Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177–185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

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Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1989.tb15220.x ◽

1989 ◽

Vol 186 (1-2) ◽

pp. 383-388 ◽

Cited By ~ 21

Author(s):

Peter HILDEBRANDT ◽

Horacio GARDA ◽

Anton STIER ◽

Galina I. BACHMANOVA ◽

Irina P. KANAEVA ◽

...

Keyword(s):

Protein Interactions ◽

Substrate Binding ◽

Protein Protein Interactions ◽

Microsomal Cytochrome ◽

Cytochrome P 450

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Thermodynamic and detailed kinetic study of the formation of orthophthalaldehyde-agmatine complex by fluorescence intensities

Journal of Analytical Science & Technology ◽

10.1186/s40543-020-00238-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Khémesse Kital ◽

Moumouny Traoré ◽

Diégane Sarr ◽

Moussa Mbaye ◽

Mame Diabou Gaye Seye ◽

...

Keyword(s):

Complex Formation ◽

Thermodynamic Parameters ◽

Initial Rate ◽

Reaction Medium ◽

Standard Entropy ◽

Step Size ◽

Formation Reaction ◽

Different Temperatures ◽

Kinetics Of ◽

Complex Formation Process

Abstract The aim of this work is to determine the thermodynamic parameters and the kinetics of complex formation between orthophthalaldehyde (OPA) and agmatine (AGM) in an alkaline medium (pH 13). Firstly, the association constant (Ka) between orthophthalaldehyde and agmatine was determined at different temperatures (between 298 K and 338 K) with a step size of 10 K. Secondly, the thermodynamic parameters such as standard enthalpy (ΔH°), standard entropy (ΔS°),and Gibbs energy (∆G) were calculated, where a positive value of ΔH° (+45.50 kJ/mol) was found, which shows that the reaction is endothermic. In addition, the low value of ΔS°(+0.24 kJ/mol) indicates a slight increase in the disorder in the reaction medium. Furthermore, the negative values of ΔG between −35.62 kJ/mol and −26.02 kJ/mol show that the complex formation process is spontaneous. Finally, the parameters of the kinetics of the reaction between OPA and AGM were determined as follows: when the initial concentration of AGM (5 × 10−6 M) is equal to that of the OPA, the results show that the reaction follows an overall 1.5 order kinetics with an initial rate of 5.1 × 10−7Mmin−1 and a half-life of 8.12 min. The partial order found in relation to the AGM is 0.8. This work shows that the excess of OPA accelerates the formation reaction of the complex.

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Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)34532-0 ◽

1982 ◽

Vol 257 (12) ◽

pp. 7023-7029 ◽

Cited By ~ 2

Author(s):

S Kawato ◽

J Gut ◽

R J Cherry ◽

K H Winterhalter ◽

C Richter

Keyword(s):

Protein Interactions ◽

Liver Microsomes ◽

Phospholipid Vesicles ◽

Protein Protein Interactions ◽

Cytochrome P 450

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Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.399-407.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 399-407

Author(s):

I J McEwan ◽

A P Wright ◽

K Dahlman-Wright ◽

J Carlstedt-Duke ◽

J A Gustafsson

Keyword(s):

Glucocorticoid Receptor ◽

Protein Interactions ◽

Core Promoter ◽

Specific Interaction ◽

Direct Interaction ◽

Dependent Manner ◽

Transactivation Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

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GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5101-5112.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5101-5112

Author(s):

J S Flick ◽

M Johnston

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Tandem Repeats ◽

Glucose Repression ◽

Molecular Data ◽

Primary Response ◽

Yeast Cells ◽

Cell Fractionation ◽

Protein Protein Interactions ◽

Yeast Saccharomyces Cerevisiae

Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

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Temperature dependence of dipalmitoyl phosphatidylcholine monolayer stability

Journal of Applied Physiology ◽

10.1152/jappl.1981.51.5.1108 ◽

1981 ◽

Vol 51 (5) ◽

pp. 1108-1114 ◽

Cited By ~ 76

Author(s):

J. Goerke ◽

J. Gonzales

Keyword(s):

Phase Transition ◽

Lung Surfactant ◽

Principal Component ◽

Water Interface ◽

Dipalmitoyl Phosphatidylcholine ◽

Phase Transition Temperatures ◽

Air Water Interface ◽

Different Temperatures ◽

Monolayer Stability ◽

Isolated Rat

Dipalmitoyl phosphatidylcholine is the principal component of lung surfactant, and knowledge of its behavior as a film spread at the air-water interface is essential for understanding how lung surfactant itself works. We therefore studied the collapse rates of very low surface tension air-water monolayers of dipalmitoyl, dimyristoyl, and palmitoyl-myristoyl phosphatidylcholines at different temperatures. In each case we found that the monolayers abruptly became unstable at temperature 3–4 degree C above their bulk lipid-water phase transition temperatures (Tc). This accords with a comparable increase in Tc occurring in bulk systems subjected to high pressure. These findings are also consistent with the behavior of isolated rat lungs, which have been found to require higher transmural pressures to maintain a given volume on deflation when kept at temperature above the Tc of dipalmitoyl phosphatidylcholine.

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