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.     

长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化方法研究

目的:研究长白山白眉蝮蛇蛇毒中类凝血酶的简单分离纯化方法。方法:采用DEAE-Sephadex A-25及Sephadex G-25层析的方法,比较二者对长白山白眉蝮蛇蛇毒中类凝血酶简单分离纯化的效果。结果:从长白山白眉蝮蛇蛇毒中分离出类凝血酶,SDS-PAGE电泳显示为一条带,分子量大约为35.5kDa,达到电泳纯。理化性质研究表明,此类凝血酶具有体外凝血活性,体外凝血酶比活力为12.57IU.mg-1,用N-苯甲酰-L-精氨酸乙酯盐酸盐测得该酶的精氨酸酯酶比活力为137.65IU.mg-1。用蛋白酶抑制剂和乙二胺四乙酸对该酶进行抑制实验,结果表明该酶属于丝氨酸蛋白酶,而不是金属蛋白酶。结论:本方法可用于长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化。     

蛋白酶抑制剂对人类肥大细胞类胰蛋白酶和类糜蛋白酶催化活性的调节(英文)

AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similarexperimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similarprocedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacrylgel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined byenzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native proteaseinhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain,benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsininhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymaticactivity of chymase. Native protease inhibitors α-antitrypsin and secretory leukocyte protease inhibitor (SLPI)inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymaticactivity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitorstested had relatively good selectivity to either tryptase or chymase.     

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.     

Purification and characterization of a prothrombin-activating protease from Nephila clavata.

We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.     

Simultaneous determination of ACE activity with 2 substrates provides information on the status of somatic ACE and allows detection of inhibitors in human blood

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of 2 homologous domains, each bearing a Zn-dependent active site. The ratio of the rates of hydrolysis of 2 synthetic substrates, Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), is characteristic for each type of ACE: somatic 2-domain 1, N-domain 4.5, and C-domain 0.7 (Williams et al, 1996). We hypothesized that inactivation or selective inhibition of the C-domain within the somatic ACE should increase the ratio from 1 toward higher values, whereas inactivation or inhibition of the N-domain should decrease the ratio to lower values. Temperatures in the 40-60 degrees C range cause preferential inactivation of the C-domain in somatic ACE and an increase in the ZPHL/HHL ratio. Determination of the ZPHL/HHL ratio in freshly 100-fold concentrated urine ruled out the existence of the N-domain fragment in human urine, which appears only as a result of long storage. Inhibition of ACE by common inhibitors also increases the ZPHL/HHL ratio for 2-domain ACE, thus providing a sensitive method for the detection of ACE inhibitors in biological fluids. Therefore, simultaneous measurements of ACE activity with 2 substrates (ZPHL and HHL) and calculation of their ratio allow us to monitor the status of the ACE molecule and detect ACE inhibitors (endogenous and exogenous) in human blood and other biological fluids. This method should find wide application in monitoring clinical trials with ACE inhibitors and in the development of the approach for personalized medicine by these effective drugs.     

New specific assays for tonin and tissue kallikrein activities in rat submandibular glands. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.

At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.     

Phospholipase A2 from Bothrops alternatus (víbora de la cruz) venom. Purification and some characteristic properties

One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.     

Glutathione S-transferase-dependent conjugation of leukotriene A4-methyl ester to leukotriene C4-methyl ester in mammalian skin.

The glutathione S-transferase (GST)-dependent conjugation of reduced glutathione (GSH) with leukotriene A4 (LTA4)-methyl ester in rodent and human skin was investigated. Incubation of [3H]LTA4-methyl ester (1 nmole, approximately 200,000 dpm) with cytosol prepared from rat, mouse and human skin or with affinity purified GST from rat skin cytosol in the presence of GSH resulted in the formation of LTC4-methyl ester. Maximum enzyme activity was observed in rat skin followed by mouse and human skin. With heat-denatured cytosol or in the absence of GSH, the product formation was negligible. GST purified from rat skin cytosol by GSH-agarose affinity chromatography exhibited a several-fold increase in the specific activity of enzyme with 1-chloro-2,4-dinitrobenzene (55-fold), ethacrynic acid (67-fold) and LTA4-methyl ester (12-fold) as substrates. Western blot analysis of the affinity purified GST indicated a predominant expression of the Pi class of GST isozyme followed by Mu and Alpha classes of isozymes. The formation of LTC4-methyl ester was established by its radioactivity profile on high pressure liquid chromatography and absorption spectroscopy. These results suggest that, in addition to xenobiotic metabolism, cutaneous GSTs may also be capable of metabolizing physiological substrates such as LTA4.     

一氧化氮合酶抑制剂和增强剂的高通量筛选

目的建立一氧化氮合酶(NOS)活性的高通量检测方法，筛选调节NOS活性的药物。方法通过NADPH荧光值的变化，间接反映NOS活性。通过对反应体系的优化，调整各反应底物(NADPH，L-Arg，NOS)及抑制剂(L-NNA)的浓度，建立高通量筛选模型并对5 600个样品进行筛选。结果方法简便、容易操作、灵敏度高、结果稳定，发现了一批对NOS具有抑制或增强作用的化合物。结论此法适用于高通量药物筛选。     

A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.     

Glucose metabolism in rat mast cells. Stimulation of the pentose phosphate pathway by compound 48/80

The glycogen content of rat peritoneal mast cells (mean: 3 nmoles/10(6) cells) was increased 15% by incubation with glucose (1 mM) and reduced 35% when incubated without glucose at 37 degrees C for 15 min. The storage capacity for glycogen is thus low. Lactate production at 37 degrees C in a substrate-free medium was low (2.5-6.3 nmoles/10(6) cells in 40 min), but was stimulated 5-fold in the aerobic medium and 10-15 fold in the anaerobic medium by glucose. Both aerobic and anaerobic glycolysis in presence of glucose can thus provide energy for histamine secretion. The initial enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, have been demonstrated in mast cells. The enzyme activity in mast cells was, however, low compared to the high activity in the other peritoneal cells. The extent of the pentose cycle activity was determined from the conversion of 14C1- and 14C6-glucose to 14CO2, expressing the specific 14CO2 yields as fractions of the total glucose utilization. The normal pentose cycle activity with 1 mM glucose was 0.4% of the glucose metabolism. This was remarkably simulated by an electron acceptor, phenazine methosulfate. The pentose cycle was enhanced to 0.71% (80% stimulation) after exposure of the mast cells to compound 48/80, causing 68% histamine release. The stimulation of the pentose cycle by compound 48/80 seems to be due to the enhancement of biosynthetic processes during the regenerative phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu.

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoelectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIHU/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degrees C and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 degrees C. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4A.     

Identification and characterization of nitric oxide synthase inSalmonella typhimurium

The presence of the nitric oxide synthase (NOS) enzyme from Salmonella typhimurium (S. typhimurium) was identified by measuring radiolabeled L-[3H]citrulline and NO, and Western blot analysis. NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially. The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis. The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between 37 degrees C and 43 degrees C. The activity was inhibited by NOS inhibitors such as aminoguanidine and N(G),N(G)-dimethyl-L-arginine. Taken together, partially purified NOS in S. typhimurium is assumed to be a different isoform of mammalian NOSs.     

Co-oxidation of acrylonitrile by soybean lipoxygenase and partially purified human lung lipoxygenase.

1. Human lung lipoxygenase (HLLO) was partially purified by concanavalin-A (Con-A) affinity chromatography that provided an easy and rapid one-step procedure for the removal (> or = 96%) of haemoglobin from cytosol. 2. HLLO exhibited dioxygenase activity towards arachidonic acid (AA) and linoleic acid (LA). The dioxygenase activity towards LA varied approximately 12-fold (48-591 nmol/min/mg protein) among different human lung samples examined. 3. Reverse-phase HPLC analysis of AA metabolites indicated the predominance of 15-lipoxygenase in human lung cytosol. 4. HLLO exhibited co-oxidase activity towards benzidine (BZD) and several other model compounds. The co-oxidase activity towards BZD was significantly inhibited by several lipoxygenase inhibitors. 5. HLLO and soybean lipoxygenase (SLO), used as a model enzyme, metabolized acrylonitrile (ACN) to 2-cyanoethylene oxide (CEO) and ultimately to cyanide. 6. HLLO was a approximately 6-fold better catalyst than SLO in converting ACN to cyanide. The generation of cyanide by HLLO was dependent on the concentration of enzyme and the reaction was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-oxidant butylated hydroxytoluene (BHT). 7. Under optimal assay conditions, the covalent binding of HLLO-generated reactive intermediate(s) from [14C]ACN to protein and DNA (nmol equivalent bound/15 min/mg HLLO/mg bovine serum albumin or calf thymus DNA) was observed at approximately 1.20+/-0.13 and 2.20+/-0.50 respectively. Both protein and DNA binding were inhibited by NDGA, butylated hydroxyanisole (BHA) and BHT.     

Purification and Characterization of a C-S-Lyase from Ramson, the Wild Garlic,Allium ursinum

A C-S-lyase preparation from ramson, ALLIUM URSINUM L., has been purified to apparent homogeneity. Separation techniques applied were hydrophobic interaction chromatography, anion exchange chromatography, and gel permeation chromatography. A 52-fold purification was obtained. The enzyme could be characterized by a molecular mass of M (r) = 150000 with subunits of 50 000. Its isoelectric point was determined to be at 4.7. The pH-optimum for the substrate-dependent turnover was found at 6.0. The temperature optimum was at 35 degrees C. (+)-Alliin as the substrate caused the highest enzymatic reaction velocity. The lowest K (m) value was observed with (+)- S-propyl- L-cysteine sulfoxide. Inhibitor constants were elaborated for the deoxy-derivatives of the substrates inserted and, likewise, for related amino acids. The protein was sensitive to low concentrations of hydroxylamine, indicating pyridoxal phosphate as a cofactor. Activation energies were determined for the cleavage of alliin, S-propyl- L-cysteine sulfoxide and S-methyl- L-cysteine sulfoxide, and were found to be in the range of 9 to 13 kJ . mol (-1).     

Stability of luteinizing hormone releasing hormone: effects of pH, temperature, pig skin, and enzyme inhibitors

The purpose of this investigation was to determine the stability of luteinizing hormone releasing hormone (LHRH) as a function of solution pH, temperature, and pig skin with and without enzyme inhibitors. LHRH, incubated with a 0.1 M phosphate buffer (pH 2.5-8.1), pig skin, and pig skin with enzyme inhibitors, was analyzed using reversed-phase high performance liquid chromatography. The solution's pH affected the rate constants of LHRH, following apparent first-order kinetics. Maximum stability was achieved at pH 6.05. Therefore, the effect of various temperatures (i.e., 65, 75, 80, and 90 degrees C) was studied on the stability of LHRH at pH 6.05. The activation energy for the overall reaction was 23.4 kcal/mol at pH 6.05. The shelf-life of LHRH at 25 degrees C and pH 6.05, calculated using the Arrhenius equation, was approximately 4 years. The rate constant of LHRH in the skin (area: 9 cm2; thickness: 0.5 mm) was 0.167 hr-1. Out of three inhibitors (i.e., aprotinin, bestatin, and leupeptin), bestatin had the best stabilizing effect on the degradation of LHRH by the skin. The rate constant of LHRH in the presence of bestatin was 0.082 hr-1. Sixty percent of LHRH was found to be degraded in the skin within 5 hr in the absence of enzyme inhibitors, whereas only 33% of LHRH was degraded in the presence of bestatin (an aminopeptidase inhibitor).     

Studies of UDP-glucuronyltransferase activities in human liver microsomes

Human liver samples from a "liver bank" which have been previously characterized by the ability to catalyze cytochrome P-450 dependent reactions were analyzed for various UDP-glucuronyltransferase activities. When stored at -80 degrees C, UDP-glucuronyltransferase activities and latency characteristics of the enzyme appeared to be stable for up to 2 years. The correlation of UDP-glucuronyltransferase activity (4-methylumbelliferone as substrate) with enzyme activities towards 4-nitrophenol and 1-naphthol was much higher (r greater than 0.8) than that (r less than 0.3) observed with other enzyme activities (4-hydroxybiphenyl, morphine, and chloramphenicol as substrates), suggesting the presence of multiple enzyme forms in human liver. Livers of three patients treated with phenytoin or pentobarbital showed significantly higher UDP-glucuronyltransferase activities towards 1-naphthol, 4-methylumbelliferone, and bilirubin than those from five patients who did not receive these inducing agents.     

Properties of NAD glycohydrolase purified from five-pace snake (Agkistrodon acutus) venom

NAD glycohydrolase (NADase) (E.C. 3.2.2.5) from five-pace snake (Agkistrodon acutus) venom was purified to electrophoretic homogeneity through a 4-step isolation procedure, including column chromatography using DEAE-Sephadex A-50, Sephadex G-75, CM Sephadex C-50 and Sephadex G-100. The final product was 11.8-fold purified with a 3.9% yield. The pure enzyme showed maximal activity at about 40 degrees C with optimal pH at 7.5. It was a glycoprotein with a pI of 7.6. Its mol. wt was respectively 98,000 as measured by gel filtration and 50,000, by SDS-PAGE. There was only one N-terminal residue, proline. NADase is thus composed of two identical subunits in each molecule. The enzyme contained copper ions. NADase activity was lost when the copper enzyme complex was treated with EDTA. The Km of the enzyme for beta-NAD, NADP and beta-NGP were 0.50 mM, 0.13 mM and 0.16 mM respectively.     

A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids.

The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.