Beta-fibrinogenase from the venom of Vipera lebetina

An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and pI approximately 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The Km and kcat values are for alpha-N-benzoyl-L-arginine ethyl ester (BAEE) 7.7 x 10(-5) M and 43.8 sec-1, for p-tosyl-L-arginine methyl ester (TAME) 3.6 x 10(-4) M and 39.8 sec-1 (pH 8.5, 25 degrees C, and for alpha-N-benzoyl-DL-arginine-4-nitroanilide (BAPNA) 1.8 x 10(-4) M and 0.94 sec-1 (pH 8.3, 25 degrees C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys12-Tyr13, Arg17-Arg18 and Arg18-Ala19. In fibrinogen it cleaves B beta-chain first and later also the A alpha-chain.     

Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.     

Purification and partial characterization of an arginine ester hydrolase from the venom of the bushmaster snake, Lachesis muta noctivaga

An arginine esterase was purified from the venom of Lachesis muta noctivaga by gel filtration on Sephadex G-100 and by affinity and DEAE cellulose chromatography. The purified enzyme preparation had an arginyl esterase specific activity of 181 mumoles/min/mg, which was 10.9-fold higher than the esterase activity found in the crude venom. The enzyme is free of kinin-releasing activity (kininogenase) and thrombin-like activity (fibrinogenase). The purified fraction showed a single band on polyacrylamide gel electrophoresis. The Km for Bz-L-Arg-O-Et is 1.14 X 10(-3)M, Vm 181.7 mumoles/min/mg and Kcat 90.9 sec-1. The pH profile indicates that the enzyme has an active region centered at pH 8.1. L. muta noctivaga arginyl esterase is reversibly inhibited by benzamidine (Ki 8.9 X 10(-4)M) and irreversibly inhibited by diisopropyl fluorophosphate.     

Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu).

A thrombin like enzyme, named elegaxobin II, with Lys-bradykinin releasing activity was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel-filtration on Sephadex G-100, and ion-exchange chromatography on the Q-Sepharose Fast Flow. By this procedure, about 9mg of purified enzyme was obtained from 1.1g of the venom. The purified enzyme showed a single protein band, the molecular weight of which was estimated to be about 35,000Da by sodium dodecyl sulfate-PAGE) under reducing condition, and this enzyme was found to contain a carbohydrate moiety. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 250 TAME units/mg of protein. This enzyme clotted only rabbit fibrinogen, whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin conversion, this enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas it did not release fibrinopeptide B. Furthermore, elegaxobin II released Lys-bradykinin when the enzyme was incubated with bovine plasma. The esterase activity was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), suggesting that this enzyme is a serine protease. The N-terminal sequence (Val-Ile-Gly-Gly) of this enzyme was identical to the typical sequence of serine proteinases.     

ISOLATION AND CHARACTERIZATION OF A NEW TRYPSIN-LIKE ENZYME FROM: Tenebrio molitor L. LARVAE

A trypsin-like enzyme (TLE) was separated and purified from Tenebrio molitor larval midgut enzyme solution by ion-exchange chromatography on a DEAE-cellulose column. The purified enzyme was found to be a homogeneous protein by electrophoresis in polyacrylamide gels and on cellulose acetate strips, by electrofocusing in polyacrylamide gels and by SDS-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 18300 by ultracentrifugal analysis and 24300 on SDS-polyacrylamide gel electrophoresis. It has an isoelectric point 8.0, it contains only four half-cystine residues per molecule and the NH₂-terminal amino acid is isoleucine. TLE possesses a high degree of specificity towards trypsin synthetic substrates such as N-α-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME) and poly-L-lysine hydrobromide. The optimal pH for TLE activity was found to be 8.0 and the optimal temperature 50° C. Its Km value when assayed on BAPNA was 0.93mM and on TAME 0.08mM. TLE is stable at neutral pHs and its activity is not affected by Ca²⁺ and by 0.01 M 1, 4-dithiothreitol (DTT). It is inactivated by DFP and tosyl-L-lysine chloromethylketone (TLCK) and is fully inhibited by the naturally occurring trypsin inhibitors such as trypsin-and α-chymotrypsin inhibitor (AA) from soybeans, basic pancreatic trypsin inhibitor (BPTI), chick peas trypsin and chymotrypsin inhibitor (CI) and crystalline soybean trypsin inhibitor (CSBTI), forming with them complexes in a molar ratio of 1:1. The Ki value for AA with BAPNA as substrate is 5.87 10^-7 M and for BPTI 7.92 10^-7M. No common antigenic determinants were noted between TLE and bovine trypsin. This finding together with the relatively low number of -S-S- bonds in the TLE molecule indicate that TLE differs in conformation from bovine trypsin.     

Purification and characterization of glucocorticoid-inducible steroid esterase in rat hepatic microsomes.

A steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was purified from the hepatic microsomes of rats treated with dexamethasone, a potent inducer of the esterase. The enzyme was solubilized by Lubrol WX and purified up to 30-fold over the microsomal fraction by ammonium sulfate fractionation and successive chromatographies with gel permeation, DEAE-cellulose and hydroxylapatite. The steroid esterase thus purified showed a single band and a molecular mass of 58 kDa on SDS-polyacrylamide gel electrophoregram. The enzyme appears likely to exist as two interconvertible forms, which can be distinguished by pI values, pI 4.9 and 5.1. The enzyme was completely inhibited by organic phosphates, indicating that it can be classed as a carboxylesterase (EC 3.1.1.1). Both negatively charged and uncharged esters of several steroids (methylprednisolone, hydrocortisone, deoxycorticosterone and dehydrotestosterone) as well as various non-steroidal esters including 4-nitrophenyl esters were hydrolysed by the enzyme, but none of the amides were substrates. The enzyme showed higher activity with increasing lipophilicity of the substrates. It is noticeable that the optimum pH for charged esters was 5.5, whereas the highest activity was observed around pH 7-8 for uncharged esters. When methylprednisolone 21-hemisuccinate (one of the charged esters) was used as substrate, the Km value was 2.8 mM and Vmax was 59.3 mumol/mg protein for 1 min at the optimum pH of 5.5. Regarding the methyl ester of methylprednisolone 21-hemisuccinate, Km and Vmax values were 1.8 mM and 193 mumol/mg protein/min, respectively, at the optimum pH of 7.0. On the basis of these results, the enzyme is most likely a carboxylesterase.     

Characterization of two arginine ester hydrolases from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0-8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-N-benzoyl-L-arginine methyl ester (BAME), p-tosyl-L-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The Km values for each enzyme with alpha-N-benzoyl-L-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.     

Purification and properties of a kininogenin from the venom of Lachesis muta (bushmaster).

An acidic kininogenin from Lachesis muta snake venom was purified to apparent homogeneity by a combination of gel filtration, isoelectric focusing and preparative gel electrophoresis. It was shown to be a highly stable serine protease (mol. wt 27,900; pI 5.4) capable of releasing bradykinin from low mol. wt bovine kininogen and of cleaving some synthetic chromogenic peptides with the following catalytic efficiencies (Kcat/Km, M-1.sec-1): N-benzoyl-Phe-Val-Arg-p-nitroanilide (1.92 x 10(4)); H-D-Val-Leu-Arg-p-nitroanilide (1.55 x 10(4)); N-acetyl-Phe-Arg-p-nitroanilide (3.98 x 10(2)); no hydrolysis was observed with N-benzoyl-Arg-p-nitroanilide. A marked and sustained hypotensive effect was recorded following i.v. injection of purified kininogenin into rats. Tachyphylaxis was observed after repeated i.v. injection of the enzyme, a phenomenon accompanied by a decrease of only 15% in the total circulating rat kininogen. Both the in vivo action and the enzymatic properties of the L. muta kininogenin indicate that this enzyme might be helpful for understanding the kinin-kininogen system.     

Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.     

Acetazolamide inhibition of renal gamma-glutamyl transpeptidase

The effect of acetazolamide (AZ) on renal gamma-glutamyl transpeptidase (EC 2.3.2.2) activity (gamma-GT) was studied with the purified enzyme, subcellular fractions, and in the isolated functioning kidney. Activity of gamma-GT was assessed using either one of two gamma-glutamyl donors, gamma-glutamyl-p-nitroanilide (gamma GpNA) or glutamine, and either the gamma-glutamyl acceptor glycylglycine (Gly-Gly) or methionine (Met). With the microsomal enzyme and beta-GpNA, AZ was shown to inhibit p-nitroaniline (p-NA) formation; however, gamma-GpNA Km remained unchanged (1.8 mM), while the Vmax was reduced significantly, 333 vs 200 mumoles . min-1 . mg-1. Adding Gly-Gly removed AZ inhibition, while AZ elevated the apparent Km from Gly-Gly from 16 to 48; AZ inhibition of gamma-GT activity resulted in a decrease in gamma-glutamyl-Gly-Gly formation consistent with interaction at the gamma-glutamyl acceptor site. With glutamine as the beta-glutamyl donor, AZ reduced NH3 and apparent gamma-glutamylmethionine formation in the purified enzyme in agreement with inhibition at the acceptor site. In the functioning kidney, perfused with 10(-3)M L- or D-glutamine, AZ (10(-3)M) markedly reduced NH3 formation and increased glutamine excretion, results consistent with AZ inhibition of the in situ gamma-GT.     

Characterization of low Km GTPase activity in rat brain: comparison of opioid and muscarinic receptor stimulation

Guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) have an essential role in mediating the actions of drugs on neurotransmitter receptors by coupling them to their effectors with the attendant hydrolysis of GTP. The resulting GTPase activity was characterized in rat brain with a view toward selecting conditions under which specific hormone-stimulated activity could be monitored. Kinetic analysis with washed membranes suggested the presence of two distinct GTPases, a low Km GTPase with an apparent Km value of 0.35 +/- 0.04 microM and apparent Vmax of 108 pmol min-1 mg protein-1, together with a much higher Km component. Low Km (but not high Km) GTPase activity is stimulated by muscarinic and opioid agonists and inhibited by a nonhydrolyzable analogue of GTP, providing further evidence that the low Km component is a distinct enzyme. The activity of the low Km component is a linear function of protein concentration (20-100 micrograms/mL), time (2-10 min), and temperature (25-37 degrees C). The specific activity of the low Km component is selectively increased by approximately 50% in purified synaptic membranes compared with the washed membrane preparation. Both carbamylcholine-stimulated and basal low Km GTPase activities, but not the high Km component, are inhibited by a nonhydrolyzable analogue of GTP but not by the comparable analogue of ATP, demonstrating the specificity of low Km GTPase for guanine nucleotides. Opioid- and muscarinic-stimulated GTPase activities are additive in brain, suggesting that the two receptor systems are associated with different domains of G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a Vmax of 2141+/-500 nmol/min/mg and a Km of 11+/-4 microM. This enzyme was inhibited by pyrazole with a KI of 3.1+/-0.57 microM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a Vmax of 115 nmol/min/mg and a Km of 15+/-2 microM and was not inhibited by pyrazole.     

Thrombin-like enzyme, flavovilase, with kinin-releasing activity from Trimeresurus flavoviridis (habu) venom

A thrombin-like enzyme, flavovilase, with kinin-releasing activity was isolated, purified, and characterized from the venom of Trimeresurus flavoviridis (habu) using Sephadex G-100, DEAE-Cellulose, and CM-Cellulose column chromatographies. The final preparation was homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing electrophoresis. The enzyme possesses a molecular weight of 26,500, an isoelectric point of 5.0, and consists of 247 total amino acid residues. Specific electrolytic activities of this enzyme on N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE) were determined to be 50.9 and 17.4 micromol/min/mg, respectively. The enzyme was inhibited by p-APMSF (p-amidinophenylmethanesulfonyl fluoride hydrochloride), beta-mercaptoethanol, and N-bromosuccinimide. Additionally, the enzyme was found stable to heat treatment. It was also observed that the enzyme cleaved a kininogen analog with the release of bradykinin.     

纳豆激酶的分离纯化及酶学性质研究

目的研究纳豆激酶分离纯化工艺及酶学性质。方法纳豆激酶发酵液的粗提物经Superdex 75凝胶色谱和聚丙烯酰胺凝胶电泳(PAGE)分离纯化,采用TAME法测定酶的活性,通过SDS-PAGE对纯化结果进行了检验。结果SDS-PAGE中显示单一色带,相对分子质量28000,以TAME为底物时纳豆激酶的米氏常数(Km)为35.47mmol/L,最适宜的温度37℃,最适宜pH为8.6。结论该分离纯化方法可以得到较纯的纳豆激酶。     

Enhanced expression, purification, and characterization of a novel class alpha glutathione S-transferase isozyme appearing in rabbit hepatic cytosol following treatment with 4-picoline.

A novel class alpha glutathione S-transferase (GST) isozyme is expressed in the hepatic cytosol of rabbits treated with 4-picoline. SDS-PAGE analysis revealed the presence of a new 28-kDa band which cross-reacted with class alpha GST-specific IgG. This new GST isozyme was isolated from the hepatic cytosol of 4-picoline-treated rabbits and purified to homogeneity using S-hexylglutathione-agarose, CM-Sepharose, and PBE118 chromatofocusing chromatography. The isozyme was determined by SDS-PAGE and gel filtration analyses to be a homodimer of approximately 28 kDa with blocked N-terminus. A heterodimer consisting of 25 and 28 kDa subunits with activity toward the substrate 1-chloro-2,4-dinitrobenzene was also purified. Immunoblot analysis revealed that the 25, 26.5, and 28 kDa bands cross-reacted with class alpha GST-specific IgG and failed to react with either class mu or class pi GST-specific antibodies. The 28 kDa enzyme had a pI of 8.2 as determined by nonequilibrium pH gel electrophoresis. The purified 28 kDa enzyme exhibited activity toward 1-chloro-2,4-dinitrobenzene (Km = 1.60 mM and Vmax = 73.5 mumol/min/mg) and cumene hydroperoxide (Km = 1.02 mM and Vmax = 6.92 mumol/min/mg). Amino acid sequence analysis of several fragments resulting from cyanogen bromide cleavage of the 28 kDa GST isozyme revealed a class alpha GST consensus sequence. In addition, proteolytic digestion with alpha-chymotrypsin yielded peptide maps which showed distinct differences between the purified 28 kDa GST and another purified class alpha GST isozyme present in rabbit liver. These results provide evidence that class alpha GST isozymes containing a novel 28 kDa subunit are expressed following treatment with 4-picoline.     

Cloning, high level expression of human paraoxonase-3 in Sf9 cells and pharmacological characterization of its product

Human paraoxnase-3 (hPON3) (EC3.1.8.1) is a lipid-associated enzyme with antioxidant activity, and can inhibit the oxidation of low-density lipoprotein (LDL), thereby inhibiting early atherogenic process. In the present study, human PON3 gene was cloned from Human Fetal Liver Marathon-Ready cDNA and expressed in insect cells using baculovirus vector. Twenty-eight milligrams of purified recombinant hPON3 (rhPON3) was obtained from 1L Sf9 cells culture. The Km and Vmax values of rhPON3, with respective to phenylacetate hydrolysis were 7.46+/-4.40 mM and 89+/-10.54 U/mg (n=3). The kinetic parameters of Vmax and Km for dihydrocoumarin hydrolysis by rhPON3 were 698+/-248 U/mg and 0.84+/-0.24 mM (n=3). LDL oxidation assay indicated that rhPON3 could effectively protect LDL against copper-induced oxidation in vitro.     

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl-N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2-oxoquinolyl oxy)] -butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 micrograms/ml collagen was 5 microM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 microM, respectively. We purified over 480-fold the soluble low Km form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sepharose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a Km value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 microM respectively, and a Vmax value of 85.3 and 19.8 nmole/min/mg protein, respectively. Ki value of the OPC-13135 for the enzyme was 0.015 microM and was of competitive fashion against cyclic AMP.     

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.     

Biochemical characterization of the soluble alkaline phosphatase isolated from the venomous snake W. aegyptia

A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP.     

Anti-inflammatory and immuno-modulatory effects of novel retinoid-like 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10-23 and the level of plasma fibrinogen (MED approximately 25 mumoles/kg). When given therapeutically (75 mumoles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25-75 mumoles/kg). Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12-15) responded poorly to the T cell mitogen, Con A (2.5 micrograms/ml) and the B cell mitogen, LPS (10 micrograms/ml). The responses were partially restored (approximately 30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 mumoles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.