Purification of the constituent enzyme fractions of mycobacillin synthetase.

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.     

Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.     

Isolation of Active cAMP Dependent Protein Kinases from Calf Ovaries: Gel Electrcphoresis vs. Gel Electrofocusting

Calf ovarian CAMP dependent protein kinase A was isolanted by adsorntion on to DEAF-cellulose, gel chromatography on agarosepolyacrylamide copolymer, electrophoresis in a 6% polyacrylamide gel, 0.2% in Iriton X-100, and DEAF-chromatography. The yield was 3.3 mg, representinw 22% of the starting material.

Purification was 400-fold. The product appears homogeneous on gel electropheresis at p 10.2, but DEAF-chromatography, gel elecectro focusing and gel electrophoresis at pH 8.5 and 7.5 reveal two charge isomeric forms of the enzyme.

Optimization of gel concentration for the separation of the enzyme from its closest migrating contaminant pointed to gel electrofocusing, rather than electrophoresis, as the appropriate Separation tool. However, that electrophoresis, as the inactivate the enzyme when conducted on wide-diameter preparative gels, if allowed to proceed to the steady-state, using either Ampholine or buffers as the carrier ampholytes, and etyleneglycol to repress no I yacrylami decopolyner, electrophorus is in a 6 po lyacrylaniide el, 0.7 in TM on -1 00, and DSAF-chromatography. The yield was 3.1 me, representing 787% of the starting material Petrification was 400-fold. The product appears homos jeneousonge I electrophoresis at pM 10.7, hut DEAF- chromatography, gel electron focus inn and ruels ectroohores is at pH 6.5 and 7.5 reveal two charred is on ericforirs of the enzyme, Opticalization of feel concentration for the separation of the enzyme from its closest ml floating cont eminent pointed to jel electro focusing, rather then elect rophoresis, as the appropriate separation tool. However, that method proved to inactivate the enzyme when c on ducted on wide-diameter preparative gels, if all owed to proceed to the steady-state, usinp; either Ampholine or buffers as the carrier ampholytes, and ethyl eneglycol to repress isoelectric precipitation. Only buffer electrofoucing on ultrogel Aca 54 if stoppert prior to the attainment of the isoelectric endpoint of the enzyme succeeded in recovering substantial (65%) activity, albeit at the price of resolution. Thus, a non-optimal concentration in polyacrylamide gel electrophoresis was applied in preference to preparative gel electro focusing.

Preparative methods for the isolation of Proter Kinase 8 and CAMP Binding Pritein A in homogeneous form were also developed, using nodifications of the above-stated procedure.     

Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

Stabilization and purification of ornithine transcarbamylase from Neisseria gonorrhoeae.

Ornithine transcarbamylase was stabilized in cell-free extracts by the presence of either carbamyl phosphate or glycerol. The enzyme was rapidly purified by a procedure consisting of ion-exchange chromatography and electrofocusing. The native molecular weight of the enzyme was determined by gel filtration to be 110,000. A subunit molecular weight of 36,000 was determined by polyacrylamide electrophoresis under dissociating conditions. These findings indicated a trimeric quaternary structure for the enzyme. The isoelectric point of the purified enzyme was 4.75, and no evidence of multiple forms of active enzyme was found in either crude or purified preparations. An inactive form of the enzyme appeared upon storage in the absence of stabilization buffer.     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Purification and properties of the nuclease inhibitor of Aspergillus oryzae and kinetics of its interaction with crystalline nuclease O.

A nuclease inhibitor found in the mycelia of Aspergillus oryzae has been purified 158,000-fold by ammonium sulfate precipitation, chromatography on Sephadex G-75, DEAE-Sephadex A-50 and Bio-Gel p-60 columns, preparative disc electrophoresis on acrylamide gel, and electrofocusing in ampholite. The purified inhibitor is nearly homogeneous as judged by disc electrophoresis. It shows a typical ultraviolet absorption curve for protein, and the inhibitory activity is inactivated by chymotrypsin. The inhibitor and nuclease O (EC 3.1.4.9, a crystalline enzyme from the mycelia of the same organism) form a stable enzyme inhibitor complex. The molecular weights of nuclease O, the inhibitor and the enzyme inhibitor complex are estimated to be 46,000, 22,000 and 73,000 respectively, by Sephadex G-100 gel filtration. The isoelectric points of the enzyme and the inhibitor are 10.0 and 4.09, respectively, as determined by electrofocusing in ampholite. The inhibition is noncompetitive, and the inhibitor constant (K1) is 3.2 X 10(-12) M, whereas the Michaelis constant (Km) for DNA is 2.2 X 10(-8) M. The inactive enzyme-inhibitor complex is reactivated by chymotrypsin through inactivation of the inhibitor. The reactivated enzyme can be inactivated again by the inhibitor, which shows that desensitization of the enzyme does not occur by the action of chymotrypsin.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans.

Fructose transport in Streptococcus mutans LG-1 is mediated by at least two distinct phosphoenolpyruvate fructose phosphotransferase systems. One system is constitutive and consists of membrane components enzyme II as well as enzyme I and heat-stable protein. The other system is inducible and requires, in addition to enzyme I and heat-stable protein, a soluble substrate-specific protein for catalytic activity. This protein factor, designated IIIfru, was purified by DEAE-cellulose chromatography, hydroxylapatite chromatography, molecular sieving on Sephadex G-75, and preparative electrophoresis. The purified preparation showed only one protein band, with a molecular weight of 12,600, on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, on gel electrophoresis with the discontinuous buffer Tris-glycine, and after electrofocusing in gel (pI congruent to 3.7). The molecular weight of the native protein determined by gel filtration at 4 degrees C was 51,000. Immunodiffusion experiments performed with immunoglobulins prepared against the purified IIIfru from S. mutans LG-1 suggested that other S. mutans strains possessed a IIIfru. No precipitin bands, however, were detected with extracts from S. salivarius, S. sanguis, S. lactis, S. faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus casei, and Escherichia coli.     

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.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Purification and Properties of Three Forms of α-Glucosidase from Germinated Green Gram (Phaseolus vidissimus Ten.)

Three forms of α-glucosidase have been isolated from 5-day-old green gram (Phaseolus vidissimus Ten.) seedlings, by a procedure including fractionation with ammonium sulfate and polyethylene glycol 6000, DEAE-cellulose column chromatography, SP-Sephadex column chromatography, preparative gel electrofocusing and preparative disc gel electrophoresis. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II–1 and α-glucosidase II–2. They were homogeneous on polyacrylamide disc gel electrophoresis. Their molecular weights were 145,000, 105,000 and 65,000, respectively. The three enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch liberating glucose, but did not act on sucrose. Their enzymes hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. They hydrolyzed amylose liberating α-glucose. Maltotriose was the main α-glucosyltransfer product formed from maltose by the three α-glucosidases.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Purification and properties of human serum cholinesterase

Cholinesterase was purified from human serum by a three-stage procedure involving chromatography on DEAE-cellulose at pH4.0, an electrofocusing technique and gel filtration on Sephadex G-200. The final product was purified 13000-fold with a yield of 54%, and only one protein and one cholinesterase band could be demonstrated by polyacrylamide-disc electrophoresis. The catalytic properties appeared to be unchanged by the purification procedure. The molecular weight was determined by both ultracentrifugation in a density gradient and gel filtration, and values close to 366000 were obtained. The isoelectric point of cholinesterase was estimated to be pH3.99. The method appears suitable for the preliminary purification of the rare genetic variants of human cholinesterase.     

Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from human liver

Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange chromatograhy and electrofocusing The native molecular weight of the enzyme was determined by gel filtration to be 190,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a monomeric molecular weight of 165,000. The isoelectric point of the purified enzyme was 6.05, and only one species of active enzyme was observed during electrofocusing of both purified enzyme preparations and crude liver homogenates. The enzyme exhibited a pH optimum of 7.8. The apparent Michaelis constants for NH4+, HCO3-, MgATP, and the activator, N-acetyl-L-glutamic acid, were 0.8, 6.7, 1.1, and 0.1 mM, respectively.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

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.