scholarly journals Characterization of microsomal choloyl-coenzyme A synthetase

1977 ◽  
Vol 163 (2) ◽  
pp. 357-362 ◽  
Author(s):  
D A Vessey ◽  
D Zakim
Keyword(s):  
Bile Salts ◽  
Substrate Inhibition ◽  
Active Form ◽  
Microsomal Membrane ◽  
Lipid Phase ◽  
Bivalent Cation ◽  
Absolute Requirement ◽  
High Concentrations ◽  
Triton X ◽  
Membrane Treatment

Choloyl-CoA synthetase (EC 6.2.1.7) was characterized for the first time under appropriated assay conditions. The p/ optimum for the reaction is pH 7.2.-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The KAppm (apparent Km) for CoA, extrapolated from kinetic data, is 50 micronM, but in fact the rate of reaction is increased little by concentrations of CoA above 25 micronM. The KAppm for ATP is 600 micronM. High concentrations of ATP appear to cause substrate inhibition. The KAppm for cholate was 6 micronM. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.

scholarly journals Synthesis of retinyl phosphate mannose and dolichyl phosphate mannose from endogenous and exogenous retinyl phosphate and dolichyl phosphate in microsomal fraction. Specific decrease in endogenous retinyl phosphate mannose synthesis in vitamin A deficiency

1982 ◽  
Vol 208 (1) ◽  
pp. 159-170 ◽  
Author(s):  
L M De Luca ◽  
M R Brugh ◽  
C S Silverman-Jones ◽  
Y Shidoji
Keyword(s):  
Vitamin A ◽  
Microsomal Fraction ◽  
Vitamin A Deficiency ◽  
Bivalent Cation ◽  
Dolichyl Phosphate ◽  
Absolute Requirement ◽  
Syrian Golden Hamsters ◽  
Bivalent Metal Ions ◽  
Undecaprenyl Phosphate ◽  
Triton X

Rat liver microsomal fraction synthesized Ret-P-Man (retinyl phosphate mannose) and Dol-P-Man (dolichyl phosphate mannose) from endogenous Ret-P (retinyl phosphate) and Dol-P (dolichyl phosphate). Ret-P-Man synthesis displayed an absolute requirement for a bivalent cation, and also Dol-P-Man synthesis was stimulated by bivalent metal ions. Mn2+ and Co2+ were the most active, with maximum synthesis of Ret-P-Man occurring at 5-10 mM: Mg2+ was also active, but at higher concentrations. At 5mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 microM-GDP-mannose in 15 min at 37 degrees C was approx. 3 pmol/mg of protein. In the same assays about 7-10 pmol of endogenous Dol-P was mannosylated. Bivalentcation requirement for Ret-P-Man synthesis from exogenous Ret-P showed maximum synthesis at 2.5 mM-Mn2+ or -Co2+. In addition to Ret-P-Man and Dol-P-Man, a mannolipid co-chromatographing with undecaprenyl phosphate mannose was detected. Triton X-100 (0.5%) abolished Ret-P-Man synthesis from endogenous Ret-P and caused a 99% inhibition of Ret-P-Man synthesis from exogenous Ret-P. The presence of detergent (0.5%) also inhibited Dol-P-Man synthesis from endogenous Dol-P and altered the requirement for Mn2+. Microsomal fraction from Syrian golden hamsters was also active in Ret-P-Man and Dol-P-Man synthesis from endogenous Ret-P and Dol-P. At 5 mM-Mn2+ about 2.5 pmol of endogenous Ret-P and 3.7 pmol of endogenous Dol-P were mannosylated from GDP-mannose per mg of protein in 15 min at 37 degrees C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2 pmol of Ret-P and 14.1 pmol of Dol-P available for mannosylation. Since GDP-mannose: Ret-P and GDP-mannose: Dol-P mannosyltransferase activities were not affected, depletion of vitamin A must affect Ret-P and Dol-P pools in opposite ways.

scholarly journals Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Robert G. SPIRO ◽  
Vishnu D. BHOYROO
Keyword(s):  
Periodate Oxidation ◽  
Maximal Activity ◽  
Sialic Acid Residue ◽  
Lymphoid Tissues ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Selectin Ligands ◽  
Strict Requirement ◽  
Lectin Chromatography ◽  
Triton X

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

scholarly journals Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Juan L. Rendón ◽  
Mauricio Miranda-Leyva ◽  
Alberto Guevara-Flores ◽  
José de Jesús Martínez-González ◽  
Irene Patricia del Arenal ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Active Form ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Hysteretic Behavior ◽  
Low Concentrations ◽  
Mechanistic Basis ◽  
High Concentrations ◽  
Thioredoxin Glutathione Reductase

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

scholarly journals Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 9-18 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Mitochondria ◽  
Exchange Chromatography ◽  
Glycerophosphate Dehydrogenase ◽  
Pig Brain ◽  
Purification And Properties ◽  
High Concentrations ◽  
Triton X

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics

2018 ◽  
Vol 475 (11) ◽  
pp. 1979-1993 ◽  
Author(s):  
Andreas Sukmana ◽  
Zhaomin Yang
Keyword(s):  
Atpase Activity ◽  
Enrichment Culture ◽  
Physiological State ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Enzyme Analysis ◽  
Type Iv Pilus ◽  
Type Iv ◽  
High Concentrations

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Measurement of Isocitric Dehydrogenase Activity in Body Fluids

1959 ◽  
Vol 5 (6) ◽  
pp. 509-518 ◽  
Author(s):  
George N Bowers
Keyword(s):  
Standard Deviation ◽  
Dehydrogenase Activity ◽  
Substrate Inhibition ◽  
Body Fluids ◽  
Isocitric Dehydrogenase ◽  
Factors Influencing ◽  
Activity Curve ◽  
High Concentrations ◽  
Healthy Males

Abstract 1. Factors influencing the isocitric dehydrogenase (ICD) activity of serum were studied. Substrate inhibition by high concentrations of isocitrate was observed. 2. The temperature-activity curve of serum ICD was determined. The importance of controlling the temperature of the reaction was stressed. 3. Using this information, a method of measuring the ICD activity in 0.2 ml. of serum is described. 4. The range of ICD activity in 50 healthy males was 30 to 192 units with a mean of 90 units and a standard deviation of ± 34.3 units.

scholarly journals Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression

1985 ◽  
Vol 227 (3) ◽  
pp. 753-757 ◽  
Author(s):  
N Allison ◽  
M J O'Donnell ◽  
M E Hoey ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Cytoplasmic Membrane ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Acinetobacter Calcoaceticus ◽  
Regulation Of Expression ◽  
Ionic Detergents ◽  
Membrane Bound ◽  
Lactate Dehydrogenases ◽  
Triton X

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.

scholarly journals The permeability to calcium of pigeon erythrocyte ‘ghosts’ studied by using the calcium-activated luminescent protein, obelin

1975 ◽  
Vol 152 (2) ◽  
pp. 255-265 ◽  
Author(s):  
Anthony K. Campbell ◽  
Robert L. Dormer
Keyword(s):  
Pyruvate Kinase ◽  
Time Course ◽  
Small Concentration ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Experimental Conditions ◽  
Bivalent Cation ◽  
Low Concentration ◽  
Triton X ◽  
Obelia Geniculata

1. Obelin, the Ca2+-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ‘ghosts’ in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca2+ within the ‘ghosts’ were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ‘ghosts’ during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ‘ghosts’. Less than 10% of the inulin or pyruvate kinase sealed within the ‘ghosts’ was released under any of the experimental conditions. 3. Triton X-100 (0.1–10%, v/v) made the ‘ghosts’ highly permeable to Ca2+. In the presence of 1mm-Ca2+ and Triton, 95–100% of the obelin was utilized within 10–20s. 4. A time-course of resealing ‘ghosts’ at 37°C showed that over a period of 90min, the ‘ghosts’ became gradually less permeable to Ca2+. ‘Ghosts’ which remained at 0°C retained only a small concentration of obelin and ATP, and were highly permeable to Ca2+. 5. Erythrocyte ‘ghosts’ resealed for 30min at 20°C rather than 37°C were more permeable to Ca2+, as shown by the fact that 92% of the obelin in the ‘ghosts’ was utilized during the first 60s after the addition of 1mm-Ca2+, as opposed to 44% for ‘ghosts’ resealed at 37°C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ‘ghosts’ which were highly permeable to Ca2+ after resealing for 60min at 37°C. Of the obelin in the ‘ghosts’, produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca2+ compared with 23% for ‘ghosts’ produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ‘ghosts’ to Ca2+. Maximum effects of the ionophore (16μg/ml) were obtained by preincubating the ‘ghosts’ with the ionophore A23187 (16μg/ml) in the presence of a low concentration of Mg2+ and in the absence of Ca2+.

scholarly journals Membranes and bile formation. Composition of several mammalian biles and their membrane-damaging properties

1979 ◽  
Vol 178 (1) ◽  
pp. 201-208 ◽  
Author(s):  
R Coleman ◽  
S Iqbal ◽  
P P Godfrey ◽  
D Billington
Keyword(s):  
Guinea Pig ◽  
Bile Salts ◽  
Cell Damage ◽  
Membrane Damage ◽  
Total Content ◽  
Hepatic Cell ◽  
Membrane Composition ◽  
Bile Formation ◽  
Composition And Structure ◽  
High Concentrations

The total content and profile of bile salts and phospholipids are reported for several mammalian biles. Rabbit and guinea-pig biles are characterized by high proportions of conjugated dihydroxy bile salts with respect to trihydroxy bile salts, but contain relatively little phospholipid. Both rabbit and guinea-pig biles exhibit little evidence of hepatic cell damage, even though they are able to cause membrane damage (as evidenced by lysis of human erythrocytes) at low (2–3 mM) concentrations of bile salts; this lytic behaviour is also a property of their predominant bile salts. Addition of phosphatidylcholine to the bile or bile salt is able to decrease the lytic behaviour. Perhaps the most significant observation is that these biles, and their predominant bile salts, are dramatically less lytic towards sheep erythrocytes, indicating that some factor(s) in membrane composition and structure may partly explain the resistance of membranes of the biliary tract to the presence of high concentrations of potentially membrane-damaging bile salts.

scholarly journals The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous

1982 ◽  
Vol 201 (3) ◽  
pp. 515-521 ◽  
Author(s):  
P S J Cheetham ◽  
P Dunnill ◽  
M D Lilly
Keyword(s):  
Cell Membrane ◽  
Membrane Lipids ◽  
Cholesterol Oxidase ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Solubilized Enzyme ◽  
High Concentrations ◽  
Triton X ◽  
Catalytic Functions ◽  
Hydrophobic Anchor

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.

Sign in / Sign up

Export Citation Format

Share Document