Nucleoside diphosphatase from pig liver: purification and some properties

A procedure is described for purification of nucleoside diphosphatase from pig liver microsomes which avoids exposure of the enzyme to potentially denaturing conditions. The purest fractions obtained have specific activities of approximately 100 units/mg and appear to contain approximately 35% NDPase on a protein basis. Pig liver nucleoside diphosphatase resembles the enzyme obtained from other mammalian tissues in its substrate specificity and in its interaction with MgATP^2? as an allosteric modifier. However the molecular weight of the pig liver enzyme appears higher than that reported for other nucleoside diphosphatases, and activation by MgATP^2? is attributable to an increase in the maximal rate of nucleoside diphosphate hydrolysis rather than to a decrease in K_m. These differences in properties seem to be due to a species difference since similar properties were found with pig liver enzyme prepared by a different extraction procedure. The kinetic parameters which describe the reaction catalyzed by pig liver nucleoside diphosphatase are insensitive to changes in [H⁺]over the range pH 6.5–8.6. The intracellular location of nucleoside diphosphatase is microsomal in both pig and chicken liver.     

Nitrite oxidoreductase from Nitrobacter hamburgensis: redox centers and their catalytic role

Nitrite oxidoreductase was isolated from mixotrophically grown cells of Nitrobacter hamburgensis. The enzyme purified from heat treated membranes was homogeneous by the criteria of polyacrylamide gel electrophoresis and size exclusion chromatography. The monomeric form consisted of two subunits with M_r 115000 and 65000, respectively. The dimeric form of the enzyme contained 0.70 molybdenum, 23.0 iron, 1.76 zinc, and 0.89 copper. The catalytically active enzyme was investigated by visible and electron paramagnetic resonance spectroscopy (EPR) under oxidizing (as isolated), reducing (dithionite), and turnover (nitrite) conditions. As isolated the enzyme exhibited a complex set of EPR signals between 5–75 K, originating from several ironsulfur and molybdenum (V) centers. Addition of the substrate nitrite, or the reducing agent dithionite resulted in a set of new resonances. The molybdenum and the iron-sulfur centers of nitrite oxidoreductase from Nitrobacter hamburgensis were involved in the transformation of nitrite to nitrate.Abbreviations EPR electron paramagnetic resonance - ICP-AES inductively coupled plasma-atomic emission spectrometry - NaP_i sodium phosphate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition,catalytic properties,and sequence alignments

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating and structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent K _m for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent V _max of 64 U/mg (at 65°C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70°C). The temperature optimum was above 90°C. At 90°C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the -subunit had similarities to the N-terminal amino acid sequence of the -subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the -subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate: ferredoxin oxidoreductase from Halobacterium halobium.Abbreviations CoASH Coenzyme A - F ₄₂₀ Coenzyme F₄₂₀     

Alkaline phosphatase from pig kidney. Method of purification and molecular properties

Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH₄)₂SO₄ precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s⁻¹ per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.     

The purification and some properties of pig liver hyaluronidase

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) has been isolated from pig liver and purified 1720-fold with an overall yield of 9.5%. The enzyme was purified using an acid-extraction technique followed by successive chromatography on DEAE-cellulose, two boronate affinity columns and Sephadex G-75. This final preparation, which was essentially homogeneous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70 000 with an isoelectric point of 5.0. No contaminant enzymes capable of degrading glycosaminoglycans could be detected in the final preparation. The substrate specificity of the enzyme was the same as for bovine testicular hyaluronidase; however, both the Km and V values were significantly lower for the pig liver enzyme with all of the substrates tested (hyaluronate, chondroitin 4-sulphate, chondroitin 6-sulphate). A full kinetic analysis of the enzyme using hyaluronate as a substrate showed that the activity of pig liver hyaluronidase was uncompetitively activated by either protons or NaCl.     

Rat liver cysteine sulfinate decarboxylase: purification, new appraisal of the molecular weight and determination of catalytic properties.

Rat liver cystein sulfinate decarboxylase (L-cystein sulfinate carboxylase) was purified approximately 500-fold. By cellulose acetate and polyacrylamide gel electrophoresis or by analytical ultracentrifugation, the purified enzyme appears to be nearly homogeneous. The Stokes radius (3.4 nm) and sedimentation coefficient (6.5 S) were determined. The molecular weight, calculated and experimentally estimated is around 100 000 and the enzyme is constituted of two identical subunits whose molecular weights are 55 000. The role of pyridoxal phosphate as coenzyme was demonstrated and the requirement for free sulhydryl groups for activity was studied. The ability of native pure cysteine sulfinate decarboxylase to also decarboxylate cysteate was stressed: therefore, we concluded that in rat liver a single protein catalyzed both reactions, although only the decarboxylation of cysteine sulfinate is of physiological interest.     

Electron transfer flavoprotein from pig liver mitochondria. A simple purification and re-evaluation of some of the molecular properties.

Electron transfer flavoprotein (ETF) from pig liver mitochondria has been purified to homogeneity by a three-step procedure with approx. 10-fold higher yields than previously reported. The purified ETF exhibits an absorption coefficient for the bound FAD of 13.5 mM-1.cm-1 at 436 nm and an isoelectric point of 6.75. Gel filtration, sodium dodecyl sulphate gel electrophoresis and flavin analysis indicate that pig liver ETF is a dimer, composed of non-identical subunits (Mr 38 000 and 32 000) with only one FAD/dimer. Anaerobic reduction by dithionite produces anionic flavin semiquinone as a stable intermediate and establishes the flavin to be the only redox-active chromophore in ETF.     

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.     

DNA topoisomerase from Agrobacterium tumefaciens: purification and catalytic properties.

The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. The enzyme is a single polypeptide of about 100,000 in molecular weight. No apparent separation of the nicking and sealing activities could be obtained in attempts to separate the two activities by a variety of methods, including limited protease digestion, thermal denaturation, and differential inhibition. Monoclonal antibodies obtained from hybridomas likewise did not preferentially inhibit one of the two activities. These results suggest that the two catalytic functions are carried by the same essential residues of the active enzyme site.     

Identification, purification, and characterization of a thermophilic imidase from pig liver

This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.     

Isolation, purification, and characterization of glucosamine-6-phosphate-N-acetylase from pig liver

The procedure of isolation, purification, and characterization of glucosamine-6-phosphate acetylase from the pig liver is described. The steps of purification were as follows: adsorption on hydroxylapatite, fractionation with ammonium sulfate, chromatography on cellulose phosphate, electrofocusing, and preparative gel electrophoresis. A highly purified (about 3000-fold) preparation of GlcN-6-P acetylase, with a yield of 23%, was obtained. It was found that GlcN-6-P acetylase from pig liver is heterogeneous and exists in two active forms. The characteristic features of the preparation were established: Mr, about 24 kDa; temperature optimum at 37 degrees; pH optimum at 7.45; and Km (GlcN-6-P) 3.7 x 10(-3) M and Km (AcCoA) 1.4 x 10(-3) M. The ions K+, Na+, NH4+, Mg2+, Mn2+, and CH3COO- do not stimulate the acetylase activity. The product of acetylase reaction (GlcNAc-6-P) inhibits this reaction according to the feedback process. The highly purified preparation of GlcN-6-P acetylase is unstable during storage and it is protected by ampholine or glycine from enzyme inactivation, but it is not protected by 2-mercaptoethanol.     

Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

The cerebroside sulfate activator from pig kidney: purification and molecular structure.

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.     

Heterogeneity of pig plasma amine oxidase: molecular and catalytic properties of chromatographically isolated forms

Pig plasma amine oxidase was resolved into several fractions by ion-exchange and hydroxyapatite chromatography. These fractions were separately purified, and each fraction was analyzed for catalytic and structural properties. The relative amount of these fractions varied between preparations. Each fraction was composed of a unique set of bands on isoelectric focusing, as revealed by activity and protein staining. All the fractions contained 2 mol of Cu2+ and one "active-carbonyl" cofactor per 195 000 g of protein. There was no detectable difference in the amino acid contents of the fractions. The fractions all had similar catalytic properties using benzylamine as the substrate. The chromatographically resolved fractions had differing carbohydrate contents as revealed by gas chromatographic analysis and interaction with lectins. Further, some of the isoelectric focusing bands interacted with lectins of differing affinities. The results suggest that the heterogeneity may be due to variable carbohydrate content. Further, the practice of pooling the various chromatographic fractions may yield misleading results under certain circumstances.