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.     

Studies on puff adder (Bitis arietans) venom. I. Purification and properties of protease A

The proteases from puff adder venom were separated by gradient elution from CM-cellulose, and protease A was further purified by Sephadex G-75 gel filtration. It was found to have a molecular weight of 21,400 and it showed a tendency to associate particularly at higher concentrations. The amino-acid composition and physical properties of protease A are reported. It had no esterase activity towards ATEE or TAME and could not be inhibited by DFP, TPCK, TLCK or PMA. The enzyme requires the presence of calcium to prevent autolysis.     

Isolation and some biochemical properties of a paralysing toxin from the venom of the wasp Microbracon hebetor (Say)

A toxin with paralysing activity was isolated from crude preparations of Microbracon hebetor (Say) venom by ion exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, electrophoresis on polyacrylamide gel and gel chromatography on Sephadex G-75. The active component is very labile. A 28-fold purification was obtained with a recovery of about 2.5% of the original biological activity. The toxin shows one single protein band after disc electrophoresis. The molecular weight was assessed at 61,000 by gel chromatography, and at 62,700 by the sedimentation equilibrium method. The isoelectric point was at pH 6.8. The purified toxin was inactivated by a number of proteolytic enzymes.     

Purification and properties of a lethal,hemorrhagic protein, “Mucrotoxin A”, from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Mucrotoxin A was purified from the lyophilized venom of Trimeresurus mucrosquamatus using gel filtration on a Sephadex G-100 column, followed by chromatography on CM-Sephadex C-50 and DEAE-Sephadex A-50. By these procedures, 14 mg of purified preparation could be obtained from 1 g of crude venom. The purified preparation was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. Mucrotoxin A possessed both lethal and hemorrhagic activities, but it did not show caseinolytic activity. Its molecular weight was approximately 94,000 and the isoelectric point was 4.3. Mucrotoxin A contains approximately 3 moles of Ca and 2 moles of Zn per mole of toxin. The amino acid composition of Mucrotoxin A was determined. No carbohydrate was present.     

Binding of amino beta-lactam antibiotics to soluble protein from rat intestinal mucosa--I. Purification of drug-binding protein

Several amino beta-lactam antibiotics, including ampicillin, amoxicillin, cyclacillin, cephalexin, cephradine and cefadroxil, were found to bind in vitro to specific components in 105,000 g supernatant of homogenate obtained from rat intestinal mucosa. The major binding component (fraction b) was purified by chromatography on DEAE-cellulofine and by gel filtration on Sephadex G-50. The molecular weight of fraction b was determined by SDS polyacrylamide gel electrophoresis (15,000 Da). The binding behaviour of these amino beta-lactam antibiotics to fraction b were estimated by equilibrium dialysis. There were significant high affinities of all tested amino beta-lactam antibiotics which were well absorbed from intestine, but there was not a good correlation between binding and absorption of these drugs. It was also found that poorly absorbed cephalosporins which lack aminobenzyl group in their structure, cefazolin and cephaloridine, did not bind to fraction b.     

Enzymes and toxins of the scorpion venom Palamneus gravimanus

Hyaluronidase activity (300 units per mg protein) and alkaline phosphatase activity (0.62 × 10^-3 units per mg protein) were found in the venom of the scorpion, Palamneus gravimanus. In addition, two toxins have been isolated from the crude venom by gel filtration on Sephadex G-100 and Sephadex G-25, and by isoelectric focusing. The molecular weight of the toxins was estimated to be about 7000, and the isoelectric pH was basic.     

Kinin-generating and esterolytic activity of purified human urinary kallikrein (urokallikrein)

Urinary kallikrein (urokallikrein), as defined by its capacity to generate kinin from heat-inactivated plasma or from purified human kininogen, was isolated from fresh concentrated male human urine and shown to be an antigenically unique urinary p-tosyl-L-arginine methyl ester HCl (TAMe) esterase. The isolation procedure achieved a 400- to 576-fold purification of the kinin-generating activity/mg of protein and yielded a product with albumin as the only significant contaminant at the isoelectric focusing step. The purified urokallikrein, defined by its kinin-generating activity, exhibited an isoelectric point with a range from pH 3.9 to 4.2 with charge heterogeneity, an apparent molecular weight of 25,000–40,000 on Sephadex gel filtration, and an anodal mobility on alkaline disc gels. Urokallikrein eluted from disc gels and identified by kinin-generating activity elicited monospecific antiserum in the rabbit. That purified urokallikrein is a TAMe esterase was evident from the concordance of kinin-generating activity, antigenic reactivity with a donkey antipancreas serum shown to recognize urokallikrein and esterolytic capacity as assessed after isoelectric focusing. There was suppression of the esterolytic activity of purified urokallikrein by increasing doses of TAMe or benzoyl-L-arginine methyl ester HCl (BAMe), and analysis of these data with Dixon plots indicated substrate inhibition.     

Purification and characterization of alpha-mucrofibrase,a novel serine protease with alpha-fibrinogenase activity from the venom of Chinese Habu (Trimeresurus mucrosquamatus)

A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.     

PURIFICATION AND HETEROGENEITY OF HUMAN KININOGEN.

Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five-step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14–20) from pooled human plasma, and containing approximately 30% alpha-2HS-glycoprotein and 2.8% albumin. Alpha-2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE-Sephadex using various linear gradients and gel filtration on Sephadex G-100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7–12 S sieve fractions from Sephadex G-200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption-desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha-2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti-kininogen. With 200 μg of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6–8 weeks in rabbits. With a polymer prepared with 4 ml anti-kininogen serum (1:8) and incubated with 800 μg highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption-desorption procedure.     

Isolation and characterization of a protein from Mendole (Spicara maena) eggs that binds to DNA and inhibits its replication as well as its acid precipitation

Proteins that bind and protect nucleic acids from acid precipitation have been characterized from human and mouse plasma. In the present study, one protein from Mendole (Spicara maena) eggs was purified to homogeneity, by means of acetone fractionation and Sephadex G-100 gel filtration. The protein inhibited DNA replication, exerted by various DNA polymerases. Amino-acid sequence analysis in the amino terminus revealed a unique sequence. Its possible physiological role is discussed.     

Isolation and identification of a collagenolytic enzyme from the venom of the western diamondback rattlesnake (Crotalus atrox)

A collagenolytic enzyme, with a molecular weight of 58,000 daltons and isoelectric point of 5.1, was isolated and purified from the venom of the rattlesnake Crotalus atrox by Sephadex G-100 gel filtration followed by chromatography on DEAE-Bio-gel A. The enzyme released alpha-chains from beta-chains of the native collagen and cleaved in the helical region similar to other animal collagenases. The enzyme also hydrolyzed the PZ-peptide, however, it did not hydrolyze synthetic substrates for serine protease (such as TAME or ATEE). The enzyme had no hemorrhagic activity. Immunocross-reactivity suggested that only the venom from Crotalidae contain the enzyme.     

Purification and partial characterization of rat intestinal cefuroxime axetil esterase

An esterase which hydrolyses the cephalosporin antibiotic, cefuroxime axetil has been isolated from rat intestinal washings and purified. Closely related cefuroxime esters were extremely poor substrates, but p-nitrophenyl acetate and alpha-naphthyl acetate were slowly hydrolysed by the purified enzyme. Analysis by gel filtration gave an Mr = 51,000 and on SDS-polyacrylamide gel electrophoresis the esterase resolved into two main bands of Mr = 31,500 and 26,800. Analytical isoelectric focusing resolved purified esterase into multiple forms active toward alpha-naphthyl acetate, the isoelectric points of which ranged from pH 4.5 to 6.3. The esterase bound specifically to Con A-Sepharose suggesting it could be a glycoprotein. Esterase activity was unaffected by the presence of dihydroxy bile salts (1-8 mM) and inhibition studies using organophosphates and eserine salicylate have classified the enzyme as a carboxylesterase.     

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.     

Purification of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

During the isolation of a capillary permeability-increasing enzyme from the venom of A. caliginosus, a kininogenase was also purified from the venom by gel filtration on Sephadex G-100, ion-exchange chromatographies on CM-Sephadex C-50 and DEAE-Sephadex A-50, and gel filtration on Sephadex G-75. By this procedure, 11 mg of the purified enzyme were obtained from 4 g of the venom. The purified enzyme was homogeneous by polyacrylamide disc gel electrophoresis at pH 8.3 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and did not show any caseinolytic or clotting activity. The purified enzyme released bradykinin from purified bovine high mol.wt kininogen. The capillary permeability was increased by injection of the purified enzyme into the depilated skin of the back of a rabbit. It is supposed that the capillary permeability-increasing activity exerted by the enzyme is due to the release of bradykinin.     

Extraction and Purification of Curcain,a Protease from the Latex of Jatropha curcas Linn

Abstract— A proteolytic enzyme, curcain, has been extracted from the latex of Jatropha curcas Linn. The enzyme was purified by chromatography on carboxymethyl cellulose and gel filtration on Sephadex G-200. The homogeneity of protein associated with curcain was established by non-denatured polyacrylamide gel electrophoresis using a discontinuous buffer system. The molecular weight of curcain was estimated by Sephadex G-100 gel filtration using a calibration curve of standard proteins to be around 22000 daltons.     

大鲵皮肤分泌液中抗菌肽的鉴定及生物活性研究

目的鉴定大鲵皮肤分泌液中抗菌肽,研究其部分生物活性。方法 5%醋酸浸提和Sephadex G-50、G-25凝胶过滤色谱等方法分离纯化抗菌肽;采用抑菌圈法检测抗菌活性,Tricine-SDS-PAGE电泳和等电聚焦电泳鉴定其抗菌活性成份。结果大鲵皮肤分泌液中含有抗菌活性物质。对革兰阴性菌、革兰阳性菌和真菌均具有较强的抗菌活性;电泳检测显示该小分子多肽相对分子质量约为4 300,具有较强的碱性。结论首次从大鲵皮肤分泌液中分离纯化到一种抗菌肽,此抗菌肽可能是一个具有较强阳离子特征的碱性肽。     

Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of beta-lactams in Cephalosporium acremonium

The catalytic properties of the partially purified deacetoxycephalosporin C (DAOC)-synthetase and DAOC-hydroxylase from an industrial strain of Cephalosporium acremonium were studied. After mechanical breakage of the cells, purification was achieved by fractional (NH4)2SO4 precipitation, gel chromatography on Sephadex G-75, ion exchange chromatography on DEAE-Trisacryl M and two isoelectric focusing steps. The two enzyme activities could not be separated. Indirect evidence was obtained from SDS-polyacrylamide gel electrophoresis of the purest fractions obtained by isoelectric focusing that the two reactions are catalyzed by a single enzyme with a molecular weight of 33,000 +/- 2,000 and a pI of 4.6 +/- 0.1. Both reactions require alpha-ketoglutarate, FeSO4, ascorbate and O2, whereas additional ATP shows only a slight stimulation.     

Purification and some properties of 3 alpha-hydroxysteroid dehydrogenase from pig adrenal cytosol]

A 3 alpha-reducing activity of 5 alpha-dihydrotestosterone (5 alpha-DHT) was found in pig adrenal cytosol. The enzyme (3 alpha-hydroxysteroid dehydrogenase: 3 alpha-HSD) has been purified to homogeneity from pig adrenal cytosol by ammonium sulfate precipitation followed by DEAE-cellulose, 2', 5'-adenosine diphosphate-Sepharose and Sephadex G-100 column chromatographies. The molecular weight was estimated to be 33,000 and 39,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point was estimated to be 8.5 by isoelectric focusing. The Km and Vmax values for 5 alpha-DHT in the reduction were 10.2 microM and 10.6 nmol/min/mg. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotine amide adenine dinucleotide (NADH) in the reduction as a cofactor, but it preferentially required NADPH rather than NADH. Furthermore, the purified enzyme catalyzed not only 3 alpha-reduction of 5 alpha-DHT (9.65 nmol/min/mg), but also catalyzed 20 alpha-reduction of 17 alpha-hydroxyprogesterone (0.58 nmol/min/mg). The enzyme activity of 3 alpha-HSD was strongly inhibited by Hg2+, but it was not inhibited by medroxyprogesterone acetate and some anti-inflammatory agents. No remarkable differences was demonstrated between 3 alpha-HSD and 20 alpha-HSD activity under the influence of heat treatment, divalent cation, anti-inflammatory agents and some inhibitory steroids. These results strongly suggest that 3 alpha-HSD purified from pig adrenal cytosol is a bi-functional enzyme catalyzing 3 alpha- and 20 alpha-HSD activities.     

Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi)

Two thrombin-like enzymes were purified from the venom of Agkistrodon caliginosus. Both were homogeneous on polyacrylamide gel electrophoresis, hydrolyzed $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester and did not show any caseinolytic activity. The molecular weights of the enzymes were estimated to be about 34,000–37,000 and 42,000–43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel-filtration on Sephadex G-100 column. One enzyme was purified from the venom by gel-filtration on Sephadex G-100 column, CM-Sephadex C-50 and DEAE-Sephadex A-50 chromatographies, affinity chromatography on p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B and gel-filtration on Sephadex G-100 column. The second enzyme was separated from the first enzyme by the DEAE-Sephadex A-50 chromatography mentioned in the above procedure and was isolated by affinity chromatography on arginine-Sepharose 4B and gel-filtration on Sephadex G-100 column. From 4 g of the venom, 0.88 mg of the first enzyme and 1.18 mg of the second enzyme were obtained, with specific activities of 118 and 139 NIH units per mg of protein, respectively.     

A low-molecular-weight cadmium-binding substance in human and rat livers and human blood

A cadmium-binding substance with a molecular weight even lower than that of metallothionein was demonstrated on Sephadex G-75 gel filtration chromatography of the soluble fractions from newborn human and adult rat liver homogenates and adult human hemolysate which were mixed with CdCl2 in vitro. This substance was purified from rat liver extracts by gel filtration and ion-exchange column chromatography and characterized by 6 M guanidine X HCl thin-layer gel filtration chromatography and N-terminal and total amino acid analyses. The results showed that the isolated low-molecular-weight cadmium-binding substance was a cadmium-reduced glutathione complex, whose molecular weight was found to be approximately 1400 by Sephadex G-15 gel filtration.