Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues

We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.     

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.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Specific modification of the functional arginine residue in soybean trypsin inhibitor (Kunitz) by peptidylarginine deiminase

In order to elucidate the specificity of rabbit muscle peptidylarginine deiminase, which catalyzes the conversion of arginyl to citrullyl residues (Takahara, H., Oikawa, Y., and Sugawara, K. (1983) J. Biochem. (Tokyo) 94, 1945-1953), we examined the action of this enzyme on a variety of trypsin inhibitors by assay of residual trypsin-inhibiting activity. The enzyme rapidly abolished the activity of soybean trypsin inhibitor (Kunitz) (STI) in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 5.0 X 10(4) M-1 S-1), whereas no detectable changes in activity were noted for other inhibitors tested. Inactivation of STI was due to the conversion of 1 arginine to a citrulline residue and was accompanied with a 0.2 unit decrease of the isoelectric point. There was no alteration of the molecular size and overall conformation of STI. Furthermore, analysis of modified STI indicated that arginine 63, known as the reactive site of STI, is the residue modified by peptidylarginine deiminase. Thus, peptidylarginine deiminase selectively catalyzes the deimination of the functional arginine residue of STI.     

Purification and subunit structure of recBC DNase from Escherichia coli harboring a recB and recC genes-inserted plasmid

recBC DNase of Escherichia coli has been purified from the transformant, HB101/pFS11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography. The purified enzyme was obtained in an overall yield of 3%. The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis. The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125,000 and 135,000, and isoelectric points of 5.6 and 5.7, respectively.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Purification de l'alcool deshydrogénase de tomate par chromatographie d'affinité

Tomato alcohol dehydrogenase has been purified 99-fold by affinity chromatography on Blue Sepharose CL-6B with 37% yield. The enzyme so obtained is homogenous in polyacrylamide gel electrophoresis. By adding 20% glycerol to the extraction and purification buffers, an enzyme is obtained which is stable for several months at 4°. The molecular weight values determined by gel filtration (Sephadex G 200) and polyacrylamide gradient gel electrophoresis on one hand and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on the other, show that the enzyme exists in dimeric form.     

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.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Purification and properties of acid phosphatase from cultured tobacco cells

One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.     

Identification of a plasma gelatinase in preparations of fibronectin

Preparations of fibronectin purified from human plasma according to conventional methods was found to contain a latent gelatinolytic activity. The protease was activated by exposure to trypsin or electrophoresis in sodium dodecyl sulfate. Zymography of the enzyme under nonreducing conditions gave an estimated Mr of 72,000. Reducing agents destroyed the activity of the enzyme. The gelatinase co-purified with fibronectin in chromatography on Sepharoses conjugated with gelatin, arginine, and heparin but could be separated from fibronectin by gel filtration in a physiological buffer. This protease was found to be a normal constituent of plasma and was probably not derived from the blood cells since the 72-kDa protease was not detected in lysates of these cells.     

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.     

Purification and some properties of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Purification and ligand-directed association-dissociation.

N-Acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8), the second enzyme of arginine biosynthesis, was purified over 2000-fold from Pseudomonas aeruginosa. The purification procedure involved a heat treatment, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, Sephadex G-150, and hydroxyapatite. The purified enzyme was greater than 90% pure as judged by analytical polyacrylamide gel electrophoresis. A molecular weight of approximately 230000 was obtained by gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 29000. Due to the capacity for self-association, the enzyme can exist in different states of aggregation depending on the nature of ligands and the concentrations of phosphate buffer. As estimated by gel filtration, the molecular weight was about 230000 in the presence of N-acetyl-L-glutamate. With L-arginine, the feedback inhibitor, and MgATP forms of smaller molecular weight (minimum of approximately 65000) were found. A concurrent change in the sedimentation coefficient as a function of ligands was demonstrated by sucrose gradient centrifugation. The synthesis of N-acetylglutamate 5-phosphotransferase was not repressed by exogenous L-arginine or its precursors.     

Purification and characterization of the sex steroid-binding protein of rabbit serum. Comparison with the human protein

The sex steroid-binding protein (rSBP) of immature rabbit serum was purified to homogeneity by the sequential use of DEAE-cellulose chromatography, affinity chromatography on 5alpha-dihydrotestosterone-17 beta-succinyl-diaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, agarose (Bio-Gel-A-0.5m) gel filtration, and preparative polyacrylamide gel electrophoresis. The cumulative yield is 13%. Homogeneity of rSBP was shown by the equilibrium sedimentation ultracentrifugation in 6 M guanidine HCl containing 0.1 M mercaptoethanol which yields an average molecular weight of 36,475 +/- 865. Analytical gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on agarose yield a molecular weight of 57,000 and 120,000, respectively. The variation is due to a 30% carbohydrate content. The amino acid composition is reported. Comparison of the rabbit and human SBP indicate that they are different in both their molecular and functional properties.