Purification and characterization of dioscin-alpha-L-rhamnosidase from pig liver

Dioscin-alpha-L-rhamnosidase was isolated, purified and partially characterized from pig liver. The maximum activity was reached at pH 7, 42 degrees C, 24 h, and 2% of substrate concentration. Fe3+ and Cu2+ inhibited the enzyme; the ion Ca2+ activated it. Mg2+ was an inhibitor at 100 mM, but it was an activator at 200 mM. Zn2+ could be a weak activator of the enzyme. The molecular weight of dioscin-alpha-L-rhamnosidase was about 47 kDa as determined by the method of SDS-polyacrylamide gel electrophoresis.     

Purification and characterization of befunolol reductase from rabbit liver

An enzyme (befunolol reductase) which catalyzes the reduction of befunolol to dihydrobefunolol was purified from the cytosolic fraction of rabbit liver to homogeneity by various chromatographic techniques. Befunolol reductase had molecular weights of 29000 on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and 34000 on gel filtration. The enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and showed an optimal pH of 6.5. The apparent Km and Vmax values of the enzyme for the reduction of befunolol were 1.7 mM and 4.4 units/mg, respectively. Flavonoids, sulfhydryl reagents, heavy metals and coumarins strongly inhibited the enzyme. The enzyme catalyzed the reduction of a variety of aromatic ketones. In addition to befunolol, some ketone-containing drugs such as daunorubicin and levobunolol were efficiently reduced by the enzyme. On the basis of substrate specificities for steroids, befunolol reductase purified from the cytosolic fraction of rabbit liver appeared to be a 3 alpha-hydroxysteroid dehydrogenase.     

Purification and characterization of a major glycoprotein in rat liver lysosomal membrane

A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.     

Purification of clathrin assembly protein from rat liver

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.     

Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain

N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn²⁺, Cu²⁺, Sn²⁺, Co²⁺, Zn²⁺ and Hg²⁺, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.     

Purification and characterization of transglutaminases from the genital tract of the male rat.

In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.     

Purification and some properties of squalene-2,3-epoxide: lanosterol cyclase from rat liver

Squalene-2,3-epoxide: lanosterol cyclase was purified from rat liver in five steps as a soluble and homogeneous protein. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 75 kD. In its native state it behaved as a homo-dimer. The isoelectric point of 5.5 and the apparent Km value for (3S)-squalene-epoxide of 55 microM were estimated for the cyclase.     

Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid

The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by successive gamma-deoxygenation through the cyclocarbonates, separation from the (alphaS,betaR)-isomer by means of flash chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNA(Phe) was isolated on a scale of 100 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P(1), followed by reverse-phase column chromatography, complete digestion with nuclease P(1)/alkaline phosphatase, and reverse-phase HPLC. Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (alphaS,betaS)-11.     

Purification and characterization of theD-hydantoinase from bacillus circulans

AD-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity fromBacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0–10.0 and a temperature of 75‡C. The enzyme is the most stable in a pH range of 8.5–9.5 and up to 60‡C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Purification and characterization of ginsenoside-beta-glucosidase from ginseng.

The ginsenoside-beta-glucosidase that hydrolyzes the beta-(1-->2)-glucoside of the ginsenoside Rg3 sugar moiety to ginsenoside Rh2 was isolated from the ginseng root, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 59 kDa. The optimum temperature of the ginsenoside-beta-glucosidase was 60 degrees C, and the optimum pH was 5.0. Ca2+ ion had positive effect on ginsenoside-beta-glucosidase, while Cu2+ had negative effect on it. The ginsenoside-beta-glucosidase may be a special beta-glucosidase that is different from the original exocellulase such as beta-glucosidase (EC 3.2.1.21).     

Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Purification and characterization of gamma-enolase from various mammals.

The gamma subunit of enolase (gamma-enolase) was purified from the brain tissues of cow, dog, goat, pig, rabbit, and rat. The purification was achieved in only three steps: ammonium sulfate-precipitation, DE 53 cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis (PAGE) in a preparative mode. The purification procedure was comparatively more simple than previously reported methods, and the yield of gamma-enolase was sufficient for subsequent structural and immunological analyses. In all mammals, the purified gamma-enolase migrated in sodium dodecyl sulfate-PAGE (SDS-PAGE) with a molecular mass of 46 kilodaltons (kDa), and the immunological cross-reactivity between those gamma-enolases was very strong. The structural homology of these gamma-enolases was examined by peptide mapping using cyanogen bromide cleavage and subsequent two-dimensional electrophoresis. The resulting peptide patterns were highly similar and in cow, dog, and goat, the patterns were almost identical. These results indicate that structural homology, that is, the species non-specificity of gamma-enolase, appears to be very high.