The activity and kinetic properties of mevalonate kinase in superovulated rat ovary.
Ontology highlight
ABSTRACT:
SUBMITTER: Flint AP
PROVIDER: S-EPMC1179578 | biostudies-other | 1970 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Sterol, glyceride and phospholipid were found to account for more than 90% (w/w) of the lipid extracted from whole superovulated rat ovaries. These lipids, together with non-esterified fatty acids, were assayed in slices of the tissue after incubation for various times. Whereas the concentrations of triglyceride, diglyceride and phospholipid did not change significantly during incubation, that of sterol ester markedly decreased and those of free sterol, monoglyceride and non-esterified fatty acid increased. Evidence is presented that in this tissue (in contrast with other mammalian tissues) the main endogenous substrate for respiration is fatty acid derived from sterol ester.
| S-EPMC1185326 | biostudies-other
Project description:1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.
| S-EPMC1176917 | biostudies-other
Project description:Pyruvate kinase in rat pancreatic islets was characterized immunologically and kinetically. It is concluded that this activity is predominantly if not totally of the M(2) type.
| S-EPMC1158390 | biostudies-other
Project description:Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.
| S-EPMC1168063 | biostudies-other
Project description: Not available
| S-EPMC3952732 | biostudies-literature
Project description:A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM-1 s-1 in solution to 32.23±3.20 or 24.46±4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.
| S-EPMC4547157 | biostudies-literature
Project description:1. Superovulated rat ovary slices from rats treated with 20mug. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Delta(4)-3-oxo steroids (0.2mumole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90.0+/-4.6mumoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78.0+/-2.9mug. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-(14)C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60.7+/-0.9, 2.4+/-0.1, 18.0+/-1.1 and 0.7+/-0.1mug. atom of carbon/g. wet wt./hr. and accounted for 104.5+/-1.9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [(14)C]carbon dioxide were increased by approx. 25%, and 108.4+/-3.2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the (14)C incorporated into this fraction during incubation with [U-(14)C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of (14)C were incorporated into these lipid fractions from [1-(14)C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [(14)C]lactate and glucose 6-phosphate (C-1) derived from [1-(14)C]-, [6-(14)C]- and [U-(14)C]-glucose, and the ratio of [(14)C]carbon dioxide yields from [1-(14)C]glucose and [6-(14)C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of (14)C from [1-(14)C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [(3)H]water, [(14)C]sorbitol and glucose (1mg./ml.), the total water space (865+/-7.1mul./g.) and the extracellular water space (581+/-22mul./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540+/-23.6mul./g. to 639+/-31.3mul./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.
| S-EPMC1187697 | biostudies-other
Project description:Mevalonate kinase-associated diseases (MKAD) are caused by pathogenic mutations in the mevalonate kinase gene (MVK) and encompass several phenotypically different rare and hereditary autoinflammatory conditions. The most serious is a recessive systemic metabolic disease called mevalonic aciduria, and the most recently recognized is disseminated superficial actinic porokeratosis, a dominant disease limited to the skin. To evaluate a possible correlation between genotypes and (1) the different MKAD clinical subtypes or (2) the occurrence of severe manifestations, data were reviewed for all patients with MVK variants described in the literature (N = 346), as well as those referred to our center (N = 51). The genotypes including p.(Val377Ile) (homozygous or compound heterozygous) were more frequent in mild systemic forms but were also sometimes encountered with severe disease. We confirmed that amyloidosis was more prevalent in patients compound heterozygous for p.(Ile268Thr) and p.(Val377Ile) than in others and revealed new associations. Patients homozygous for p.(Leu264Phe), p.(Ala334Thr) or compound heterozygous for p.(His20Pro) and p.(Ala334Thr) had increased risk of severe neurological or ocular symptoms. All patients homozygous for p.(Leu264Phe) had a cataract. The variants associated with porokeratosis were relatively specific and more frequently caused a frameshift than in patients with other clinical forms (26% vs. 6%). We provide practical recommendations focusing on phenotype-genotype correlation in MKAD that could be helpful for prophylactic management.
| S-EPMC8067830 | biostudies-literature
Project description:DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.
| S-EPMC1798436 | biostudies-literature
Project description:Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.
| S-EPMC1168057 | biostudies-other