Separation of soluble mouse liver 21-desoxy-20α-hydroxysteroid: NADP 20α-oxidoreductase from 21-hydroxy-20α-hydroxysteroid: NADP 20α-oxidoreductase

1968 ◽  
Vol 46 (8) ◽  
pp. 715-724 ◽  
Author(s):  
Marvin Darrach ◽  
Ruth E. Krehbiel ◽  
Leslie A. Deeth
Keyword(s):  
Mouse Liver ◽  
High Speed ◽  
Supernatant Fraction ◽  
Kinetic Properties ◽  
Enzymatic Method ◽  
High Speed Supernatant

At least two 20α-faydroxysteroid dehydrogenases occur in the dialyzed high-speed supernatant fraction of mouse liver. These enzymes have been separated from each other with good yields of each and some of their kinetic properties have been studied. With corticosterone, 11-dehydrocorticosterone, and 11β-hydroxyprogesterone as substrates, it has been shown that the specificity of each enzyme is determined, in part, by the nature of the substituent at C-21, hence the enzymes have been designated 21-desoxy- or 21-hydroxy-20α-hydroxysteroid 20α-dehydrogenase. An enzymatic method is described for the preparation of 11β,20α-dihydroxy-4-pregnen-3-one.

scholarly journals Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver

1989 ◽  
Vol 263 (2) ◽  
pp. 355-363 ◽  
Author(s):  
L Shaw ◽  
R Schauer
Keyword(s):  
Submandibular Gland ◽  
Mouse Liver ◽  
High Speed ◽  
Metal Salts ◽  
Supernatant Fraction ◽  
Inhibitory Influence ◽  
Hydroxylase Activity ◽  
Acetylneuraminic Acid ◽  
Liver Homogenates ◽  
High Speed Supernatant

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.

scholarly journals BRAIN MITOCHONDRIA

1963 ◽  
Vol 19 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Diana S. Beattie ◽  
Howard R. Sloan ◽  
R. E. Basford
Keyword(s):  
High Speed ◽  
Brain Cortex ◽  
Lactate Production ◽  
Mitochondrial Fraction ◽  
Brain Mitochondria ◽  
Glycolytic Enzymes ◽  
Supernatant Fraction ◽  
Glycolytic Activity ◽  
High Speed Supernatant ◽  
Stimulation Of

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.

Phospholipid metabolism in the molluscs. II. Activities of choline kinase, ethanolamine kinase, and CTP:phosphorylethanolamine cytidyltransferase in the mollusc Helix lactea

1970 ◽  
Vol 48 (5) ◽  
pp. 580-584 ◽  
Author(s):  
Chi-Rong Liang ◽  
M. Segura ◽  
K. P. Strickland
Keyword(s):  
Digestive Gland ◽  
High Speed ◽  
Enzyme Preparation ◽  
Phospholipid Metabolism ◽  
Maximum Activity ◽  
Supernatant Fraction ◽  
Choline Kinase ◽  
Lipid Phosphorus ◽  
Ethanolamine Kinase ◽  
High Speed Supernatant

The distribution of phospholipids in the mollusc Helix lactea was investigated. The results showed that ethanolamine and choline phosphoglycerides are the major phospholipids in this species, amounting to 49% and 26% of the total lipid phosphorus. The amount of ceramide 2-aminoethylphosphonate was 9% of the total phospholipids and this compound was the only phosphonolipid detected in this species. Ethanolamine and choline kinase activities were shown to be present in the high-speed supernatant fraction of snail digestive gland. The maximum activity of choline kinase was found to be higher than that of ethanolamine kinase in the same enzyme preparation. The enzyme CTP:phosphorylethanolamine cytidyltransferase was also demonstrated in the high-speed supernatant fraction of snail digestive gland. The enzymic reaction product was identified as CDP-ethanolamine by paper chromatography and radioautography. The enzymes phosphorylcholine cytidyltransferase and aminoethylphosphonate cytidyltransferase were not detected under the conditions employed in the phosphorylethanolamine cytidyltransferase assays.

scholarly journals GENETIC ANALYSIS OF MUTANTS WITH A REDUCED Ca2+-DEPENDENT K+ CURRENT IN PARAMECIUM TETRAURELIA

Genetics ◽  
1985 ◽  
Vol 111 (3) ◽  
pp. 433-445
Author(s):  
Robert D Hinrichsen ◽  
Ed Amberger ◽  
Yoshiro Saimi ◽  
Anthony Burgess-Cassler ◽  
Ching Kung
Keyword(s):  
Genetic Analysis ◽  
High Speed ◽  
Behavioral Responses ◽  
Supernatant Fraction ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
K Current ◽  
High Speed Supernatant ◽  
K Currents

ABSTRACT Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.

scholarly journals Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation

1989 ◽  
Vol 263 (2) ◽  
pp. 565-572 ◽  
Author(s):  
D Riendeau ◽  
D Denis ◽  
L Y Choo ◽  
D J Nathaniel
Keyword(s):  
Lipid Peroxidation ◽  
High Speed ◽  
Chemical Reduction ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Lipoxygenase Activity ◽  
Supernatant Fraction ◽  
Acid Oxidation ◽  
Thiol Compounds ◽  
Stimulation Of

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.

scholarly journals Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts.

1982 ◽  
Vol 94 (2) ◽  
pp. 287-296 ◽  
Author(s):  
J A Cooper ◽  
T Hunter
Keyword(s):  
Protein Kinase ◽  
High Speed ◽  
Divalent Cations ◽  
Rous Sarcoma ◽  
Nonionic Detergent ◽  
Primary Location ◽  
Hypotonic Buffer ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
High Speed Supernatant

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high-speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.

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