be 56 and 204 kDa, respectively. The iminopeptidase was decomposed during SDS electrophoresis to four subunits of 50 kDa. Minor impurities of myrosinase-associated protein (approximately 70 kDa) were found in both preparations. Preliminary data of their amino acid sequences showed similarities to those of aminopeptidases N (family M1) and proline iminopeptidases (family S33).

Brain angiotensin receptors and binding proteins

This review addresses classical and novel aspects of the brain angiotensin system. The brain contains both the AT1 and AT2 angiotensin II (Ang II) receptor subtypes which are well-characterized guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs). Like other GPCRs, novel signal transduction pathways and protein interactions are being described for Ang II receptors. For brain AT1 receptors, there is a controversy regarding the identity of the active angiotensin peptide in the brain which is addressed in this review. This review also summarizes a recent discovery of a novel, membrane-bound, non-AT1, non-AT2 binding site for angiotensin peptides that appears to be brain-specific. This binding site is unmasked by a limited concentration range of the organometallic sulfhydryl-reactive agent p-chloromercuribenzoic acid (PCMB) suggesting that functional expression of this binding site may depend on the redox state of the milieu of the brain. While this binding site has similarities to a previously described soluble angiotensin-binding protein found in liver that is unmasked by PCMB, it has many different characteristics. The possible functional significance of this novel non-AT1, non-AT2 binding site for angiotensin peptides as a mediator of non-traditional actions of Ang II in the brain, e.g., stimulation of dopamine release from the striatum, as a peptidase, or as a clearance receptor, and the importance of the state of the internal environment of the brain to its function is reviewed.

Purification and Biochemical Characterization of Cell Wall-bound Trehalase from Pericarp Tissues of Actinidia deliciosa

A trehalase (EC 3.2.1.28) was purified from the cell walls of Actinidia deliciosa fruit. The purified trehalase had optimal pH of around 5, Km of 0.25 mM and Vmax of 5667 pkat/mg protein, and was relatively heat stable. The enzyme showed highly specific activity to trehalose and weak activity to maltose and maltotriose, but did not hydrolyze any other disaccharides. Trehalase activity was unaffected by Ca2+, Na+, K+, Li+, Mn2+, Co2+, and Mg2+ ions and EDTA, but markedly inhibited by Hg2+ and Fe3+ ions, iodoacetic acid, tris(hydroxymethyl)aminomethane (Tris), p-chloromercuribenzoate (PCMB), glucose and glucosamine. This cell wall-bound enzyme seems to degrade apoplastic trehalose. Another possibility is that this trehalase has additional functions such as defence against insects.

Proteolytic activation and glycosylation of N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme involved in the endocannabinoid metabolism

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme hydrolyzing bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. Previously, we suggested that NAAA is glycosylated and proteolytically cleaved. Here, we investigated the mechanism and significance of the cleavage of human NAAA overexpressed in human embryonic kidney 293 cells. Western blotting with anti-NAAA antibody revealed that most of NAAA in the cell homogenate was the cleaved 30-kDa form. However, some of NAAA were released outside the cells and the extracellular enzyme was mostly the uncleaved 48-kDa form. When incubated at pH 4.5, the 48-kDa form was time-dependently converted to the 30-kDa form with concomitant increase in the N-palmitoylethanolamine-hydrolyzing activity. The purified 48-kDa form was also cleaved and activated. However, the cleavage did not proceed at pH 7.4 or in the presence of p-chloromercuribenzoic acid. The mutant C126S was resistant to the cleavage and remained inactive. These results suggested that this specific proteolysis is a self-catalyzed activation step. We next determined N-glycosylation sites of human NAAA by site-directed mutagenesis addressed to asparagine residues in six potential N-glycosylation sites. The results exhibited that Asn-37, Asn-107, Asn-309, and Asn-333 are actual N-glycosylation sites. The glycosylation appeared to play an important role in stabilizing the enzyme protein.

A novel alpha-glucosidase from the moss Scopelophila cataractae.

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.

The Role of Monocarboxylate Transporter 2 and 4 in the Transport of γ-Hydroxybutyric Acid in Mammalian Cells

Monocarboxylate transporter 1 (MCT1) is an important determinant of the renal transport of the drug of abuse, gamma-hydroxybutyric acid (GHB). The objective of this study was to investigate the role of MCT2 and MCT4, present in tissues including intestine, kidney, skeletal muscle, and brain, in the membrane transport of GHB and the MCT substrate l-lactate. mRNA and protein of MCT2 and MCT4 were expressed in MDA-MB231 cells, as detected by reverse transcription-polymerase chain reaction and Western blot analysis; MCT1 and MCT3 were not detected. The uptake of GHB or l-lactate by MDA-MB231 cells was pH-dependent but not sodium-dependent. The concentration-dependent uptake of GHB was best fitted to a single-transporter model with a diffusional clearance component (K(m) of 17.6 +/- 1.5 mM, V(max) of 50.6 +/- 9.0 nmol x mg(-1) min(-1) and diffusional clearance of 0.20 +/- 0.07 microl x mg(-1) min(-1)). On the other hand, the concentration-dependent uptake of l-lactate was best fitted to a two-transporter model (K(m) of 21 +/- 2.5 and 3.0 +/- 1.5 mM, and V(max) of 268 +/- 72 and 62.9 +/- 42.2 nmol x mg(-1)min(-1), respectively). The uptake of GHB and l-lactate was inhibited by MCT inhibitors alpha-cyano-4-hydroxycinnamate (CHC), phloretin, and p-chloromercuribenzoic acid; CHC inhibited GHB and l-lactate uptake with IC(50) values of 1.71 +/- 0.39 and 0.71 +/- 0.11 mM, respectively. Small interfering RNA treatment to silence MCT2 or MCT4 significantly decreased their protein expression and the uptake of l-lactate and GHB; however, the decrease in GHB uptake with MCT2 inhibition was smaller than that for MCT4. This investigation demonstrated that GHB is a substrate for both MCT2 and MCT4; these transporters may be important in the nonlinear disposition of GHB, as well as influencing its tissue distribution.

Purification of serine carboxypeptidase from the hepatopancreas of Japanese common squid Todarodes pacificus and its application for elimination of bitterness from bitter peptides

An acidic serine carboxypeptidase (CPase Tpa) from the hepatopancreas of Japanese common squid Todarodes pacificus was purified. Purified CPase Tpa had a molecular mass of 36 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 6.0. The optimum pH of CPase Tpa was pH 4.0. In investigating the specificity of CPase Tpa for several peptide substances, it was found that peptides with hydrophobic or bulky amino acid residues at the P1 position reacted well. The enzymatic activity was almost completely inhibited by p-chloromercuribenzoic acid, monoiodoacetic acid, diisopropylfluorophosphate and HgCl2. This is the basis for its grouping in the serine carboxypeptidase family (EC 3. 4. 16. 5). The substrate specificity of CPase Tpa can be used to eliminate the bitterness of bitter peptides. In this study, the bitterness-reductive effect using bitter peptides prepared by hydrolyzing soy protein, casein and corn gluten with pepsin or trypsin was tested. The bitterness of soy peptide digested with pepsin was completely eliminated by treatment with CPase Tpa, whereas the bitterness of casein digested with trypsin and corn peptide digested with pepsin were somewhat less efficient. On the basis of these results, it is anticipated that CPase Tpa would be effective in eliminating the bitterness of some bitter peptides.

Identification of a novel non-AT1, non-AT2 angiotensin binding site in the rat brain

Efforts to protect radiolabeled angiotensins from metabolism during receptor binding assays date back more than 30 years. However, this continues to be a problem. This study focused on the effects of a protease inhibitor, p-chloromercuribenzoate (PCMB), on the binding of 125I-Ang II to rat brain membranes. Addition of PCMB to the incubation medium revealed a high affinity binding site for 125I-Ang II in brain membranes ( K d = 1–4 nM) with a greater amount of binding than revealed in previous studies of brain Ang II receptors. Further characterization of this binding, revealed it to be insensitive to inhibition by losartan (an AT 1 receptor antagonist) and PD123319 (an AT 2 receptor antagonist). This non-AT1, non-AT2 binding site was not present in liver or adrenal membranes. It was activated by a limited range of concentrations of PCMB, with maximal activation at 0.3–1 mM. This binding site was equally abundant in cerebral cortex (a brain region with few Ang II receptors) and the hypothalamus (a brain region with abundant Ang II receptors). The binding site was also present in mouse brain, but not mouse liver. The binding site shows high affinity for Ang I, Ang II and Ang III ( K i ∼ 40–100 nM), but lesser affinity for smaller angiotensin fragments and other neuropeptides. This binding site shares some characteristics with the liver cytosolic Ang II binding proteins, later identified as endopeptidases EC 3.4.24.15 and/or EC 3.4.24.16. However, some unique characteristics of this non-AT1, non-AT2 binding site suggest that it may be a novel angiotensin binding substance.

설프히드릴 변형 화합물질들에 의한 양배추 포스포리파제 D의 시스테인 잔기의 특성

SH group modifying chemicals were used to characterize the eight cysteine residues of cabbage PLD. 5,5-dithiobis(2-nitrobenzoate)(DTNB) was used to titrate the SH group of cysteine residues . Based on the optical density at 412nm due to the reduced DTNB, 4 SH groups are found to be present in a native PLD while 8 SH groups in the denatured PLD whose tertiary structure was perturbed by 8M urea. The results imply that among the 8 cysteine residues of PLD, the half(4) are exposed on the surface whereas the other half are present at the interior of the enzyme tertiary structure. The PLD was inactivated by SH modifying reagents such as p-chloromercuribenzoate(PCMB), iodoacetate, iodoacetamide, and N-ethylmaleimide. At the addition of dithiothreitol(DTT) only the PCMB inhibited PLD activity was recovered reversibly. The micro-environment of the exposed SH group of cysteine residues was examined with various disulfide compounds with different functional groups and we found that anionic or neutral disulfides appear to be more effective than the positively charged cystamine for inactivating the PLD activity. The effect of redox state of cysteine residues on the PLD activity was further explored with H2O2. The oxidation of SH groups by H2O2 inhibited the PLD activity more than 70%, which was mostly recovered by DTT. From these results, we could confirm chemically that all the cysteine residues of PLD are present as in their reduced SH forms and the 4 SH groups exposed on the surface of the enzyme may play important roles in the regulation of PLD activity.

Monocarboxylate transporter 1 (MCT1) plays a direct role in short‐chain fatty acids absorption in caprine rumen

Despite the importance of short-chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT-PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen >/= reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double-immunofluorescence confocal laser-scanning microscopy confirmed the co-localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p-chloromercuribenzoate (pCMB) and p-chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.

Monocarboxylate transporter 1 (MCT1) mediates transport of short‐chain fatty acids in bovine caecum

The present study was undertaken to investigate the functional role of monocarboxylate transporter 1 (MCT1) in the ruminant large intestine. Messenger RNA encoding for MCT1 was verified by reverse transcriptase-polymerase chain reaction in caecum, proximal colon and distal colon of adult cattle. Both immunohistochemistry and confocal laser microscopy verified that the MCT1 protein was abundant in the surface epithelium of the large intestine, and the amount decreased from the opening of the crypt to its base. In the immunopositive cells, MCT1 was primarily localized in the basolateral membranes of epithelium lining the large intestine. Western blotting indicated that the levels of MCT1 protein were highest in the caecum, followed by proximal colon and then distal colon. In vitro studies were conducted to elucidate the possible involvement of MCT1 in the transport of short-chain fatty acids (SCFA) across the isolated mucosal sheets of cattle caecum using the Ussing chamber technique. Acetate absorption was found to be pH dependent, and the rate of acetate absorption increased as pH decreased. The serosal application of the MCT1 inhibitor 'p-chloromercuribenzoic acid (pCMB)' significantly reduced the transport of acetate across the caecal epithelium of cows. In addition, the transport of acetate was significantly reduced in the presence of its analogue, propionate, indicating that acetate and propionate compete for binding to the same transporter. The results show that MCT1 is a major route for SCFA efflux across the basolateral membrane of bovine large intestine and that it could play a role in the regulation of intracellular pH.

Inhibitory kinetics of mercuric ion on the activity of β- N-acetyl- d-glucosaminidase from green crab ( Scylla serrata)

Chemical modification of p-chloromercuribenzoate (PCMB) on β- N-acetyl- d-glucosaminidase (NAGase, EC 3.2.1.52) from green crab ( Scylla serrata) has been studied. The results show that sulfhydryl group is essential for the activity of the enzyme. Inhibitory kinetics of the enzyme by mercuric chloride (HgCl 2) has been studied using the kinetic method of the substrate reaction during inhibitor of enzyme. The kinetic results show that the inhibition of the enzyme by mercuric ion (Hg 2+) at lower than 1.0 μM is a reversible reaction with residual activity and the inhibition belongs to be competitive. The inhibition kinetics model of Hg 2+ on the enzyme was set up and the microscopic rate constants were determined and the data obtained were well fitted with the model. It was also turned out that only one molecule of HgCl 2 binds to the enzyme molecule to lead the enzyme lose its activity. The above results suggest that the cysteine residue is essential for activity and is situated at the active site of the enzyme.

Irreversible kinetics of β- N-acetyl- d-glucosaminidase from prawn ( Penaeus vannamei) inactivated by mercuric ion

Cysteine residues in prawn ( Penaeus vannamei) β- N-acetyl- d-glucosaminidase (NAGase, EC 3.2.1.52) have been modified by p-chloromercuribenzoate (PCMB). The results show that sulfhydryl group is essential for the activity of the enzyme. Inactivation kinetics of the enzyme by mercuric chloride (HgCl 2) has been studied using the kinetic method of the substrate reaction during inactivation of enzyme previously described by Tsou. The kinetic results show that the inactivation of the enzyme is an irreversible reaction. The microscopic rate constants for the reaction of Hg 2+ with free enzyme and with the enzyme–substrate complex are determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by Hg 2+. The above results suggest that the cysteine residue is essential for activity.

Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors

The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC 50 values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 °C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB ( p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC 50 values of PCMB and bestatin for VACV and GVACV were found to be 3.81 ± 0.94 and 0.34 ± 0.08 μM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.

Effect of Arachidonic Acid Reacylation on Leukotriene Biosynthesis in Human Neutrophils Stimulated with Granulocyte-macrophage Colony-stimulating Factor and Formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.