scholarly journals Peroxisomal localization of glucose-6-phosphate dehydrogenase and pyrophosphate-stimulated dihydroxyacetone-phosphate acyltransferase in mouse kidney

1987 ◽  
Vol 244 (2) ◽  
pp. 443-448 ◽  
Author(s):  
B N Patel ◽  
M I Mackness ◽  
M J Connock
Keyword(s):  
Subcellular Localization ◽  
Dehydrogenase Activity ◽  
Inorganic Phosphate ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Mouse Kidney ◽  
Dihydroxyacetone Phosphate ◽  
Ph Optimum ◽  
Dependent Activity ◽  
Glucose 6 Phosphate Dehydrogenase

1. The subcellular localization of dihydroxyacetone-phosphate acyltransferase (DHAPAT) (assayed in the presence of pyrophosphate) and glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in mouse kidney was investigated by density-gradient centrifugation. 2. DHAPAT has a predominantly peroxisomal distribution, and the activity in purified peroxisomes is stimulated by various organic and inorganic phosphate-containing compounds. The pH optimum is acid. 3. Approx. 10% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and may provide a source of NADPH for the peroxisomal reduction of acyl-dihydroxyacetone phosphate formed by DHAPAT activity.

Density Increase and Ageing of Erythrocytes in Stored Blood

1989 ◽  
Vol 17 (5) ◽  
pp. 461-466 ◽  
Author(s):  
M. Rocchigiani ◽  
M. Pescaglini ◽  
S. Sestini ◽  
V. Micheli ◽  
C. Ricci
Keyword(s):  
Dehydrogenase Activity ◽  
Whole Blood ◽  
Storage Time ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Acid Citrate Dextrose ◽  
Citrate Dextrose ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Stored Blood ◽  
Citrate Phosphate

An increase in the density of erythrocytes was observed after storage of whole blood for 30 days at 4°C in either acid citrate – dextrose or citrate – phosphate – dextrose – adrenaline. Glucose- 6-phosphate dehydrogenase activity in unfractionated red blood cell lysates did not vary with the storage time. Enzyme activity in the lighter fraction separated by density gradient centrifugation was higher than that in heavier fractions. The decline in glucose-6-phosphate dehydrogenase activity with density was less marked after storage of whole blood for 30 days. It is suggested that density modifications are not related to the ageing of erythrocytes and additional mechanisms may be involved.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

Subcellular localization of oxytocin in the ovine corpus luteum

1985 ◽  
Vol 63 (4) ◽  
pp. 309-314 ◽  
Author(s):  
G. E. Rice ◽  
G. D. Thorburn
Keyword(s):  
Subcellular Localization ◽  
Physicochemical Properties ◽  
Corpus Luteum ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Dense Granules ◽  
Oxytocin Release ◽  
Midluteal Phase

The subcellular localization of oxytocin within the corpus luteum of sheep was investigated using differential and density gradient centrifugation. Oxytocin was associated with a particulate fraction which sedimented to a density of 1.054 – 1.061 g/mL. The exclusion of [3H]oxytocin from this particulate fraction is indicative that particulate oxytocin represents endogenous compartmentalization. Particulate oxytocin, incubated in buffered medium at 37 °C, was stable for up to 1 h and the release of oxytocin was not affected by the pH of the incubation medium, over the range 5.5 – 8.5. Oxytocin release, however, was stimulated by incubating particle-bound oxytocin in buffered medium of low osmolality (<200 mosmol). These data are similar to the physicochemical properties reported for peptide-containing neurohypophysial secretory granules. Ultrastructural analysis of oxytocin-containing fractions revealed the presence of electron-dense granules (diameter, 200–250 nm). These data are suggestive that oxytocin, in the corpus luteum of sheep, is contained within a population of secretory granules which occur in high numbers during the midluteal phase of the oestrous cycle.

scholarly journals Studies of dihydroxyacetone phosphate acyltransferase in rat small intestine. Subcellular localization and effect of partially hydrogenated fish oil and clofibrate

1992 ◽  
Vol 282 (2) ◽  
pp. 565-570 ◽  
Author(s):  
B Ruyter ◽  
J S Lund ◽  
M S Thomassen ◽  
E N Christiansen
Keyword(s):  
Small Intestine ◽  
Subcellular Localization ◽  
Fish Oil ◽  
Gradient Centrifugation ◽  
Dihydroxyacetone Phosphate ◽  
Rat Small Intestine ◽  
Specific Activities ◽  
Fold Stimulation ◽  
Effect Of Feeding ◽  
Stimulation Of

The subcellular localization of dihydroxyacetone phosphate acyltransferase (DHAPAT) activity in rat small intestine was investigated by Nycodenz-gradient centrifugation. We found that DHAPAT had a predominant peroxisomal distribution, with a separate enzyme activity located in the microsomal fraction, the same distribution as found in rat liver. The effect of feeding rats on a diet with 20% (w/w) partially hydrogenated fish oil (PHFO) or 0.3% clofibrate on the activity of DHAPAT in rat small intestine and liver was studied. Both 20% PHFO and 0.3% clofibrate gave a 1.8-fold stimulation of the specific activities of DHAPAT in peroxisomes of the small intestine, whereas in the liver 20% PHFO gave a 1.4-fold stimulation and 0.3% clofibrate a 1.6-fold stimulation of the total DHAPAT activities in the postnuclear supernatant. The specific activities of DHAPAT in liver were not affected.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

Adenine Phosphoribosyltransferase Activity in Mitochondria of Catharanthus roseus Cells

1982 ◽  
Vol 37 (11-12) ◽  
pp. 1288-1289 ◽  
Author(s):  
Fumiko Hirose ◽  
Hiroshi Ashihara
Keyword(s):  
Subcellular Localization ◽  
Density Gradient ◽  
Catharanthus Roseus ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Adenine Phosphoribosyltransferase ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient

Abstract Subcellular localization of adenine phosphoribosyltrans­ferase was examined in Catharanthus roseus cells. At least 13% of the activity was observed in the particulate fraction with the rest observed in the cytosol. Both differential and Percoll density gradient centrifugation indicated that particulate adenine phosphoribosyltransferase was predominantly located in the mitochondria.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

scholarly journals The effects of pH and citrate on the activity of nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria

1969 ◽  
Vol 113 (5) ◽  
pp. 813-820 ◽  
Author(s):  
G. F. Cox ◽  
D. D. Davies
Keyword(s):  
Density Gradient ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Substrate Concentration ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Nicotinamide Adenine ◽  
Ph Optimum ◽  
Effects Of Ph ◽  
Metabolic Significance

1. The effect of pH on the co-operative activation of the NAD-specific isocitrate dehydrogenase from pea mitochondria by isocitrate is shown. 2. The interlinked effects of pH on the affinity of the NAD-specific isocitrate dehydrogenase for isocitrate and the dependence of the pH optimum on the substrate concentration are presented. 3. A consideration of the conditions of pH and substrate concentration under which citrate activates the NAD-specific isocitrate dehydrogenase demonstrates similarities between the binding of isocitrate and citrate. 4. A comparison of the effects of citrate and pH on the gross structure of the enzyme is investigated by density-gradient centrifugation. 5. The kinetic interpretations of these results are briefly considered. 6. The metabolic significance of these studies is discussed.

scholarly journals Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 157 (3) ◽  
pp. 635-641 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Isoelectric Focusing ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Organ Distribution ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Amino Donor

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.

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