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.

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 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 The ability of adenosine to decrease the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. A cyclic AMP-mediated effect

1984 ◽  
Vol 218 (1) ◽  
pp. 157-163 ◽  
Author(s):  
R Bartrons ◽  
E Van Schaftingen ◽  
H G Hers
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pyruvate Kinase ◽  
High Speed ◽  
Opposite Effect ◽  
Marked Decrease ◽  
Isolated Hepatocytes ◽  
Adenosine Transport ◽  
High Speed Supernatant ◽  
Stimulation Of

The presence of adenosine (25-250 microM) or of 2-chloroadenosine (2.5-100 microM) in the incubation medium caused a marked decrease in the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. This effect was accompanied by an increase in the concentration of cyclic AMP, an activation of phosphorylase and of fructose 2,6-bisphosphatase, and an inactivation of pyruvate kinase and of 6-phosphofructo-2-kinase. As a rule, the changes in the fructose 2,6-bisphosphate-modifying system were slower but more persistent than those in the activities of phosphorylase and pyruvate kinase. The effect of the nucleoside to decrease the concentration of fructose 2,6-bisphosphate was not affected by an inhibitor of adenosine transport and could not be obtained in a liver high-speed supernatant. These data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenosine to membranous receptors. Like glucagon, 2-chloroadenosine stimulated gluconeogenesis in isolated hepatocytes, whereas adenosine had an opposite effect.

Phosphofructokinase and glycolysis in the livers of rats fed a thrombogenic diet

1970 ◽  
Vol 48 (2) ◽  
pp. 222-224
Author(s):  
N. Simard-Duquesne
Keyword(s):  
Lactic Acid ◽  
High Speed ◽  
Mitochondrial Fraction ◽  
Pasteur Effect ◽  
Phosphofructokinase Activity ◽  
Reconstituted System ◽  
High Speed Supernatant

Liver supernatant phosphofructokinase activity is increased in rats fed a thrombogenic diet at about the same time as the incidence of endotoxin-induced thrombosis is known to increase. In the livers of these rats, the Pasteur effect, as determined by the production of lactic acid in a reconstituted system containing a high-speed supernatant with and without the mitochondrial fraction, is significantly decreased.

ACTIVATION OF SOLUBILIZED STEROID-TRANSFORMING ENZYMES OF ADRENAL MICROSOMAL ORIGIN BY SERUM PROTEINS

1972 ◽  
Vol 54 (2) ◽  
pp. 297-315 ◽  
Author(s):  
MARGOT A. HAMILTON ◽  
R. W. McCUNE ◽  
SIDNEY ROBERTS
Keyword(s):  
High Speed ◽  
Microsomal Fraction ◽  
Adrenal Glands ◽  
Serum Proteins ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fraction ◽  
Rat Serum ◽  
Microsomal Preparation ◽  
Stimulation Of

SUMMARY The reactions which result in the conversion of pregnenolone to progesterone and of progesterone to deoxycorticosterone in undisrupted microsomal preparations from rat adrenal glands were stimulated by homologous serum. The active materials were shown to be firmly associated with serum proteins. The dialysable fraction of serum was either without effect on these transformations or was inhibitory. The enzyme systems involved were partially solubilized by exposure of the microsomal preparation to prolonged sonic treatment or to 1% Triton N-101. After either treatment, 35–40% of the original specific activity of the steroid 21-hydroxylase system responsible for the conversion of progesterone to deoxycorticosterone was found in the supernatant fraction after high-speed centrifugation. However, this solubilized system did not respond to serum preparations. The same procedures also resulted in a supernatant fluid which showed about 50–60% of the initial specific activity of the multi-enzyme system involved in the conversion of pregnenolone to progesterone. In these instances, the stimulatory effect of serum was retained or accentuated. Acetone powders prepared from the adrenal microsomal fraction were also active in the conversion of pregnenolone to progesterone and responded to serum with enhanced activity. Earlier observations that activation of steroid 21-hydroxylase by homologous rat serum was specific for the β-globulin fraction were confirmed in the present investigations. In contrast, stimulation of the conversion of pregnenolone to progesterone was apparently due principally to the albumin fraction. Albumin preparations from a number of other sources, as well as whole human serum protein, were also effective in this regard. The active protein preparations selectively stimulated 4-ene-3β-hydroxysteroid dehydrogenase activity, but did not activate 5-ene-3-oxosteroid isomerase in the microsomal fraction. This finding suggested that activation of 5-ene-3β-hydroxysteroid dehydrogenase was responsible for stimulation of progesterone synthesis from pregnenolone. The results indicate that the protein-bound factor in rat serum which was capable of stimulating the conversion of progesterone to deoxycorticosterone in microsomal preparations from rat adrenal glands was different from that which activates the conversion of pregnenolone to progesterone. Moreover, these diverse factors appeared to act by different mechanisms.

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 Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1967 ◽  
Vol 105 (3) ◽  
pp. 927-936 ◽  
Author(s):  
R. Vrba
Keyword(s):  
Particulate Matter ◽  
High Speed ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Axoplasmic Flow ◽  
Brain Lipids ◽  
Mitochondrial Fractions ◽  
High Speed Supernatant

1. Rats were injected with [U−14C]glucose and the content of 14C in proteins and lipids of the cerebral P1 (‘nuclear’), P2 (‘mitochondrial’), P3 (‘microsomal’) and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P2) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the 14C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the 14C assimilated by brain proteins was found in the P2 (‘mitochondrial’) fraction. About 68–70% of the 14C assimilated by brain lipids was also recovered from the lipids of the P2 fraction. 4. Between 22 and 93hr. after injection of [U−14C]glucose both the amount of 14C in the protein of the P2 (‘mitochondrial’) fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P1, P2 and P3) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P2) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U−14C]glucose. The amount of 14C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of 14C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U−14C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.

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