Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils.

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.     

Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies.

Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20--24 U/mg protein at 25 degree C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84 000. The enzyme is specific for fatty acids with 14--18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.     

Purification and properties of the phosphorylated form of guanylate cyclase

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.     

Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207

An extracellular lipase from Aspergillus niger NCIM 1207 has been purified to homogeneity using ammonium sulfate precipitation followed by phenyl sepharose and Sephacryl-100 gel chromatography. This protocol resulted in 149 fold purification with 54% final recovery. The purified enzyme showed a prominent single band on SDS-PAGE. The purified enzyme is a monomeric protein of 32.2 kDa molecular weight and exhibits optimal activity at 50 degrees C. One interesting feature of this enzyme is its highly acidic pH optimum. The isoelectric point (pI) of lipase was 8.5. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Chemical modification studies revealed that His, Ser, Carboxylate and Trp are involved in catalysis.     

Amplification and purification of exonuclease I from Escherichia coli K12

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.     

Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species

Melatonin is synthesized by a series of enzymes, the penultimate one, serotonin N-acetyltransferase, catalyzing the limiting reaction. In the present study, we compared the recombinant serotonin N-acetyltransferases from rat, ovine, and human. The human protein is particularly difficult to purify because it interacts strongly with a putative chaperone protein from bacteria whereas the rat and sheep enzymes, which interact less strongly with this protein, have been purified close to homogeneity. We identified the contaminating protein as GroEL, the bacterial equivalent of Hsp60. We present numerous catalytic activities (substrate and cosubstrate specificities as well as inhibitor specificities) measured on the three species enzymes from which we deduced that the presence of the chaperone might partly explain the differences between the various species enzyme characteristics, beside the inter-species ones resulting from sequence differences. Despite several trials reported in the literature, a purification to homogeneity of the human (recombinant) enzyme has never been described. We present a new purification method, by using an original denaturation/renaturation process in which the enzyme is immobilized on an affinity chromatography column. The enzyme is then eluted in an active and pure form (i.e., absence of chaperone). The up-scaled system permitted us to perform the necessary experiments for the measurement of more accurate affinities of human serotonin N-acetyltransferase towards its main natural substrates, showing that only the activity of the enzyme towards phenylethylamine was modified.     

一种具有抑制糜蛋白酶活性的血清DNA结合蛋白质的纯化和理化特性

 本文采用离子交换层析,DNA亲和层析和硫酸铵盐析三步从人血清中分离纯化了一种肿瘤相关DNA结合蛋白质(64DP)。本方法较简便,产率提高。经SDS聚丙烯酰胺凝胶电泳和免疫电泳鉴定纯度符合要求。SDS聚丙烯酰胺凝胶电泳测定分子量为64,000。等电聚焦电泳测得等电点在4.2左右。醋酸纤维膜电泳和转移电泳表明其为一种α_1球蛋白。过碘酸西夫氏糖蛋白染色呈阳性反应。氨基酸分析和酶抑制试验证实64DP与α_1抗縻蛋白酶很相似。     

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.     

A beta-D-galactosidase from nasturtium (Tropaeolum majus L.) cotyledons. Purification, properties, and demonstration that xyloglucan is the natural substrate

beta-D-Galactosidase activity has been detected previously in the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds and has been linked to the hydrolysis in vivo of storage xyloglucan (amyloid) (Edwards, M., Dea, I. C. M., Bulpin, P. V., and Reid, J. S. G. (1985) Planta (Berl.) 163, 133-140). The major beta-D-galactosidase present in extracts from the cotyledons of 9-day seedlings has now been purified to apparent homogeneity. The enzyme (Mr 97,000, no subunits) comprised a range of closely related molecular species ranging in isoelectric point from pH 6.6 to 7.1. Further purification to give a single protein band on isoelectric focusing (pI = 7.1) was achieved by chromatofocusing. The pH optimum of the enzyme (mixed molecular species) was 4.0-5.0 (stable from pH 3-10), and the temperature optimum was 50 degrees C (stable to 50 degrees C). It hydrolyzed lactose and beta-D-galactopyranosides but not melibiose and alpha-D-galactopyranosides. It did not release the terminal nonreducing alpha-D-galactopyranosyl residues from seed galactomannans, but catalyzed the rapid removal of terminal nonreducing beta-D-galactopyranosyl residues from xyloglucans. On the basis of the ability of the enzyme to hydrolyze xyloglucans, the kinetics of xyloglucan hydrolysis, and an experimental demonstration of a clear correlation between xyloglucan depletion and the activity in vitro of this enzyme, it is argued that the cell-wall storage xyloglucan of the nasturtium seed is its natural substrate.     

Novobiocin affinity purification of a mitochondrial type II topoisomerase from the trypanosomatid Crithidia fasciculata

A mitochondrial type II DNA topoisomerase (topoIImt) has been purified to near homogeneity from the trypanosomatid Crithidia fasciculata. A rapid purification procedure has been developed based on the affinity of the enzyme for novobiocin, a competitive inhibitor of the ATP-binding moiety of type II topoisomerases. The purified enzyme is capable of ATP-dependent catenation and decatenation of kinetoplast DNA networks as well as catalyzing the relaxation of supercoiled DNA. topoIImt exists as a dimer of a 132-kDa polypeptide. Immunoblots of whole cell lysates show a single predominant band that comigrates with the 132-kDa polypeptide, indicating that the 264-kDa homodimer represents the intact form of the enzyme. Localization of the enzyme within the single mitochondrion of C. fasciculata (Melendy, T., Sheline, C., and Ray, D. S. (1988) Cell, in press) suggests an important role for topoIImt in kinetoplast DNA replication.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.     

Purification of the autotransporter protein Hbp of Escherichia coli

The enzyme Hbp (hemoglobin protease) of the pathogenic Escherichia coli strain EB1 has been purified to homogeneity by gel filtration chromatography. The purified protein is capable of binding heme and shows hemoglobin protease activity. Our method of purification is applicable not only to Hbp but also to other autotransporter proteins and will contribute to a better understanding of the function-structure relationship of this family of proteins.     

Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart.

Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) was 24 microM. The molecular weight of the enzyme was about 31000 on SDS-polyacrylamide gel electrophoresis and was about 60000 by gel filtration. The enzyme was separated from glutathione (GSH) S-transferase by DEAE-Toyopearl column chromatography, and did not exhibit any GSH S-transferase activity towards four different substrates. The purified enzyme was active in the absence of GSH, but it was activated by various SH-reducing reagents including dithiothreitol, GSH, or beta-mercaptoethanol. This is the first reported purification of membrane-bound PGE synthase to apparent homogeneity.     

Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond

A peptide N-glycosidase that catalyzes the hydrolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 degrees C for several months in 10 mM sodium acetate, pH 5.0, buffer. The peptide N-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66 800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) alpha-helix content. The amino acid and carbohydrate contents of the enzyme are presented. When a convenient new assay with a turkey ovomucoid glycopeptide as a substrate is used, the enzyme preparation exhibits a broad pH optimum centered between pH 4 and pH 6. The enzyme is readily inactivated by SDS and guanidine hydrochloride, but it is stable in the presence of moderate concentrations of several other protein denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterisation of chicken brain hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase enzyme (EC 2.4.2.8) from chicken brain has been purified 10 000-fold to homogeneity. The molecular mass of the native enzyme is 85 kDa, with four subunits, each of 26 kDa, and exerts its maximum activity at pH 10.0. The Km values for hypoxanthine and guanine are 5.2 and 1.8 microM, respectively. The half-life of the enzyme is 30 min at 85 degrees C. Monoclonal antibodies were raised against the native purified enzyme and were used for purification of enzyme to homogeneity. The monoclonal antibody did not bind to the active centre of the enzyme.     

Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland.

1. Acetyl-Coa carboxylase from lactating-rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. Use of phosphate buffer throughout the purification gave low recovery of enzyme. Consequently, Tris buffers were used in the extraction and in selected stages of the purification procedure. 3. The purified enzyme had a specific activity of 5.15 +/- 0.3 mumol of bicarbonate incorporated/min per mg of protein (mean +/- S.E.M. of five preparations). This represents a purification of 257 +/- 16-fold and a yield of 4.3 +/- 0.13%. 4. The kinetic parameters of the purified enzyme were similar to those reported for the enzyme from other tissue sources. 5. The enzyme was assayed by a spectrophotometric assay and by a [14C]bicarbonate-fixation assay. Short incubation were used in the radio-chemical assay to avoid substantial loss of [14C]bicarbonate.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Purification of phosphoprotein phosphatase from bovine cardiac muscle that catalyzes dephosphorylation of cyclic AMP-binding protein component of protein kinase.

A phosphoprotein phosphatase that catalyzes the dephosphorylation of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase from bovine cardiac muscle has been purified to homogeneity by a modification of the procedure of Brandt et al. (Brandt, H., Capulong, Z.L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044). Treatment of the enzyme preparation with ethanol during the early stages of purification results in activation concomitant with reduction in molecular weight to 30,000. The purified activated enzyme has a Km for phospho-protein kinase in the presence or absence of 1.2 mM Mn2+ of 5 and 22 micronM, respectively. Phosphatase activity on phospho-protein kinase but not on other phosphoprotein substrates was cAMP-dependent. This selective activation by cAMP reflects the preference of the phosphatase for the free, phosphorylated cAMP-binding protein rather than the phosphoholoenzyme.     

Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells

Recently we have identified a mitogen-activated S6 kinase from Swiss 3T3 cells (Jen?, P., Ballou, L. M., Novak-Hofer, I., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 406-410). Here we describe the detailed purification of this enzyme from high-speed supernatants (400,000 x g) of vanadate-treated cell extracts. The enzyme is purified through six sequential steps including cation- and anion-exchange, sizing, and affinity chromatography. At each step, the enzyme behaves as one entity and, on the final step of purification, is revealed on silver-stained sodium dodecyl sulfate-polyacrylamide gels as a single protein of Mr 70,000. As reported earlier, the overall purification factor is 3,000-fold, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein. However, recovery of total activity is only 0.2%. This large loss of activity appears to be due to freeze-thawing the enzyme between each step of purification. The purified kinase does not phosphorylate casein, histones 2A and 3S, or phosvitin. It has a Km for ATP of 28 microM and a broad optimum for Mg2+ between 5 and 20 mM. Mn2+ does not affect the basal level of kinase activity, and at concentrations as low as 1 mM, it completely suppresses the effect of 20 mM Mg2+ on kinase activity. The relationship of this enzyme to two other purified S6 kinases is discussed.