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.

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 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.

FRACTIONATION OF MOUSE LIVER MICROSOMAL ESTERASES

1965 ◽  
Vol 43 (1) ◽  
pp. 97-104 ◽  
Author(s):  
C. Carruthers ◽  
E. Heins ◽  
A. Baumler
Keyword(s):  
Esterase Activity ◽  
Mouse Liver ◽  
Liver Homogenate ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Nonionic Detergent ◽  
Liver Homogenates ◽  
Two Peaks ◽  
Total Esterase Activity ◽  
Glycyl Glycine

Mouse liver microsomal esterases were fractionated on DEAE-cellulose after the solution of these enzymes in a solution containing 0.1 M glycyl glycine buffer, pH 7.0, and 5 × 10−4 M Lubrol W, a nonionic detergent. Elution of the enzymes from the DEAE-cellulose was accomplished by using NaCl in the glycyl glycine – Lubrol W solution. Microsomes isolated from sucrose and glycerol homogenates have three esterase peaks in common and two peaks not in common. The glycerol supernatant fraction from whole liver homogenate, which contains about 50% of the total esterase activity, and the sucrose supernatant fraction, which contains about 6% of the total esterase activity, have four common esterase peaks, one of which is different from both the microsomes isolated from glycerol and sucrose liver homogenates. Incorporation of Lubrol W in the solution that was employed for the elution of the esterases from the DEAE-cellulose was necessary to prevent extensive loss or inactivation of these enzymes. The nature of the changes induced by glycerol on liver esterases is briefly discussed.

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.

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