Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Purification and characterization of a beta-glucosidase from Trichoderma reesei

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.     

Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C4 to C16 fatty acids with decreasing V and increasing Km for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an "active serine" enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.     

Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.     

Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

Biochemical characterization of H9/25, an allospecificity encoded by the Ly-6 region

H9/25, an allospecificity encoded by the Ly-6 region, was biochemically characterized. It was sensitive to pepsin and heat treatment, but was resistant to periodate oxidation. Its apparent molecular weight was approximately 12 000 daltons by gel filtration. The antigenic molecule was partially purified by gel filtration and antibody affinity chromatography. The partially purified antigen molecule was radioiodinated, immunoprecipitated with monoclonal antibody H9/25, and analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiograph showed the molecular weight of H9/25 to be approximately 15000 daltons under reducing conditions. These results indicate that H9/25 is a protein with a single polypeptide chain of 12000–15000 daltons molecular weight, and the antigenic specificity is carried by a peptide but not a carbohydrate moiety.     

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

Human alpha-N-acetylglucosaminidase. 1. Purification and properties.

alpha-N-Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. alpha-N-Acetylglucosaminidase is a glycoprrotein with an extensive charge heterogeneity. The molecular weight determined by gel filtration is 307000. Polycarylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 +/- 0.3 and KM and V values of 0.14-0.74 mM, and 1.04-3.68 mumol mg-1 min-1 for three aryl 2-acetamido-2-deoxy-alpha-D-glucosides and UDP-N-acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. --SH-protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65 degrees C and pH values below 5 inactivate the enzyme rapidly.     

Purification and properties of glucoside 3-dehydrogenase from Flavobacterium saccharophilum

A membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270,000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66,000. The enzyme reacted with various artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromercuribenzoate. D-Glucose and methyl-alpha- and beta-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.     

Some characteristics of the coagulation Factor Xa purified from human serum

1. Coagulation Factor X was purified from human serum to apparent homogeneity in disc gel electrophoresis, sodium dodecyl sulphate-polacrylamide-gel electrophoresis, immunoelectrophoresis and analytical ultracentrifugation. The method used was a modification of that described by Gladhaug & Prydz (1970). 2. The method permits the isolation of an activated form of Factor X (Xa) which has a molecular weight of about 25000. 3. Factor Xa is a glycoprotein containing about 14% carbohydrate. A preliminary report of the amino acid composition is given.     

The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure.

Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and properties of the human placental uracil DNA glycosylase

Human placental uracil DNA glycosylase was purified 3700-fold to apparent homogeneity as defined by SDS gel analysis. Its immunological characteristics were examined using three monoclonal antibodies prepared against partially purified human placental uracil DNA glycosylase. Immunoblot analysis demonstrated that, even in crude isolates, only one glycosylase species of molecular weight 37,000 could be detected. Each of the three monoclonal antibodies quantitatively recognized the highly purified enzyme by ELISA. The glycosylase is a single polypeptide with a molecular weight of 37,000 as defined by both Sephadex gel filtration and by SDS-polyacrylamide gel electrophoresis analysis. The enzyme is heat-stable, with a t 1/2 of greater than 30 min at 42 degrees C or at 45 degrees C. Surprisingly, inhibitor analysis demonstrated that the glycosylase was inhibited by preincubation with either 5-fluorouracil or 5-bromouracil. However, no significant inhibition was observed when either compound was added directly to the enzyme assay.     

Human liver alpha-L-fucosidase. Purification, characterization, and immunochemical studies.

Human liver alpha-L-fucosidase has been purified 6300-fold to apparent homogeneity with 66% yield by a two-step affinity chromatographic procedure utilizing agarose epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all six isoelectric forms of the enzyme were purified. Polyacrylamide gel electrophoresis of the purified alpha-L-fucosidase demonstrated the presence of six bands of protein which all contained fucosidase activity. The purified enzyme preparation was found to contain only trace amounts of seven glycosidases. Quantitative amino acid analysis was performed on the purified fucosidase. Preliminary carbohydrate analysis indicated that only about 1% of the molecule is carbohydrate. Gel filtration on Sepharose 4B indicated an approximate molecular weight for alpha-L-fucosidase of 175,000 +/- 18,000. High speed sedimentation equilibrium yielded a molecular weight of 230,000 +/- 10,000. Sodium dodecyl sulfate polyacrylamide gels indicated the presence of a single subunit of molecular weight, 50,100 +/- 2,500. The enzyme had a pH optimum of 4.6 with a suggested second optimum of 6.5. Apparent Michaelis constants and maximal velocities were determined on the purified enzyme with respect to the 4-methylumbelliferyl and the p-nitrophenyl substrates and were found to be 0.22 mM and 14.1 mumol/mg of protein/min and 0.43 mM and 19.6 mumol/mg of protein/min, respectively. Several salts had little or no effect on fucosidase activity although Ag+ and Hg2+ completely inactivated the enzyme. Antibodies made against the purified fucosidase were dound to be monospecific against crude human liver supernatant fluids and the pure antigen. No cross-reacting material was detected in the crude liver supernatant fluid from a patient who died with fucosidosis.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.