scholarly journals The characterization of two reduced nicotinamide–adenine dinucleotide phosphate-linked aldehyde reductases from pig brain

1972 ◽  
Vol 130 (3) ◽  
pp. 765-772 ◽  
Author(s):  
A. J. Turner ◽  
K. F. Tipton
Keyword(s):  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Aldehyde Reductase ◽  
Enzyme Preparations ◽  
Pig Brain ◽  
Michaelis Constants ◽  
Probable Role ◽  
Reduced Nicotinamide Adenine Dinucleotide

1. NADPH-linked aldehyde reductase from pig, ox and rat brain exhibits non-linear reciprocal plots when partially purified enzyme preparations are studied. 2. In pig brain this non-linearity is due to the presence of two distinct aldehyde reductases, which can be separated by DEAE-cellulose chromatography. 3. These two enzymes can be distinguished by several criteria, including pH optima, Michaelis constants for substrates and their inhibitor sensitivity. 4. The probable role of these enzymes in the metabolism of the aldehydes derived from the biogenic amines is discussed.

NAD(P)H oxidase–dependent platelet superoxide anion release increases platelet recruitment

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 917-924 ◽  
Author(s):  
Florian Krötz ◽  
Hae Young Sohn ◽  
Torsten Gloe ◽  
Stefan Zahler ◽  
Tobias Riexinger ◽  
...  
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Thrombus Formation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Platelets ◽  
Nicotinamide Adenine ◽  
Irreversible Aggregation ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Specific Peptide

Abstract Platelets, although not phagocytotic, have been suggested to release O2−. Since O2−-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O2−. We further studied which agonists cause platelet O2−release and whether platelet-derived O2− influences thrombus formation in vitro. Collagen, but not adenosine 5′-diphosphate (ADP) or thrombin, increased O2− formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)–dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47phox and p67phoxand inhibition of platelet O2− formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O2− did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O2−increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase–dependent O2− release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.

scholarly journals Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities

1970 ◽  
Vol 118 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M. F. Thompson ◽  
H. S. Bachelard
Keyword(s):  
Cerebral Cortex ◽  
Deae Cellulose ◽  
Kinetic Properties ◽  
Sucrose Solutions ◽  
Michaelis Constants ◽  
Ammonium Sulphate Fractionation ◽  
Triton X ◽  
Two Peaks ◽  
Basic Similarity

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, Km(ATP) 0.60mm, Km(glucose) 0.042mm; peak II, Km(ATP) 0.66mm, Km(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, Km(ATP) 0.56mm, Km(glucose) 0.048mm; peak II, Km(ATP) 0.68mm, Km(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

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