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.     

Inhibition of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi by silver nitrate.

The purified NADP-linked glutamate dehydrogenase from Trypanosoma cruzi was strongly inhibited by silver nitrate. The inhibition was reversed by reduced glutathione, and was modified by the presence of the substrates during preincubation of the enzyme with the inhibitor.     

Purification of calf thymus adenosine diphosphate ribose polymerase]

Calf thymus poly ADPR polymerase has been purified to electrophoretic homogenity. The enzyme has a molecular weight of 120,000 +/- 10,000 dalton. The substrate affinity is very high (apparent Km 82.5 micrometer). The presence of exogenous DNA does not appear to be a requisite for enzymatic activity of the purified enzyme.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

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.     

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.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

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.     

Medium- and short-chain dehydrogenase/reductase gene and protein families

The structure-function relationships of alcohol dehydrogenases from the large family of short-chain dehydrogenase/reductase (SDR) enzymes are described. It seems that while mammals evolved with a medium-chain alcohol dehydrogenase family (MDR), fruit flies utilized an ancestral SDR enzyme. They have modified its function into an efficient alcohol dehydrogenase to aid them in colonizing the emerging ecological niches that appeared around 65 million years ago. To the scientific community, Drosophila has now served as a model organism for quite some time, and Drosophila alcohol dehydrogenase is one of the best-studied members of the SDR family. The availability of a number of high-resolution structures, accurate and thorough kinetic work, and careful theoretical calculations have enabled an understanding of the structure-function relationships of this metal-free alcohol dehydrogenase. In addition, these studies have given rise to various hypotheses about the mechanism of action of this enzyme and contribute to the detailed knowledge of the large superfamily of SDR enzymes.     

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     

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.     

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).     

Inhibition of the NADP-linked glutamate dehydrogenase fromTrypanosoma cruzi by silver nitrate

Summary The purified NADP-linked glutamate dehydrogenase fromTrypanosoma cruzi was strongly inhibited by silver nitrate. The inhibition was reversed by reduced glutathione, and was modified by the presence of the substrates during preincubation of the enzyme with the inhibitor.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and the Secretaría de Estado de Ciencia y Technología de la República Argentina. JJC is a member of the Carrera del Investigador Científico, and SMJ a Research Fellow, of the former Institution.     

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.     

Isolierung eines hochgereinigten Polypeptides mit uterusrelaxierender Wirksamkeit

Summary A highly purified polypeptide with a powerful uterus relaxing activity has been isolated from pregnant sow ovaries. The homogenity has been checked, using various electrophoretic and chromatographic techniques.     

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.     

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.     

Purification of a nuclease from human serum

Summary The purification procedure for a nuclease from human serum is described. It includes ammonium sulfate precipitation, chromatography on DEAE-Sephadex and on Sephacryl-S 200, and preparative electrophoresis. The enzyme, purified about 2000-fold, is homogeneous in a sodium dodecyl sulfate electrophoretic system, where it has a mol. wt of 78,000. The pH optimum lies around pH 6.5; it is a sugar-nonspecific endonuclease.Acknowledgment. This work has been supported by a research grant from Stiftung Volkswagenwerk through the Akademie der Wissenschaften und der Literatur, Mainz. We thank Mrs R. Nehrbass and Mrs C. Wolpert for technical assistance.     

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.