scholarly journals The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

1970 ◽  
Vol 120 (1) ◽  
pp. 145-150 ◽  
Author(s):  
A. P. F. Flint
Keyword(s):  
Luteinizing Hormone ◽  
Total Activity ◽  
Partial Purification ◽  
Kinetic Properties ◽  
Mevalonate Kinase ◽  
Farnesyl Pyrophosphate ◽  
Rat Ovary ◽  
High Concentrations ◽  
Geranyl Pyrophosphate

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6±0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The Km of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6±0.5μm; its Km for MgATP2− was 120±7.7μm. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3′-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP2−. The Ki for inhibition by geranyl pyrophosphate was 1.3±0.2μm; the Ki for inhibition by farnesyl pyrophosphate was 1.0±0.3μm. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.

Partial purification of galactinol synthase from leaves of Cucurbita pepo

1982 ◽  
Vol 60 (7) ◽  
pp. 1054-1059 ◽  
Author(s):  
John A. Webb
Keyword(s):  
Cucurbita Pepo ◽  
Partial Purification ◽  
Galactinol Synthase ◽  
Inositol 1 ◽  
Mature Leaves ◽  
Galactosyl Transferase ◽  
Naturally Occurring ◽  
High Concentrations ◽  
Uridine Nucleotides

An enzyme synthesizing galactinol, UDP-D-galactose:myo-inositol-1-α-D-galactosyl transferase (galactinol synthase), has been isolated and partially purified from mature leaves of Cucurbita pepo. The enzyme showed optimal activity between pH 7.5 and 8.0 and required Mn2+ and the presence throughout isolation, storage, and assay of a sulfhydryl protectant (β-mercaptoethanol). EDTA was completely inhibitory. From a range of metal ions only Mg2+ partially replaced Mn2+, while Co2+, Zn2+, Cu2+, and Ni2+ were inhibitory. The uridine nucleotides and UDP-glucose were from 40 to 80% inhibitory and probably constitute part of the in vivo control system. High concentrations of galactose, melibiose, and xylose were partially inhibitory. The enzyme appeared highly specific for myo-inositol and showed no ability for galactosyl transfer to any other naturally occurring sugar or sugar alcohol. Some reactivity was obtained with the isomeric scyllo-inositol but the product was not identified. A range of other sugar nucleotides were unreactive.

scholarly journals Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases

2000 ◽  
Vol 350 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Carolyn A. BEETON ◽  
Edwin M. CHANCE ◽  
Lazaros C. FOUKAS ◽  
Peter R. SHEPHERD
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Membrane Lipid ◽  
Kinetic Properties ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
Functional Roles ◽  
Wide Range ◽  
High Concentrations

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.

scholarly journals Sesquiterpene α-Farnesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Development in Apples

2000 ◽  
Vol 125 (1) ◽  
pp. 111-119 ◽  
Author(s):  
H.P. Vasantha Rupasinghe ◽  
Gopinadhan Paliyath ◽  
Dennis P. Murr
Keyword(s):  
Metal Ion ◽  
Maximal Activity ◽  
Exchange Chromatography ◽  
Cytosolic Fraction ◽  
Skin Tissue ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Farnesyl Pyrophosphate ◽  
Superficial Scald

To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg2+ or Mn2+ for activity. It exhibited allosteric kinetics, S(0.5) for farnesyl pyrophosphate being 84±18 μmol·L-1, and a Hill coefficient (nH) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.

scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

scholarly journals Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger

1983 ◽  
Vol 209 (3) ◽  
pp. 669-676 ◽  
Author(s):  
A Habison ◽  
C P Kubicek ◽  
M Röhr
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Distinct Species ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Maximal Velocity ◽  
High Concentrations

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran-Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis-Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.

scholarly journals Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei

2011 ◽  
Vol 77 (21) ◽  
pp. 7772-7778 ◽  
Author(s):  
Yuliya A. Primak ◽  
Mai Du ◽  
Michael C. Miller ◽  
Derek H. Wells ◽  
Alex T. Nielsen ◽  
...  
Keyword(s):  
Mevalonate Pathway ◽  
Feedback Inhibition ◽  
Mevalonate Kinase ◽  
Farnesyl Pyrophosphate ◽  
Content Type ◽  
Dimethylallyl Diphosphate ◽  
Methanosarcina Mazei ◽  
Geranyl Pyrophosphate ◽  
Inhibition Analysis

ABSTRACTThe mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeonMethanosarcina mazei. The inhibition of theM. mazeiMVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate.M. mazeiMVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that theM. mazeiMVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.

STEROIDOGENIC EFFECTS OF LUTEINIZING HORMONE AND PROLACTIN ON THE RAT OVARY IN VIVO

1967 ◽  
Vol 38 (4) ◽  
pp. 423-430 ◽  
Author(s):  
K. YOSHINAGA ◽  
SUSAN A. GRIEVES ◽  
R. V. SHORT
Keyword(s):  
Luteinizing Hormone ◽  
Venous Blood ◽  
The Other ◽  
Progesterone Secretion ◽  
Rat Ovary

SUMMARY Progesterone and 20α-dihydroprogesterone were measured in the ovarian venous blood of rats in early pro-oestrus, late dioestrus, day 5 of pseudopregnancy, and in androgen-treated animals with persistent oestrus. Little progesterone was secreted in early pro-oestrus or persistent oestrus (0·1 μg./hr.), but the secretion rose in dioestrus (2·1 μg./hr.) and in pseudopregnancy (7 μg./hr.). The 20α-dihydroprogesterone secretion was low in the persistent oestrous group; in all the other groups it was 1·5–3 μg./hr. Treatment with luteinizing hormone increased progesterone secretion within 30 min. in pro-oestrus and persistent oestrus, had no significant effect in dioestrus, and depressed it markedly in pseudopregnancy. It appeared to have no significant effect on 20α-dihydroprogesterone secretion except during pseudopregnancy, when it was depressed. Treatment with prolactin produced no effect within 30 min.in pro-oestrous, dioestrous or pseudopregnant animals.

THE ENTRY OF ASCORBIC ACID INTO THE CORPUS LUTEUM IN VIVO AND IN VITRO AND THE EFFECT OF LUTEINIZING HORMONE

1967 ◽  
Vol 39 (1) ◽  
pp. 27-35 ◽  
Author(s):  
D. A. STANSFIELD ◽  
A. P. FLINT
Keyword(s):  
Ascorbic Acid ◽  
Exchange Rate ◽  
Luteinizing Hormone ◽  
Corpus Luteum ◽  
Plasma Ascorbic Acid ◽  
Progesterone Synthesis ◽  
Rat Ovary ◽  
Energy Dependent

SUMMARY Judged from the exchange rate between luteal and plasma ascorbic acid there appears to be no compartmentalization of ascorbic acid within the corpus luteum. Evidence is presented to show that the uptake of ascorbic acid into slices of superovulated rat ovary is an energy-dependent process which is inhibited by luteinizing hormone (LH) by means of its stimulatory effect on progesterone synthesis. The results are discussed in relation to the adrenal cortex and methods involving ascorbic acid depletion used in the assay of corticotrophin and LH.

Oligomeric Forms Of Pyruvate Kinase From Chlorella With Different Kinetic Properties

1991 ◽  
Vol 46 (5-6) ◽  
pp. 416-422 ◽  
Author(s):  
G. Ruyters ◽  
N. Grotjohann ◽  
W. Kowallik
Keyword(s):  
Pyruvate Kinase ◽  
Total Activity ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Resting Cells ◽  
Carbohydrate Degradation ◽  
Enzyme Species ◽  
Oligomeric Forms

Fast protein liquid chromatography on Superose 6 of crude extracts from the chlorophyllfree mutant no. 20 of the unicellular green alga Chlorella kessleri reveals two possibly oligomeric forms of pyruvate kinase (2.7.1.40). Their occurrence is markedly altered in the course of heterotrophic growth with changing levels of exogenous glucose as carbon source with only one enzyme species with a MW of 400 kDa existing in growing cells, two forms of 400 and 580 kDa in resting cells. Substrate affinity towards PEP of the 400 kDa form is better than that of the 580 kDa species; responses to the effector AMP are different as well. In vitro, addition of PEP or of AMP leads to the formation of higher MW enzyme species with MW of 730, 1050 and 1400 kDA without affecting the total activity. In vivo alterations in the levels of several metabolites including PEP upon addition of glucose have been shown to occur. Therefore, it is discussed, whether changes in the concentration of intermediates and effectors may provide the mechanism for the increased rate of carbohydrate degradation by affecting the occurrence and/or ratio of the various PK forms with different kinetic and regulatory properties. Upon blue light irradiation, which also stimulates carbohydrate breakdown of the Chlorella mutant cells, the distribution of PK is shifted towards the species with higher substrate affinity, a result being in accordance with the above conception

scholarly journals Isolation and properties of cholesterol esterstorage granules from ovarian tissues

1973 ◽  
Vol 134 (2) ◽  
pp. 399-406 ◽  
Author(s):  
David T. Armstrong ◽  
Anthony P. F. Flint
Keyword(s):  
Endoplasmic Reticulum ◽  
Luteinizing Hormone ◽  
Cholesterol Ester ◽  
Microsomal Fraction ◽  
Cholesterol Esterase ◽  
Interstitial Tissue ◽  
Unesterified Cholesterol ◽  
Rat Ovary ◽  
Soluble P

Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [32P]Piin vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [32P]Piin vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.

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