Biomedical subjects

K Ruckpaul

Publications and source records attributed to K Ruckpaul.

At least 37 records · Page 2Linked to original sources

PubMed1988

A simple determination of the sideness of the NH2-terminus in the membrane bound cytochrome P-450 LM2.

A simple and fast procedure for determining the sideness of the NH2-terminus in membrane proteins using FITC as label has been developed and applied to microsomal cytochrome P-450 LM2. The main advantages of FITC, which was shown not to penetrate lipid membranes, as compared to other labels are that it is fluorescent and that it can be used for manual microsequencing of proteins. By use of FITC it was directly shown that the NH2-terminus of P-450 LM2 is localized at the cytoplasmic side of the endoplasmic reticulum membrane. Based on this result the validity of recently published models of the membrane topology and the membrane insertion of P-450 are discussed.

Animals↗

PubMed1988

Comparative studies on the accessibility and functional importance of tyrosine residues in cytochrome P-450 isozymes.

Cytochromes P-450 LM2 and P-450 LM4 from rabbit liver microsomes were chemically modified with tetranitromethane. Nitration of two tyrosine residues of both isozymes inhibits the benzphetamine N-demethylase activity of P-450 LM2 as well as the p-nitrophenetole O-deethylase activity of P-450 LM4 by about 80%. For identification of the modified tyrosine residues the inactivated enzymes were digested with trypsin, and the labeled peptides were separated by HPLC. Sequencing of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM2 showed that the tyrosine residues at positions 235 and 380 were nearly fully nitrated, while Tyr-348, Tyr-484 and Tyr-111 were only partially labeled (about 40-50%). In the presence of the heme-binding inhibitor metyrapone, Tyr-380 is partially protected against modification, and the extent of inactivation is diminished as well. These results suggest that Tyr-380 of cytochrome P-450 LM2 presents a catalytically essential amino acid residue at its active center. Sequence analyses of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM4 revealed that mainly Tyr-243 and Tyr-271 were labeled, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent (about 30-45%). Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are suggested to be functionally involved in the interaction with NADPH-cytochrome P-450-reductase.

Amino Acid Sequence↗

PubMed1987

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues.

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.

Amino Acid Sequence↗

PubMed1987

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron.

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.

Animals↗

PubMed1987

Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2.

Carboxyl groups of NADPH-cytochrome P-450 reductase have been modified with the water-soluble carbodiimide EDC. Although there is no significant loss in DCPIP reduction the activity with cytochrome c and cytochrome P-450 LM2 as electron acceptors was inhibited by about 60 and 85%, respectively (1 h incubation time, 20 mM EDC). The inactivation by EDC was nearly completely prevented in the presence of cytochrome P-450 LM2, but not by bovine serum albumin. These results and crosslinking studies suggest that carboxyl groups of NADPH-cytochrome P-450 reductase are involved in charge-pair interactions to cytochrome c and to at least two amino groups of cytochrome P-450 LM2.

Animals↗

PubMed1986

Reduction of lactoperoxidase by the dithionite anion monomer.

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped-flow spectrophotometry in an anaerobic system. Under pseudo-first-order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO2- as the reducing species. The second-order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase. The second-order rate constant for the reduction of lactoperoxidase at pH 7.0 (kappa 1 = 1.3 X 10(5) M-1 s-1) was about three times higher than the rate constant for the reduction of cyanide-bound lactoperoxidase and two times the rate constant for the reduction of the fluoride-lactoperoxidase complex.

Dithionite↗

PubMed1986

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR.

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of 31P-NMR, 1H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by 31P-NMR. This method is more sensitive than 31P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.

Animals↗

PubMed1985

Relation between the structure of benzphetamine analogues and their binding properties to cytochrome P-450 LM2.

Twelve substrates of a homologous series of tertiary amines (type I substrates) have been reacted with cytochrome P-450 LM2 incorporated into unilamellar liposomes and in soluble form. The apparent spectral dissociation constants (Ks) of the substrate enzyme complexes and the induced high-spin shifts have been correlated with the electron density of distinct carbon atoms as monitored by 13C-NMR chemical shifts, the solubility of the amines and steric parameters of the substrate molecules. The results obtained led to the conclusion that two different intrinsic properties of the substrates can be discriminated in relation to the substrate-enzyme interaction. A diminished electron density at the nitrogen atom is accompanied by an increased binding affinity. The steric structure of the respective substrate determines its capability to shift the spin equilibrium to the high-spin state. Some characteristics of the active center of the enzyme are derived from the evidenced properties of the substrates.

Animals↗

PubMed1985

Interaction of hexane phosphonic acid diethyl ester with phospholipids in hepatic microsomes and reconstituted liposomes as studied by 31P-NMR.

By use of 31P-NMR, quasi-elastic light scattering and freeze-fracture electron microscopy it is shown that hexane phosphonic acid diethyl ester (PAE) is incorporated in hepatic microsomes without any alteration of the bilayer structure at two different sites. These findings proved that PAE can be used as molecular 31P-NMR probe in microsomes to get information about lipid-protein interactions. Extensive studies on reconstituted liposomal systems which contained cytochrome P-450 and cytochrome P-450 reductase showed that both proteins influence the localization of incorporated PAE. The results indicate a specific interaction of phosphatidylethanolamine (PE) with cytochrome P-450 in microsomes.

Animals↗

PubMed1985

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues.

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.

Animals↗

PubMed1985

[Regulation mechanisms of the endoplasmic cytochrome P-450 systems of the liver].

Regulation mechanisms of the cytochrome P-450 dependent monooxygenase from the hepatic endoplasmic reticulum at 3 different integrational levels are discussed. At the molecular level the activity of the system is regulated by a substrate dependent shift of an equilibrium of cytochrome P-450 spin conformers in favour of the high spin component. A correlation between the extent of the spin shift, the reduction rate and the substrate turnover has been evidenced. The regulation at the membrane level is based on interactions between the 3 essential components of the system: cytochrome P-450, reductase and lipid. The formation of the cytochrome P-450-reductase-complex necessary for oxygen activation by transfer of electrons is dependent on the charge of the phospholipids. The binding constant of the donor-acceptor complex increases with the acidity of the phospholipid head group, thus enhancing the velocity of the electron transfer.

Animals↗

PubMed1985

Chemical modification of cytochrome P-450 LM2 with N-acetylimidazole. Evidence for the functional involvement of tyrosyl residues.

Cytochrome P-450 LM2 has been reacted with N-acetylimidazole. About three tyrosyl residues of cytochrome P-450 LM2 are accessible to O-acetylation. The analysis of the spectral dissociation constants, substrate binding kinetics, reduction kinetics, N-demethylase activity and substrate conversion by artificial oxygen-donating agents of differently acetylated enzyme provides evidence for the existence of two groups of accessible tyrosines. One tyrosyl residue is located in the immediate environment of the heme iron and is involved in the binding of type II substrates. This tyrosine is not necessary for N-demethylation. Acetylation of two further tyrosyl residues, however, causes an almost complete inhibition of enzymatic activity. The results strongly suggest tyrosine(s) to be involved in NADPH-cytochrome P-450 reductase dependent N-demethylation.

Acetylation↗

PubMed1984

Mechanism of rate control of the NADPH-dependent reduction of cytochrome P-450 by lipids in reconstituted phospholipid vesicles.

The NADPH-supported reduction of cytochrome P-450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two-exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P-450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1:1 complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 microM for a microsomal lipid extract, 0.051 microM for a 3:1 (w/w) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 microM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase/P-450 ratio the amount of the rapidly reduced P-450 exceeds the equilibrium concentration of a 1:1 complex. Preformed 1:1 associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P-450 association does correspond to the data.

Cytochrome P-450 Enzyme System↗

PubMed1984

Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside, especially for measurements of protein diffusion in membranes.

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200-300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octyl-glucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.

Animals↗

PubMed1984

Chemical modification of cytochrome P-450 LM2. Characterization of tyrosine as axial heme iron ligand trans to thiolate.

Phenobarbital-inducible isozyme cytochrome P-450 LM2 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes has been modified with N-acetylimidazole and tetranitromethane. Up to four tyrosine residues of cytochrome P-450 LM2 are accessible to O-acetylation and to nitration. N-Demethylase activity, spectral dissociation constants and substrate binding kinetics of differently acetylated enzyme indicate the existence of two groups of accessible tyrosines also differing in their reactivity towards N-acetylimidazole. The fast-reacting tyrosine residue representing the first group is involved in the binding of the type II substrate aniline and appears to be located near the heme as shown by the protecting effect of the inhibitor metyrapone against modification, but obviously is not necessary for N-demethylation. Acetylation of one further tyrosine residue, however, caused an almost complete inhibition of the enzyme, indicating its involvement in the catalytic mechanism at the active center. Nitration of two tyrosine residues inactivates to about 20%. Obviously the third and fourth tyrosine residue are without functional importance. The experiments evidencing two functionally linked tyrosines are in line with HPLC analyses of tryptic peptides of cytochrome P-450 LM2 nitrated in the presence of metyrapone which gave evidence for the location of two distinct tyrosine residues in the active center. Nitration of tyrosine residues results in the partial formation of a hyperporphyrin spectrum of cytochrome P-450 LM2. Its appearance is prevented in the presence of metyrapone and can be reversed by reduction of the nitrotyrosinate .

Acetylation↗

PubMed1984

Selective chemical modification of a functionally linked lysine in cytochrome P-450 LM2.

Fluorescein isothiocyanate (FITC) has been selectively bound to the epsilon-amino group of lysine-382 in cytochrome P-450 LM2 (RH, reduced-flavoprotein: oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) at pH 8.15. Benzphetamine N-demethylase activity of the reconstituted FITC-modified cytochrome P-450 LM2 was inhibited by 25%. This inhibition has been shown to be due to an impaired electron transfer from the NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) to the haemoprotein. The data indicate that cytochrome P-450 interacts with the flavoprotein via electrostatic interactions.

Animals↗

PubMed1984

Temperature-dependent lateral diffusion of phospholipids in hepatic microsomes as studied by 31P-NMR.

The 81 MHz 31P-NMR spectra of isolated rabbit liver microsomes before and after trypsin treatment and of the total microsomal lipid extract were recorded in the 4-40 degrees C temperature range. In both treated and untreated microsomes at 4 degrees C most of the phospholipids gave rise to typical bilayer spectra whereas the lineshape of the latter in the 25-37 degrees C temperature range becomes narrower and more symmetrical. Quasi-elastic light scattering (QELS) measurements revealed that the microsomes maintain their size in the temperature region of the measurements. We interpret the lineshape changes for untreated microsomes above 25 degrees C as being determined by lateral diffusion. This is supported by lineshape calculations as a function of the lateral diffusion coefficient. The different spectral behavior of enzymatically active (untreated) and inactive (treated) microsomes suggests that the membrane proteins influence the lateral diffusion of the phospholipids.

Animals↗

PubMed1984

Motional dynamics of a spin labeled substrate analogue bound to cytochrome P-450: saturation transfer EPR studies.

The rotational motion of the spin labeled substrate analogue n- propylisocyanide bound to the active center of cytochrome P-450 was studied by saturation transfer EPR. The observed motional rate characterized by an effective rotational correlation time tau R of about 40 ns at 20 degrees C is at least 3 orders of magnitude higher than the macromolecular rotational diffusion of cytochrome P-450 in the microsomal membrane and represents a considerable motion in relation to the whole enzyme molecule. The tau R value is independent on the degree of purification of the enzyme system as revealed by measurements of (1) liver microsomes, (2) partially purified cytochrome P-450, and (3) cytochrome P-450 LM2 but shows a characteristic temperature dependence in the case of microsomes resulting in breaks in the Arrhenius plot at temperatures which correspond to phase transitions of the phospholipids. The results indicate that the mobility of the bound substrate analogue reflecting a relatively high conformational flexibility of the substrate binding region which depends on the state of the lipids and can therefore be influenced by them. These results support the assumption that cytochrome P-450 is capable of forming manifold binding to substrate molecules differing in stereochemical structures because of the conformational flexibility of its binding region.

Animals↗