Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.     

Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).     

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A280/A260 less than 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

Purification of a retinol binding protein from the hen-oviduct cytosol and its immunological cross-reactivity with those from the nucleus

Cellular retinol-binding protein was purified from the cytosol of the oviducts of laying hens by ammonium sulphate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex A-50 columns. Analysis of the purified retinol-binding protein on 10% SDS-polyacrylamide gel revealed the presence of a doublet representing very similar molecular sizes. Antiserum was prepared against the purified cellular retinol-binding protein, and on the basis of (a) immunodiffusion test and (b) immunoneutralization of 3H-labelled retinol-cellular retinol-binding protein complex on a column of Sephadex G-75, the antiserum appeared to be specific. The antiserum showed cross-reactivity with the nucleosol and a 0.4 M NaCl extract of the chromatin of the oviduct nuclei, while it did not react with the major egg-white proteins such as ovalbumin, conalbumin and ovomucoid.     

Purification and properties of the anticoagulant principle of Trimeresurus gramineus venom.

By means of DEAE-Sephadex A-50 column chromatography, Trimeresurus gramineus venom was separated into 12 fractions. Fraction 8 had marked anticoagulant action in the tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. Fraction 8 was rechromatographed on Sephadex G-100, then on DEAE-Sephadex A-50 again, and finally on Sephadex G-100, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single symmetrical boundary with 1.70 Svedberg units was obtained by ultracentrifugation. The estimated molecular weight was 19 500. The isoelectric point was pH 4.5. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was 3.5 times higher than that of the crude venom. Fraction 5 potentiated its anticoagulant activity to 10 times higher than that of the crude venom. This principle did not possess caseinolytic, tosyl-L-arginine methyl ester esterase, phospholipase A, phosphodiesterase, alkaline phosphomonoesterase, fibrinolytic, hemorrhagic or local irritating activities. The purified anticoagulant principle did not destroy fibrinogen, induce fibrinolysis, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen. However, a marked inhibition of prothrombin activation was caused by the anticoagulant principle. The inhibition of prothrombin activation was not due to the destruction of prothrombin or its activation factors, but due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom.     

Human placental anticoagulant protein: isolation and characterization

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Partial purification and characterization of membrane-bound and cytosolic phosphatidylinositol-specific phospholipases C from murine thymocytes

Membrane-bound and cytosolic phosphatidylinositol (PI)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographies on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolic enzyme increased more than 1,800- and 1,400-fold, respectively, compared with those of microsomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70,000 by gel filtration. These purified enzymes also hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2 in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphatidylcholine enhanced the activity of the membrane-bound enzyme, while phosphatidylcholine (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.     

膜荚黄芪种子中2种几丁质酶的分离纯化及活性测定

运用传统的层析技术对膜荚黄芪种子中两种几丁质酶进行分离纯化,并对分离纯化中各个步骤得到的蛋白进行了活性研究.结果表明:(1)粗提液的硫酸铵沉淀经再生几丁质亲和柱和凝胶过滤层析Sephadex G-75得到几丁质酶A.(2)粗提液的硫酸铵沉淀经离子交换色谱DE-52、CM、凝胶过滤层析Sephadex G-75得到几丁质酶B.(3)几丁质酶A、B的比活性分别为35.6 U/mg和4.1 U/mg.(4)从膜荚黄芪种子中分离纯化的几丁质酶A和B均为糖蛋白,含糖量分别为6.1%和5.8%.(5) SDS-PAGE显示,几丁质酶A、B的分子量分别为35.5 ku、39.6 ku;凝胶过滤层析测定几丁质酶A、B的分子量分别为36.9 ku、40.8 ku;表明几丁质酶A、B均为单亚基蛋白.     

Purification and activity of two phospholipase enzymes from Naja nigricolis nigricolis Reinhardt venom

Two phospholipase enzymes NN1 and NN2 were purified from the venom of Naja nigricolis nigricolis Reinhardt to apparent homogeneity. NN1 was purified by a two-step anion-exchange chromatography on DEAE-cellulose column while NN2 was purified by a combination of anion-exchange chromatography and gel filtration on Sephadex G-150. The enzyme NN1 moved homogenously on acrylamide gel as a monomer with a molecular weight of 65 kDa while NN2 was a dimer of 71 kDa. Both enzymes were clearly separated. Both enzymes hydrolyzed L-alpha-phosphatidyl choline with activities of 345.5 for NN1 and 727.8 micromol min(-1) x mg(-1) for NN2. The dimeric 71-kDa enzyme has a higher haemolytic and anticoagulant activity than the monomeric 65-kDa enzyme. It is apparent that the dimeric enzyme has a more pronounced activity than the monomer has, thus toxic activity may be related to the hydrolysis of phospholipids.     

Purification of 5 beta-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Cytochrome c3 from the sulfate-reducing anaerobe Desulfovibrio africanus Benghazi: purification and properties.

Cytochrome c3 was purified from Desulfovibrio africanus Benghazi by extraction with alkaline deoxyribonuclease, fractionation with ammonium sulfate, batch elution from carboxymethyl Sephadex followed by chromatography on the same resin, and gel filtration on Sephadex G-75. The preparation was judge homogeneous by a variety of criteria. The molecular weight was determined in an analytical ultracentrifuge, and values between 14,400 and 15,490 were obtained, depending upon the presumed value of partial specific volume. Gel filtration on a calibrated column of Sephadex G-75 gave a value of 14,900 daltons. The amino acid composition was very similar to that observed for the cytochrome from other species of Desulfovibrio, with the exception of increased levels of ThR and PhE. S-Carboxymethylation of the protein before and after heme removal by HgCl2 demonstrated eight Cys molecules involved in heme binding or four heme sites per molecule. Titration with sodium dithionite under N2 gave an electrochemical potential (E' 0) of -276 mV relative to the normal hydrogen electrode. Electrochemical titration of the cytochrome gave a Nernst plot with two linear regions with E' 0 values of -0.376 and -0.534 V. The spectra produced at various potentials exhibited shifts in isosbestic points upon reduction, suggesting changes in conformation during the reaction.     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.