[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]. / Khimicheskaia modifikatsiia steroidgidroksiliruiushchikh monooksigenaz fluorestseinizotiotsianatom.

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

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.

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.

[Mechanism of detergent interaction with hemoproteins in aqueous media]. / Mekhanizm vzaimodeistviia detergentov s gemoproteidami v vodnoi srede.

The interactions of myoglobin, cytochrome c, and cytochrome P-450 LM-2 isolated from rabbit liver microsomes with detergents of type A (TM 3-12, Tween 20, Triton N-101) and detergent of B type (sodium cholate) in aqueous media were studied. These interactions are accompanied by a decrease in the Soret band intensity for all three hemoproteins. The rate of this process depends on the nature and concentration of the detergent as well as on temperature. The rate of the Soret band decrease is maximal for the zwitter-ionic surfactant TM 3-12. The rate constants of hemoprotein transformation depend on the detergent concentration. The detergent effects on the conformation and structure of the protein were demonstrated, using CD spectra and second derivatives of the absorption spectra of the hemoproteins in the presence of the detergents. The activation energies for myoglobin transformation in the presence of various detergents are equal to 17-23 kcal/mol and possibly reflect the cleavage of the coordinative heme-apoprotein bonds. A model of detergent interaction with hemoproteins is discussed. According to this model, the bimolecular interaction of the proteins with surfactants is observed at the detergent concentrations that are much lower than those for critical micelle formation values.

Effect of the gel-liquid crystalline phase transition on the reduction of cytochrome P-450 reconstituted into dimyristoyl-phosphatidylcholine liposomes.

The isolated purified components of the hepatic endoplasmic monooxygenatic system were incorporated into liposomes (dimyristoyl-phosphatidylcholine) by gel filtration technique. The temperature dependence of the reduction reaction in the range from 11 degrees C to 37 degrees C revealed a break in the Arrhenius-diagram at the phase transition temperature of dimyristoylphosphatidylcholine. The break point is in agreement with thermodynamic parameters derived from microcalorimetric studies of the monooxygenatic system reconstituted in the same way.

Reconstitution of the liver microsomal monooxygenase system in liposomes from dimyristoylphosphatidylcholine.

Different techniques to incorporate the essential isolated and purified enzymes of the hepatic endoplasmic reticulum into monolamellar dimyristoylphosphatidylcholine vesicles are compared with respect to structural and functional parameters of the reconstituted system. By use of gel penetrating chromatography on Sepharose 4B, dynamic light scattering and electron microscopy the structural properties of the reconstituted system were proved comparing the reduction of P-450 LM2 via the NADPH dependent reductase and by dithionite as well with the microsomal reduction rates and by studying the binding of benzphetamine to P-450 LM2 in soluble and liposomal form.

Reduction of cytochrome P-450 LM2 by NADPH in reconstituted phospholipid vesicles is dependent on membrane charge.

The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.

Correlations between spin equilibrium shift, reduction rate, and N-demethylation activity in liver microsomal cytochrome P-450 and a series of benzphetamine analogues as substrates.

Cytochrome P-450 forms a thermal ferric spin equilibrium which is significantly shifted by substrate binding. Within a series of benzphetamine analogues the liver microsomal enzyme system exhibits a close correlation of the substrate induced spin equilibrium shift towards the high spin state and both the rate of P-450 reduction, and of substrate turnover, as well. The spin equilibrium regulates the first electron transfer by favoured high spin state reduction and rapid pre-equilibration with respect to the low spin fraction.

Comparison of spectral properties of 3-MC induced cytochrome P-448 from rabbits and rats.

EPR and optical spectral properties of cytochrome P-488 from 3-methylcholanthrene-induced rabbits are compared with those of rats. In the EPR spectra at 77K and in the optical absorption spectra at room temperature a considerable temperature independent high spin content of the rabbit cytochrome is observed which has been estimated to about 55% by titration with n-octylamine. On the other hand the high spin content of the rat cytochrome depends strongly on temperature and amounts to about 6% at 5 degrees C and to about 35% at 34 degrees C. The binding of substrates and ligands to the rabbit cytochrome as well as its reduction by sodium dithionite are slower as compared with the rat cytochrome but also with phenobarbital-induced cytochrome P-450 from rats and rabbits. Contrary to the 3-methylcholanthrene induced cytochrome P-448 from rats, that from rabbits does not bind 3-methylcholanthrene. A particular protein structure establishing the high spin state and an absent binding site for type I substrates is assumed to be responsible for these and other peculiarities of cytochrome P-448 from 3-methylcholanthrene-induced rabbits.

The cytochrome P-450 reaction mechanism--kinetic aspects.

The reviewed kinetic investigations at the experimentally accessible partial reactions of the reaction sequence clearly evidence that the P-450 system represents a complex reaction system, the main control points of which are shown in Scheme 3: As can be seen, control elements are the substrate, the cytochrome species (P-450/448), the reducing agent, the reductase, phospholipid and parts of the b5 system as well. Formula (see text): All of these steps can be rate limiting (rate interfering), depending on the distinct substrate and oxygen donor. The ternary complex evidently has a key position however in that is structural specificities determine rate and product distribution of the respective substrate conversion. But unfortunately the ternary complex decay (including the e2 transfer) is kinetically unresolved till now.

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. II. Temperature dependence and species differences in substrate binding reaction.

Species dependencies and the temperature function of substrate binding reaction have been studied. The solubilized P-450 preparations from rat and rabbit, respectively, exhibit similar substrate binding characteristics with respect to rate constants and substrate specificity. The rabbit P-450 is more sensitive to preparational disintegration, this holds especially for aniline. In the Arrhenius plots a normal temperature dependence without breaks is observed. Iron ligands are bound with relatively low activation energies and negative entropies. The parameters increase for benzphetamine (type 1) and further for aniline (type 2), the latter substrate being entropically favoured too.

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase, MgATP, and cyclic AMP.

Ca2+ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca2+ accumulation by microsomal preparations exhibiting a steady state Ca2+ accumulation of 25.6 nmol Ca2+/mg increased from 3.67 to 33.4 nmol Ca2+/mg - s as the free Ca2+ concentration was raised from 0.2 to 18.9 muM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca2+ accumulation rate. The amounts of Ca2+ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.

Quench-flow measurements of initial rates of Ca2+ accumulation by isolated cardiac sarcoplasmic reticulum.

Using a quench-flow technique, the initial velocity of Ca2+ accumulation by isolated cardiac sarcoplasmic reticulum was estimated at free Ca2+ ion concentrations in the range encountered in the myoplasm during the cardiac contraction cycle. With cardiac microsomes exhibiting a Ca2+ accumulative capacity of 25.6 nmol Ca2+/mg protein, initial rates were found to increase from 3.7 to 33.4 nmol Ca2+/mg protein/sec, when the free Ca2+ ion concentration was raised from 0.2 to 18.0 muM. Preincubation of the cardiac microsomes with a party purified soluble cardiac protein kinase, MgATP, and cAMP led to a significant increase in the initial Ca2+ accumulation rate.

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. I. Substrate binding to cytochrome P-450 LM.

The substrate binding step in the reaction sequence of the cytochrome P-450 enzyme system (rat liver microsomes) has been investigated. The type I/II substrate classification kinetically holds too. The rate constants are in the 10(3) to 10(5) (M-1 sec-1) range, the type I compounds are preferably bound by about one order of magnitude. The rate constants of the binding process to the reduced cytochrome are considerably decreased. The results favour the ordered reaction mechanism.