Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

Purification and characterization of phospholipase A2 released from rat platelets

It was found that phospholipase A2 and lysophospholipase, both of which were released from thrombin-stimulated rat platelets, had high affinity to insolubilized heparin. Phospholipase A2 released from rat platelets was purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW (high-performance liquid chromatography, HPLC). The enzyme was near homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, and its Mr was estimated to be 13,500. The purified enzyme was labile and lost its activity within 1 h when incubated at 37 degrees C. Phospholipids or detergent in the solution protected the enzyme against inactivation. Phospholipase activity was inhibited by p-bromophenacylbromide, but not by diisopropylfluorophosphate or iodoacetamide. Lysophospholipase, which was also released from rat platelets, was separated from phospholipase A2 by chromatography on heparin-Sepharose.     

Cloning, overexpression, and purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11891

Novobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His(6) fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PP(i) exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg(2+) or Mn(2+). Apparent K(m) values for ring A and ring B were determined as 19 and 131 micrometer respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates.     

Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties, probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipram-sensitive form between the two tissues. Since the rolipram-sensitive form detected in crude brain preparations markedly differs from the above-described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.     

Affinity purification and characterization of serine hydroxymethyltransferases from rat liver

A rapid and simple method was developed for the purification of serine hydroxymethyltransferases [EC 2.1.2.1]. The procedure involved ammonium sulfate precipitation, DEAE-cellulose column chromatography and affinity chromatography on an L-adsorbent. Through this procedure the cytosolic enzyme (s-SHMT) was purified 1,650-fold, and the mitochondrial enzyme (m-SHMT) 1,730-fold, with a yield of more than 30% in both cases. Both preparations gave a single band with a Mr of 54,000 on SDS-PAGE. The native enzymes both contained 4 mol of pyridoxal phosphate/mol of enzyme, and showed a Mr value of 220,000 on gel filtration, indicating a tetrameric structure. Several other properties of the enzymes were also studied.     

Improved purification of Aspergillus oryzae 1,2-alpha-mannosidase by using mannan gel affinity chromatography

In order to facilitate the purification of 1,2-alpha-mannosidase from an enzyme product of Aspergillus oryzae, we have devised a rapid and simple procedure. A partially purified enzyme preparation obtained from the A. oryzae enzyme product, by means of ammonium sulfate fractionation followed by CM-Sephadex C-50 chromatography, was subjected to affinity chromatography with baker's yeast mannan gel as an adsorbent. 1,2-alpha-Mannosidase was retarded and well separated from the major protein peak on the affinity column. After a second affinity chromatography under the same conditions, 1,2-alpha-mannosidase was finally purified 7,500-fold with a 22.9% yield. The enzyme preparation thus obtained was quite suitable for the structural analysis of glycoconjugates.     

Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules

A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.     

Isolation and characterization of a proteolytic enzyme from the adult hookworm Ancylostoma caninum

The adult hookworm Ancylostoma caninum releases a proteolytic enzyme which is thought to be essential for its adaption to parasitism. The protease was purified from parasite extracts by ion-exchange chromatography followed by gel filtration and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular weight of 37,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had an NH2-terminal sequence of Arg-His-His-Gln-Pro-Lys-Val-Ala-Leu-Leu-Gly-Ala-His-Gly-Gly-Ile. Using 125I-fibrin as substrate, the enzyme displayed optimal activity at pH 9-11 and was inactivated by dialysis against EDTA. The enzyme degraded [3H]elastin and both elastin and trypsin-labile glycoproteins in a rat vascular smooth muscle extracellular matrix. Antiserum raised to the protease in rabbits cross-reacted with extracts from the infective larval stage of A. caninum, suggesting that the production of the enzyme begins in an earlier developmental stage of the parasite life cycle. The role of the protease in the histolytic and anticlotting processes of the hookworm and its importance in immunity to ancylostomiasis is discussed.     

Purification and characterization of an exoglucanase from Streptomyces flavogriseus

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.     

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1.

An exo-beta-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-beta-D-cellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (greater than or equal to 300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel filtration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate- and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-beta-1,4-glucanase. Exo A required Ca2+ for maximal activity and had an apparent Km of 3.08 mM for NPC, with a Vmax of 0.298 mumol/min per mg of protein. The enzyme had an Mr of 230,000, as determined by gel filtration chromatography, and was a dimer of 118,000-Mr subunits. The N-terminal amino acid sequence of the enzyme is presented.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

Purification and characterization of a dUTPase from Acholeplasma laidlawii B-PG9

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.     

Lectin-binding domains on laminin

Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein.

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.     

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.     

Purification and properties of an exo-cellulase component of novel type from Trichoderma miride.

An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure.     

Purification of two forms of kanamycin acetyltransferase from Escherichia coli

Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.