Soybean L-(+)-lactate dehydrogenases: purification, characterization, and resolution of subunit structure

Lactate dehydrogenase (LDH) (EC 1.1.1.27) from soybean (Glycine max) was purified 2360-fold to homogeneity using ion-exchange, hydroxyapatite, affinity, and hydrophobic chromatographies. The molecular weight of the holoenzyme is 150,000 +/- 5000. Two-dimensional (isoelectrofocusing-sodium dodecyl sulfate) gel electrophoresis reveals two polypeptides subunits of 5.9 and 6.5 pI and of 36,000 +/- 1000 and 37,000 +/- 1000 Mr, respectively. Nondissociating electrophoresis and isoelectric focusing of LDH resolved five tetrameric isoenzymes with pI's between 6.0 and 6.5. The data suggest that these LDH isoenzymes are derived from random association of the products of two different, but most probably related, genes. Kinetic measurements revealed substrate inhibition at high concentrations of lactic acid and biphasic kinetics with NAD.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

An in vitro reversible interconversion of rat liver fatty acid binding protein having different isoelectric points by virtue of the fatty acid content.

Rat liver fatty acid binding protein (FABP) was purified to homogeneity by procedures including Sephadex G-100 and DEAE-cellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these two fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions has revealed that they are virtually identical or closely resemble each other. However, their fatty acid content was significantly different and heterogeneity was clearly demonstrated in the patterns of isoelectric focusing. In this communication, a single isoform (pI 5.0) from peak B FABP was further purified by successive DEAE-cellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining fatty acids. These in vitro manipulations of bound fatty acid content made clear its possible cause of the microheterogeneity of FABP.     

Purification and characterization of two distinct lipases from Geotrichum candidum

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.     

Purification of bicarbonate-sensitive sperm adenylylcyclase by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-affinity chromatography.

Anion transport inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) and heparin, inhibit reversibly the bicarbonate-sensitive adenylylcyclase of porcine sperm plasma membrane. In the light of this, SITS- and heparin-affinity chromatographies were applied in order to purify sperm adenylylcyclase. SITS-Affi-Gel 102 binds proteins extracted from the porcine cauda epididymal sperm plasma membrane by Lubrol-PX, more selectively than heparin-agarose. However, recovery of adenylylcyclase activity is higher when heparin-agarose is used. The hormone-sensitive liver adenylylcyclase, which is less sensitive to bicarbonate than sperm enzyme, has less affinity for these affinity resins than sperm enzyme. Adenylylcyclase can be purified to apparent homogeneity on two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) from the Lubrol-PX extract of the purified sperm plasma membrane by using SITS-affinity chromatography at the first step of the purification followed by preparative isoelectric focusing and gel filtration. The molecular weight and pI of the purified enzyme are 46,300 and 6.9, respectively. The purified enzyme activity is highly dependent on Mn2+. Bicarbonate activates even the purified enzyme both by decreasing Km and by increasing Vmax.     

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。     

Arylsulfatase a from normal human lung and lung tumors showed different patterns of microheterogeneity

Arylsulfatase A was purified from human lung to apparent homogeneity as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme from normal lung as well as that from lung adenocarcinoma showed considerable microheterogeneity when examined by isoelectric focussing, with an isoelectric point (pI) ranging from 5.1 to 4.6. The tumor enzyme was more heterogeneous and contained more acidic components than the normal lung enzyme. The cause of the charge heterogeneity was examined by treatment with exogenous hydrolases. Upon treatment with sialidase, phosphatase or endo-beta-N-acetylglucosaminidase H (endoglycosidase H), the acidic enzyme forms shifted to an alkaline region on isoelectric focussing gels. Combined treatment of the arylsulfatase A with endoglycosidase H and sialidase resulted in complete loss of the most acidic components to give the less acidic components with pI 5.1, 5.0, and 4.9. These results strongly suggest that the charge heterogeneity of arylsulfatase A is due not only to sialylation but also to phosphorylation at the carbohydrate moiety of the enzyme, and the extent of substitution by acidic groups is markedly increased in the tumor enzyme.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

beta-D-Glucosidase from seeds of Japanese cycad, Cycas revoluta Thunb.: properties and substrate specificity

beta-D-Glucosidase was purified from seeds of Japanese cycad by dialysis, chromatography on CM-Sepharose CL-6B, gel filtration on Biogel P-200, and chromatofocusing. By chromatofocusing, beta-D-glucosidase was separated into four components whose isoelectric points were in a very narrow range (7.43-7.68). All these components were glycoproteins. The main component (pI = 7.59) was homogeneous on gel isoelectric focusing, and was crystallized from ammonium sulfate solution. The molecular weight of the crystalline preparation was determined to be 137,000 by gel filtration, and 67,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating the main component was composed of two subunits with the same molecular weight. The amino acid composition and sugar content of the main component were also determined. All four components hydrolyzed not only o-nitrophenyl beta-D-glucopyranoside but also o-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-fucopyranoside, and o-nitrophenyl beta-D-xylopyranoside. Hydrolysis rates of each substrate by the four components were quite similar. Mixed substrate experiments using crystalline preparation proved that a single active site was responsible for the hydrolysis of these substrates.     

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.     

Isolation and characterization of alpha-fetoprotein from the mouse hepatoma BW7756.

Purification of alpha-fetoprotein from mouse hepatoma BW7756 extracts was performed using ammonium sulfate precipitations, gel filtration, ion-exchange chromatography and isoelectric focusing. These procedures produced a 5.6% yield of alpha-fetoprotein with 96% purity. Polyacrylamide slab gel electrophoresis, extended agarose electrophoresis and immunoelectrophoresis demonstrated that mouse hepatoma alpha-fetoprotein migrated at pH 8.6 as a rapid alpha1, or postalbumin globulin. Crossed antibody electrophoresis of the agarose zone containing alpha-fetoprotein failed to demonstrate microheterogeneity. Molecular weight analysis of the mouse hepatoma alpha-fetoprotein on a calibrated Sephadex G-200 column yielded a value of 72 000-73 000 for the native protein. Sodium dodecyl sulfate gel electrophoresis subsequently demonstrated a single polypeptide chain with a molecular weight of 72 000. Amino acid analysis showed the alpha-fetoprotein to be an acidic protein dominated by hydrophobic residues. The total carbohydrate content was 5.5%, and 3 mol of sialic acid were detected per mol of alpha-fetoprotein. Although neutral sugars were the principal class present, galactosamine was the most abundant single sugar detected.     

Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor.

Empty capsid species formed from the self- and extract-mediated assembly of poliovirus type 1 14S particles in vitro and procapsids isolated from virus-infected cells were subjected to isoelectric focusing in charge-free agarose gels. The empty capsid formed in the self-assembly reaction had an isoelectric point (pI) of 5.0, whereas procapsids and extract-assembled empty capsids focused at pH 6.8. Unreacted 14S particles focused at pH 4.8 to 5.0. The sedimentation coefficient (s20,w) and density of the empty capsid species were also determined. Procapsids had a density in CsCl of 1.31 g/cm3, whereas empty capsids formed by self- or extract-mediated assembly had a density of 1.29 g/cm3. Both extract-assembled empty capsids and procapsids had an s20,w of 75S, whereas self-assembled empty capsids had an s20,w of 71S. Self-assembled empty capsids were not converted to pI 6.8 empty capsids by incubation with poliovirus-infected HeLa cell extracts. The dissociated polypeptides of self-assembled empty capsids (pI 5.0) and procapsids (pI 6.8) behaved identically when analyzed by isoelectric focusing in the presence of 9 M urea and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results suggest that infected cell extracts possess a factor that influences the final conformation of the empty shell (pI 6.8, 75S) formed from 14S particles and that this influences is exerted at the initiation step or during the polymerization reaction. A small amount of this activity (less than or equal to 20% of infected extracts) was detected in uninfected cells; the significance of this remains unknown.     

An improved purification method for cytosolic malate dehydrogenase from several sources

A new purification method for cytosolic malate dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5'AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (approximately 40 hours), in large quantities, with good yields (45-54%). The specific activity of final preparations were around 1300 I.U./mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.