Determination of the mechanism of reaction for bile acid: CoA ligase

The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubstrate kinetic analysis was conducted by varying, one at a time ATP cholate and CoA. Both ATP and cholate gave parallel double reciprocal plots versus CoA, which indicates a ping-pong mechanism with either pyrophosphate or AMP leaving prior to the binding of CoA. Addition of pyrophosphate to the assays changed the parallel plots to intersecting ones; addition of AMP did not. This indicates that pyrophosphate is the first product. The end-product, AMP, was a competitive inhibitor versus ATP, as was cholyl-CoA at saturating concentrations of cholate. Both AMP and cholyl-CoA were uncompetitive inhibitors versus CoA. Based on this information, it was concluded that the reaction follows a bi uni uni bi ping-pong mechanism with ATP binding first, and with the release of the final products, AMP and cholyl-CoA, being random. CoA showed substrate inhibition at high but non-saturating concentrations and this inhibition was competitive versus ATP, which is consistent with the predicted ping-pong mechanism. The ability of cholyl-CoA, but not cholate or CoA, to bind with high affinity to the free enzyme was suggestive of a high affinity of the enzyme for the thioester link.

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The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

Biochemical Journal ◽

10.1042/bj1390109 ◽

1974 ◽

Vol 139 (1) ◽

pp. 109-121 ◽

Cited By ~ 65

Author(s):

B. Middleton

Keyword(s):

Rat Liver ◽

Substrate Inhibition ◽

Kinetic Mechanism ◽

Competitive Inhibitor ◽

Acetyl Coa ◽

Keto Form ◽

Ping Pong ◽

Substrate Inhibitor ◽

The Hill ◽

Covalent Intermediate

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg2+ inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg2+ but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a Ki of 67μm. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

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myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells

Biochemical Journal ◽

10.1042/bj2950863 ◽

1993 ◽

Vol 295 (3) ◽

pp. 863-869 ◽

Cited By ~ 11

Author(s):

G T Berry ◽

R A Johanson ◽

J E Prantner ◽

B States ◽

J R Yandrasitz

Keyword(s):

Endothelial Cells ◽

Kinetic Analysis ◽

Transport System ◽

Bovine Serum ◽

Competitive Inhibitor ◽

Aortic Endothelial Cells ◽

High Affinity ◽

Bovine Aortic Endothelial Cells ◽

Fetal Bovine ◽

Aortic Endothelial

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.

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Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

Scientific Reports ◽

10.1038/s41598-021-83265-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Avital Shushan ◽

Mickey Kosloff

Keyword(s):

Protein Interactions ◽

Structural Elements ◽

Protein Protein Interactions ◽

Immunity Protein ◽

Rational Engineering ◽

High Affinity ◽

Comparative Structure ◽

Polar Interactions ◽

Exquisite Specificity

AbstractThe interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.

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Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

The Journal of General Physiology ◽

10.1085/jgp.200308784 ◽

2003 ◽

Vol 122 (3) ◽

pp. 295-306 ◽

Cited By ~ 52

Author(s):

Sonia Traverso ◽

Laura Elia ◽

Michael Pusch

Keyword(s):

Chloride Channels ◽

Bound State ◽

Side Chain ◽

Pore Channel ◽

Kinetic Properties ◽

Wild Type ◽

High Affinity ◽

Critical Residue ◽

Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

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Determination of 4-Hydroxy-3-methoxymethamphetamine as a Metabolite of Methamphetamine in Rats and Human Liver Microsomes Using Gas Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry

Journal of Analytical Toxicology ◽

10.1093/jat/33.5.266 ◽

2009 ◽

Vol 33 (5) ◽

pp. 266-271 ◽

Cited By ~ 5

Author(s):

K. Kuwayama ◽

K. Tsujikawa ◽

H. Miyaguchi ◽

T. Kanamori ◽

Y. T. Iwata ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Liquid Chromatography ◽

Human Liver ◽

Liver Microsomes ◽

Gas Chromatography Mass Spectrometry ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

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Characterization of a Specific, High Affinity [3H]Arginine8Vasopressin-Binding Site on Liver Microsomes from Different Strains of Rat and the Role of Magnesium*

Endocrinology ◽

10.1210/endo-118-3-990 ◽

1986 ◽

Vol 118 (3) ◽

pp. 990-998 ◽

Cited By ~ 18

Author(s):

VENKAT GOPALAKRISHNAN ◽

CHRIS R. TRIGGLE ◽

PRAKASH V. SULAKHE ◽

J. ROBERT McNEILL

Keyword(s):

Binding Site ◽

Liver Microsomes ◽

High Affinity ◽

Different Strains

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Calcium pump, high-affinity Ca2+-ATPase, and other ATPases in dog antrum smooth muscle plasma membrane

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.5.c449 ◽

1985 ◽

Vol 248 (5) ◽

pp. C449-C456 ◽

Cited By ~ 36

Author(s):

A. K. Grover ◽

C. Y. Kwan ◽

P. J. Oakes

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Maximal Activity ◽

Calcium Pump ◽

Disulfonic Acid ◽

Calmodulin Antagonists ◽

High Affinity ◽

Chelex 100 ◽

Muscle Plasma Membrane ◽

Absolute Requirement

The plasma membrane-enriched fraction from dog antrum smooth muscle is enriched in ATP-dependent azide-insensitive Ca2+ uptake (0.3-0.4 microM Ca2+ required for half-maximal activity), a high-affinity Ca2+-ATPase (Km of 0.3-0.8 microM for Ca2+), a low-affinity Ca2+-ATPase (Km for 250-400 microM for Ca2+), and a Mg2+-ATPase. Studies using membranes washed with EDTA and assay media treated with Chelex 100 showed that the high-affinity Ca2+-ATPase did not depend on contaminating Mg2+. Thus, whereas the ATP-dependent Ca2+ uptake had an absolute requirement for Mg2+, the Ca2+-ATPases did not. Studies using gamma-irradiation showed that the protein responsible for the ATP-dependent Ca2+ uptake was inactivated at significantly lower doses of radiation than the three ATPases. The Ca2+ uptake and the high-affinity Ca2+-ATPase also differed in their inhibition by calmodulin antagonists and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus it is unlikely that the high-affinity Ca2+-ATPase by itself is responsible for the ATP-dependent Ca2+ uptake.

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ENZYMATISCH-FLUOROMETRISCHE BESTIMMUNG DER CORTISOLBINDUNGSKAPAZITÄT DES PLASMA

Acta Endocrinologica ◽

10.1530/acta.0.0560099 ◽

1967 ◽

Vol 56 (1) ◽

pp. 99-106 ◽

Cited By ~ 7

Author(s):

K. Leybold ◽

J. Rieper ◽

L. Weissbecker

Keyword(s):

Binding Capacity ◽

Liver Microsomes ◽

Mean Value ◽

Female Rats ◽

Plasma Samples ◽

Simple Method ◽

Adult Men ◽

The Mean ◽

Precision And Accuracy

ABSTRACT A simple method for the determination of cortisol-binding capacity is described. For saturation of the cortisol-binding proteins, plasma samples are incubated with an excess of cortisol. In the next step NADPH and liver microsomes of female rats are added. The microsomal Δ4-3-ketosteroid hydrogenase only reduces non protein-bound cortisol to tetrahydrocortisol-5α. Then the steroids are extracted by dichloromethane, and after some purification steps analyzed by fluorometry. Tetrahydrocortisol gives practically no fluorescence. The cortisol determined by this method corresponds to protein-bound cortisol and indicates the extent of cortisolbinding capacity. Precision and accuracy of the method were found to be good. The values of cortisol-binding capacity obtained by our method are compared with the results of other authors. The mean value of adult men was 25.5 ± 3.4 μg/100 ml, that of pregnant women, mens IX-X, 42.3 ± 4.2 μg/100 ml.

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