scholarly journals The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain

1974 ◽  
Vol 137 (3) ◽  
pp. 525-530 ◽  
Author(s):  
Elling Kvamme ◽  
Ingeborg Aasland Torgner
Keyword(s):  
Binding Sites ◽  
Protein Concentration ◽  
Sedimentation Coefficient ◽  
Acetyl Coa ◽  
Pig Kidney ◽  
Substrate Activation ◽  
Significant Difference ◽  
Chain Acyl ◽  
Powerful Activator ◽  
Phosphate Activated Glutaminase

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (KA about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.

Characterization and measurement of cytoplasmic and nuclear oestradiol-17β-receptor proteins in benign hypertrophied human prostate

1982 ◽  
Vol 93 (3) ◽  
pp. 305-317 ◽  
Author(s):  
G. Auf ◽  
R. Ghanadian
Keyword(s):  
Ionic Strength ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Sedimentation Coefficient ◽  
Nuclear Extract ◽  
Human Prostate ◽  
Protamine Sulphate ◽  
Significant Difference ◽  
The Mean ◽  
Binding Component

The demonstration and partial characterization of a high-affinity saturable binding component for oestradiol-17β in the cytosol and nuclear extract of the benign hypertrophied human prostate is reported. This binding component was found to precipitate with protamine sulphate and to exhibit marked specificity for oestradiol and diethyl-stilboestrol (DES) but not dihydrotestosterone (DHT) or progesterone. Analysis of oestradiol-labelled cytosol on low ionic strength glycerol gradients revealed a binding component with a sedimentation coefficient of 4S which was inhibited with DES but not DHT and destroyed either by preheating labelled cytosol at 45 °C for 30 min or by treatment with the sulphydryl blocking agent, parachloromercureobenzoate. The oestradiol-binding complex precipitated by ammonium sulphate was found to have an isoelectric point (pI) of 6·3 as analysed on polyacrylamide-gel plates. The oestradiol-binding component in the nuclear extract also exhibited similar steroid specificity and sedimented with a coefficient of 2·8S on high ionic strength glycerol gradients. Isoelectric focusing of the heparin-extracted nuclear oestradiol-binding complex revealed a pI of 6·0. The dissociation constants (Kd) and the concentrations of the cytoplasmic and nuclear oestradiol-binding sites in 19 samples of benign hypertrophied prostates were estimated by Scatchard plot analyses. The mean Kd and concentration of binding sites in the cytosol were 2·9 ± 2·6 (s.d.) nmol/l and 11·7± 8·4 fmol/mg protein respectively. The corresponding values for nuclear extract were 4·1 ±7·5 nmol/l and 347·1±415·5fmol/mg DNA respectively. There was no significant difference between the mean concentration of oestradiol-binding sites in the two cellular fractions.

Interaction of acyl coenzyme A substrates and analogs with pig kidney medium-chain acyl-CoA dehydrogenase

Biochemistry ◽  
1987 ◽  
Vol 26 (12) ◽  
pp. 3704-3710 ◽  
Author(s):  
Patricia J. Powell ◽  
Sze Mei Lau ◽  
David Killian ◽  
Colin Thorpe
Keyword(s):  
Coenzyme A ◽  
Medium Chain ◽  
Pig Kidney ◽  
Chain Acyl ◽  
Acyl Coenzyme A

scholarly journals Evidence that ACN1 (acetate non-utilizing 1) prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings

2011 ◽  
Vol 437 (3) ◽  
pp. 505-513 ◽  
Author(s):  
Elizabeth Allen ◽  
Annick Moing ◽  
Jonathan A. D. Wattis ◽  
Tony Larson ◽  
Mickaël Maucourt ◽  
...  
Keyword(s):  
Carbon Sink ◽  
Glyoxylate Cycle ◽  
Eicosenoic Acid ◽  
Primary Metabolism ◽  
Acetyl Coa ◽  
Primary Metabolite ◽  
Lipid Mobilization ◽  
Transcript Levels ◽  
Chain Acyl ◽  
Acetate Accumulation

ACN1 (acetate non-utilizing 1) is a short-chain acyl-CoA synthetase which recycles free acetate to acetyl-CoA in peroxisomes of Arabidopsis. Pulse-chase [2-13C]acetate feeding of the mutant acn1–2 revealed that acetate accumulation and assimilation were no different to that of wild-type, Col-7. However, the lack of acn1–2 led to a decrease of nearly 50% in 13C-labelling of glutamine, a major carbon sink in seedlings, and large decreases in primary metabolite levels. In contrast, acetyl-CoA levels were higher in acn1–2 compared with Col-7. The disappearance of eicosenoic acid was slightly delayed in acn1–2 indicating only a small effect on the rate of lipid breakdown. A comparison of transcript levels in acn1–2 and Col-7 showed that induced genes included a number of metabolic genes and also a large number of signalling-related genes. Genes repressed in the mutant were represented primarily by embryogenesis-related genes. Transcript levels of glyoxylate cycle genes also were lower in acn1–2 than in Col-7. We conclude that deficiency in peroxisomal acetate assimilation comprises only a small proportion of total acetate use, but this affects both primary metabolism and gene expression. We discuss the possibility that ACN1 safeguards against the loss of carbon as acetate from peroxisomes during lipid mobilization.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Increased concentrations of glycogen synthase protein in skeletal muscle of patients with NIDDM

1995 ◽  
Vol 269 (1) ◽  
pp. E27-E32 ◽  
Author(s):  
M. Lofman ◽  
H. Yki-Jarvinen ◽  
M. Parkkonen ◽  
J. Lindstrom ◽  
L. Koranyi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Metabolism ◽  
Protein Concentration ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Dependent Diabetes Mellitus ◽  
Euglycemic Hyperinsulinemic Clamp ◽  
Control Subjects ◽  
Significant Difference ◽  
Glycogen Synthase Activity

To examine whether changes in the glycogen synthase protein concentration contribute to impaired insulin-stimulated glycogen metabolism in patients with noninsulin-dependent diabetes mellitus (NIDDM), muscle biopsies were taken before and after a 4-h euglycemic hyperinsulinemic clamp to measure glycogen synthase activity and glycogen synthase protein concentrations in 14 patients with NIDDM and in 17 control subjects. Nonoxidative glucose metabolism was reduced by 64% in patients with NIDDM compared with control subjects and correlated with insulin-stimulated glycogen synthase activity (r = 0.55, P < 0.05). The concentration of glycogen synthase protein in skeletal muscle was higher in patients with NIDDM than in control subjects (6.75 +/- 0.88 vs. 4.41 +/- 0.50 counts.min-1.micrograms protein-1, P < 0.05), whereas there was no significant difference in glycogen synthase mRNA concentration between the two groups. The glycogen synthase protein concentration correlated inversely with the rate of nonoxidative glucose metabolism (r = -0.63, P < 0.05). These findings indicate that the amount of glycogen synthase protein is increased in skeletal muscle of patients with NIDDM. The increase in the glycogen synthase protein may serve to compensate for a functional defect in the activation of the enzyme by insulin.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

Solubilization and molecular characterization of the nitrobenzylthioinosine binding sites from pig kidney brush-border membranes

1994 ◽  
Vol 1191 (1) ◽  
pp. 94-102 ◽  
Author(s):  
Francisco Ciruela ◽  
Julià Blanco ◽  
Enric I. Canela ◽  
Carmen Lluis ◽  
Rafael Franco ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membranes ◽  
Pig Kidney
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