Affinity chromatography of glucose dehydrogenase

Porcine liver beta-D-glucose dehydrogenase, a multi-functional protein, has been purified to apparent homogeneity. The enzyme has been separated from the endoplasmic reticulum using Triton X-114 and further purified using NAD to release glucose dehydrogenase from a NADP-linked sepharose column. The purified enzyme is capable of producing both NADH and NADPH in vivo as indicated by kinetic studies.     

Affinity chromatography of glucose dehydrogenase

Summary Porcine liver -d-glucose dehydrogenase, a multi-functional protein, has been purified to apparent homogeneity. The enzyme has been separated from the endoplasmic reticulum using Triton X-114 and further purified using NAD to release glucose dehydrogenase from a NADP-linked sepharose column. The purified enzyme is capable of producing both NADH and NADPH in vivo as indicated by kinetic studies.     

Purfication and molecular weight of an RNA-dependant RNA polymerase from Brassicae oleracea var. Botrytis]

An RNA-dependent RNA polymerase has been completely purified from Cauliflower inflorescences. Analysis of the purified enzyme on SDS-polyacrylamide gels showed one polypeptide chain with a molecular weight of 140,000 dalton. The enzyme is monomeric in its native state. The in vitro activity was completely dependent on added RNA, the most efficient templates being poly (U), poly (U, C), poly (I) and viral RNA.     

Partial purification and characterization of acid phosphatase from sporulated oocysts of Eimeria tenella

Acid phosphatase of Eimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and Vmax values for (Peak II) were 25 mM and 1.57 mumol/min/mg protein, respectively. The enzyme (Peak II) is strongly inhibited by Hg++, Cu++, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Rat liver S-adenosyl-L-homocysteine hydrolase purification by affinity column chromatography (author's transl)]

S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been purified 240-fold from rat liver by affinity column chromatography on aminohexyl sepharose bound 6-mercaptopurine 9 D-riboside. The purified enzyme was homogeneous by gel electrophoresis.     

Purification of a membrane protein modifying the sensitivity of Na+/K+ ATPase to ouabain from a supernatant of plasma membrane treated with EDTA]

A membrane protein of M. W. 32,000 D and pI 5 has been purified from EDTA-treated plasma membrane supernatants by Page without detergents. These membranes are purified from the MF2S cell line issued from the murine plasmacytoma MOPC 173. The purified protein was able to induce a 300 fold increase in the Na+/K+ ATPase resistance to ouaba?n thus mimicking the original resistance of the enzyme to the drug.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Summary Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000×g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closley related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.We would like to thank Miss S. Fukushima for technical assistance.     

Partial purification and characterization of acid phosphatase from sporulated oocysts ofEimeria tenella

Summary Acid phosphatase ofEimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and V_max values for (Peak II) were 25 mM and 1.57 mol/min/mg protein, respectively. The enzyme (Peak II) ist strongly inhibited by Hg⁺⁺, Cu⁺⁺, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Purification de la S-adénosyl-L-homocystéine hydrolase du foie de rat par chromatographie d'affinité

Summary S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been purified 240-fold from rat liver by affinity column chromatography on aminohexyl sepharose bound 6-mercaptopurine 9 D-riboside. The purified enzyme was homogeneous by gel electrophoresis.

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Ce travail a bénéficié de l'aide du CNRS (ERA 560) et de l'INSERM (FRA 5).     

Characterization of carrot pectin methylesterase

The most alkaline form of pectin methylesterase was purified from ripe carrot roots and used for structural analysis. Determination of an N-terminal blocking group and of the primary structure allowed comparisons with other forms, and facilitated crystallographic determination of the three-dimensional structure. The mature enzyme has 319 residues and the N-terminal blocking group was shown to be a pyroglutamyl residue derived from a glutaminyl cyclization. Few other methylesterases have been isolated and assigned to exact mature forms, and together with the present enzyme, only two have been analyzed in three-dimensional structure. However, comparison of 39 forms, mainly from GenBank data, reveals clear relationships and identifies sub-groups of this enzyme type, deviating in structure but centering around two functionally important and conserved Asp residues at positions 136 and 157 in the carrot enzyme. Received 2 January 2002; accepted 4 January 2002     

Abtrennung des anaphylatoxinbildenden Prinzips aus Cobragift von anderen Giftkomponenten

Summary Cobra venom contains an anaphylatoxin-forming principle. This component has been purified by gel filtration and ion exchange chromatography. It has been obtained free from proteolytic or hemolytic activity as well as from phospholipase A. It seems to be an enzyme that splits the anaphylatoxin from its inactive precursor.     

Immunochemical studies on Rhodotorula gracilis D-amino acid oxidase.

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified from R. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

Nitric oxide and cellular respiration

The role of nitric oxide (NO) as a signalling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) has been elaborated during the last decade. Since NO has also been found to inhibit cellular respiration, we review here the available information on the interactions of NO with cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. The effect of NO on cellular respiration is first summarized to present essential evidence for the fact that NO is a potent reversible inhibitor of in vivo O2 consumption. This information is then correlated with available experimental evidence on the reactions of NO with purified COX. Finally, since COX has been proposed to catalyze the degradation of NO into either nitrous oxide (N2O) or nitrite, we consider the putative role of this enzyme in the catabolism of NO in vivo.     

Immunochemical studies onRhodotorula gracilis D-amino acid oxidase

Summary Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified fromR. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5′-nucleotidase

Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.Received 25 March 2005; received after revision 3 May 2005; accepted 13 May 2005     

Purification of rat brain guanylate cyclase]

The purification of guanylate cyclase has been achieved. After electrophoresis on polyacrylamide gel only one protein band was observed. The low factor of purification must therefore be ascribed to the loss of an activator of guanylate cyclase. Stimulation of the activity by nitroprusside is also lost during purification. The purified enzyme follows Michaelis--Menten kinetics, it is activated by Mn++ ions and inhibited by triphospho nucleotides.     

Purification and immobilization of human carbonic anhydrase B by using polyacrylamide gel.

Human erythrocyte carbonic anhydrase B was purified and immobilized in polyacrylamide gel. As compared to the soluble enzyme, the immobilized enzyme was considerably more resistant to heat and sulphanilamide action.     

Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast.

Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.     

Purification and properties of glutamine synthetase I fromRhizobium sp. UMKL 20

Summary Glutamine synthetase I was purified fromRhizobium sp. UMKL 20 following polyethylene glycol precipitation. The enzyme had a subunit molecular weight of 58 kd. Apparent K_m values for ammonia and glutamate were 5.6 and 15.2 mM, respectively. Glutamine synthetase I activity was inhibited by several end products of glutamine metabolism. The purified enzyme was highly adenylylated (E _n ^– =8.5).Acknowledgment. I would like to thank Mr J. C. Lai for technical assistance. This work was carried out with the support of Vote F 153/79 from the University of Malaya.