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Biomedical subjects
A I Archakov
Publications and source records attributed to A I Archakov.
Epitope mapping of horseradish peroxidase (isoenzyme C).
Peptide scanning (PEPSCAN) was used to determine linear antigenic determinants of horseradish peroxidase isoenzyme C (HRPC). For this purpose, we synthesized 303 overlapping hexapeptide fragments (with a step of one amino acid residue) of the protein primary structure and studied their interactions with anti-HRPC polyclonal antisera by ELISA. Experiments with various titers of antisera allowed us to determine linear antigenic determinants of HRPC; several such determinants were spatially located in regular elements of the secondary structure (alpha-helices) found both inside and outside the protein globule. A fraction of epitopes were located in loops and folds of the HRPC peptide chain with irregular shapes. These epitopes contained several functionally important residues: Arg 38, which is part of the active site of the enzyme, as well as Phe 142 and Phe 143, which form a channel allowing aromatic substrates to reach the active site. Amino acid residues that form calcium-binding sites or occur in the vicinity of disulfide bonds are not involved in these epitopes.
Amino Acid Sequence↗
Immobilization of proteins to lipid bilayers.
Phospholipid bilayers deposited on sensor surfaces are excellent substrates for immobilizing proteins via a molecular anchor. An integrated optics sensing device was used to accurately measure the binding kinetics of proteins thus anchored. By comparing the results with measurements using proteins from which the anchor had been enzymatically removed, it was shown that the anchor accounts for essentially all the irreversible binding. The insertion of the anchor into the lipid bilayer is a spontaneous process. This method of immobilization should be widely applicable to many soluble protein molecules, to which an anchor can be attached by routine methods of molecular engineering.
Animals↗
Interactions of cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by fluorescent probe.
A new method for monitoring the formation of the cytochrome P450 complexes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The method is based on the quenching of fluorescence of NCPR labelled with 7-ethylamino-3-(4'-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholipid, cytochrome P450 2B4 and NCPRcpm were shown to form 1:1 complexes with a Kd of 0.038 microM. Formation of the complex follows the kinetics of reversible second order transition with k(on) = 6.5 10(5) M-1 s-1. Application of high hydrostatic pressure induces dissociation of the complex (delta V degrees = -65 mL/mol). Succinylation of the hemoprotein increases the value of Kd to 0.5 microM primarily by decreasing k(on). In contrast to what was shown for intact 2B4, rising pressure does not take apart succinylated hemoprotein and NCPRcpm molecules, but causes some internal transition in their complex that diminishes the quenching. This transition is characterised by a very large volume change (delta V degrees = -155 mL/mol). The following conclusions were drawn: 1) a molecule of 2B4 contains two distinct contact regions involved in the interactions with NCPR. Only one of these regions is polar and highly hydrated in unbound hemoprotein; 2) interactions of the polar regions of 2B4 and NCPR are necessary to bring CPM-labelled cysteine of NCPR in short distance of the heme of 2B4; and 3) some of the lysine residues located in the proximity of the polar binding regions are apparently involved in the formation of the internal salt bridges in the molecule of 2B4.
Animals↗
Antigenic mapping of bacterial and animal cytochromes P-450.
A peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. The antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in the protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.
Animals↗
Design of an artificial hemoprotein based on human serum albumin.
For modelling cytochrome P450-catalyzed reactions, an artificial hemoprotein was designed. Upon complex formation of human serum albumin with iron protoporphyrine IX, there occurred the incorporation of heme into the protein and formation of a specific complex with the albumin to heme molar ratio 2:1. The apparent dissociation constant Kd of the complex, as determined by optical absorption spectroscopic technique, was 1.9 +/- 0.4 M-6. Based on spectral studies and molecular modelling of the complex spatial structure, it was assumed that His 31 or His 90 may be the most probable 5th ligand for the heme iron. The artificial hemoprotein was able to catalyze (in the presence of riboflavin as electron carrier) the NADH-dependent aniline hydroxylation and dimethylaniline and amidopyrine N-demethylation. The electron transfer pathway from NADH to substrate was demonstrated. Flavin appears to serve as an input center (mediator) for the rapid transfer of electrons from NADH to heme, where substrate is oxidized. The same reactions were accomplished using riboflavin photoreduction in the hemoalbumin-riboflavin system with the unfocussed laser emission at lambda = 457.9 nm. As electron donor, metallic zinc was used. The artificial hemoprotein obtained was also able to catalyze H2O2-dependent oxidase reactions.
Aminopyrine↗
Reductase and oxygenase activities of semisynthetic flavocytochromes based on cytochrome P450 2B4.
A synthetic flavocytochrome with the reductase and oxygenase activities was obtained by covalent binding of riboflavin to cytochrome P450 2B4. The reactions catalyzed by the newly synthesized flavocytochromes were studied. Formation of carboxycomplex with the reduced form of hemoprotein led to 60-80% inhibition of oxygenase reactions indicating the leading role of reduced heme iron in generating active oxygen species by flavocytochromes.
Aniline Compounds↗
[The rational computer-aided design of new drugs].
At present, searches are being made to accelerate the designing of new drugs and at the same time to considerably reduce the associated expenses. The process of designing a new drug generally consists of 3 stages: (1) search for a new agent having the required pharmacological activity; (2) experimental studies and pharmacological and toxological tests; (3) an appropriate pharmacological committee's approval for clinical tests of the agent. Stage 1 is the most expensive and labour-consuming, as it requires a half the total expenditures. Its clinical version consists in the synthesis of numerous homologues of the well-known biologically active compounds and in long-term and costly trials. At the same time, an investigator has a definite range of concepts of the structure of a new drug as a new representative of the known group that he already knows. The application of computer-aided analysis and design enables these disadvantages to be overcome. The introduction of methods for predesigning computer-aided drug design has permitted drastic reduction of expenses and accelerated designing of new drugs, by decreasing the number of unsuccessful syntheses and trials.
Computer Graphics↗
Dynamic interactions of rabbit liver cytochromes P450IA2 and P450IIB4 with cytochrome b5 and NADPH-cytochrome P450 reductase in proteoliposomes.
Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-reconstituted with cytochrome b5 or NADPH-cytochrome P450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteoliposomes catalyzed drug oxidation. Rotational diffusion of cytochrome P450 was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1 ms to a time-independent value, r3. Different rotational mobility for the two cytochrome P450s was observed. Though 20% of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecules were rotating. The rotational relaxation time, phi, of the mobile population was 237 microseconds for cytochrome P450IA2 and 160 microseconds for cytochrome P450IIB4. The two cytochrome P450s have shown very different interactions with cytochrome b5 and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P450IA2 was increased significantly from 80% to 96% (plus cytochrome b5) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociation of P450 oligomers. On the other hand, the mobility of cytochrome P450IIB4 was not considerably affected by the presence of cytochrome b5 or NADPH-cytochrome P450 reductase as judged by little difference in phi and r3, keeping the mobile population of 100%. These results imply that cytochrome P450IA2 forms a transient association with cytochrome b5 and NADPH-cytochrome P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: evidence for conformational inhomogeneity in the oligomers.
Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The amplitude of the P450-->P420 transition accounted for 67 +/- 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450-->P420 conversion and was estimated to be only about 31 +/- 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450-->P420 transition (delta V degrees = -86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450-->P420 conversion (delta V degrees = -32 ml/mol, P1/2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (delta V degrees = -49 ml/mol) and substrate-free (delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency (delta V degrees = -8 ml/mol).
Algorithms↗
Interaction of organophosphorus analogues of amino acids with P450.
1. This study deals with the oxidation of organophosphorus amino acid analogues by phenobarbital-induced rabbit liver microsomes. It has been shown that 1-aminoalkylphosphonous and 1-aminoalkylthiophosphonic acids are converted by P450 to 1-aminoalkylphosphonic acids. 2. Phosphonous analogues of amino acids cause type I spectral changes, and thiophosphonic analogues produce reverse type I changes in different spectra. 3. In the presence of NADPH, the 1-aminoalkylphosphonous acids form the corresponding 1-aminoalkylphosphonic acids by the reaction P-H-->P-OH, as monitored using 1H nmr spectroscopy. 4. Aminoalkylthiophosphonic acids have also proven to be the substrates for the NADPH-dependent monoxygenase system. During the course of oxidative desulphuration 1-aminoalkylphosphonic acids were formed by the reaction P = S-->P = O, as monitored by 31P-nmr spectroscopy. 5. Using resonance Raman (RR) spectroscopy, the interaction of 1-aminoisobutyl-phosphonous acid with P450 was investigated, and characteristic changes in spectral frequencies in the region between 1370 and 1700 cm-1 were demonstrated. These latter changes indicate that substrate binding of organophosphorus compounds leads to alterations in haem conformation and to redistribution of the electron density.
Amino Acids↗
[Surface of cytochrome P-450 2B4: structure and function].
The review is devoted to the identification and structure of one of the cytochromes P450s-cytochrome P-450 2B4 derived from the rabbit liver endoplasmic reticulum. A critical review is made of the data on this enzyme membrane topology, its active site's structure and localization of its membrane and water-exposed regions. The paper is based on the data available in the literature and the authors' own findings. Various experimental and calculating methods used to identify the topography of cytochrome P450 are covered in the paper.
Amino Acid Sequence↗
[Biomedical chemistry: yesterday, today, tomorrow].
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Biochemistry↗
[Self-inactivation of cytochrome P-450 in the catalytic cycle].
The paper deals with possible mechanisms of cytochrome p-450 self-activation during catalytic turnover. Two routes of hemoprotein inactivation are so far known. The first route studied extensively by many authors, consists in formation of active intermediates capable of modifying heme and apoenzyme. The second route revealed only lately, which results from uncoupled cytochrome P-450-catalyzed monooxygenase reactions, is yet to be clarified. Briefly, it consists in the fact that the hydrogen peroxide formed in the hemoprotein active center interacts with the enzyme-bound Fe2+, thereby generating hydroxyl radicals that bleach the heme and modify the apoenzyme. This mechanism operates with all the substrates and all cytochrome P-450 forms capable of catalyzing the partially coupled monooxygenase reactions proceeding with the formation of hydrogen peroxide as a by-product.
Animals↗
The comparative study of peroxidase activity and substrate binding properties of cytochrome P450 2B4 incorporated into phospholipid vesicles with the use of two different methods.
The comparative study of peroxidase activities and substrate binding properties of cytochrome p450 2B4 in reconstituted vesicles prepared with the use of two different techniques, and microsomal cytochrome P450 was carried out. The data obtained show that the two types of cytochrome P450 2B4-containing proteoliposomes do not differ substantially from each other with respect to H2O2- or cumene hydroperoxide-dependent substrate hydroxylation activities as well as substrate binding properties of hemoprotein reconstituted. However, some parameters measured with proteoliposomal cytochrome P450 are markedly different from those measured with microsomal hemoprotein, suggesting the existence of conformational differences between the molecules of these two cytochromes or the differences in the depth of their immersion into lipid bilayer.
Animals↗
Determination of membrane-bound fragments of cytochrome P-450 2B4.
Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.
Amino Acid Sequence↗
Catalytic activity of cytochrome P4501A2 in reconstituted system with Emulgen 913.
Physicochemical properties and catalytic activity of cytochrome P4501A2 in the reconstituted system with Emulgen 913 were studied. The formation of cytochrome P4501A2 monomers was shown by gel filtration at an Emulgen concentration of 8 g/liter. The catalytic activity of the monomeric monooxygenase system was low. The maximum rate of the 7-ethoxyresorufin O-deethylation reaction, 150 pmol resorufin min-1 nmol-1 cytochrome P4501A2, was observed at an Emulgen concentration of 0.1 g/liter when cytochrome P4501A2 pentamers predominated. The effects of Emulgen on cytochrome P4501A2 cumene hydroperoxide-dependent peroxidase activity, on its affinity to substrate and to cytochrome b5, and on the rate constants of dithionite-dependent reduction were insignificant. Study of the NADPH-dependent reduction of cytochrome P4501A2 in the reconstituted system showed that the rate constant and reduction level of cytochrome P4501A2 were always higher when the reaction was initiated by NADPH than when it was initiated by NADPH-cytochrome P450 reductase. This indicated that the reduction reaction initiated by the reductase was limited by a step in the cytochrome P4501A2 and reductase interaction. Correlation between 7-ethoxyresorufin O-deethylase activity and reduction level of cytochrome P4501A2 was found.
Animals↗
Semi-artificial hydroxylating enzymes created by flavins binding to cytochrome P450 2B4 and by bleomycin binding to NADPH-cytochrome P450 reductase.
To create a semi-artificial monomolecular oxygenase system, FAD or FMN were covalently bound to cytochrome P450 2B4 as electron donor centers and bleomycin to NADPH-cytochrome P450 reductase as a generator of active oxygen species. The most catalytically active was the conjugate of cytochrome P450 with FMN, able to initiate the reactions of dimethylaniline and aminopyrine demethylation along with the reaction of aniline p-hydroxylation. The conjugate of cytochrome P450 with FAD oxidized these substrates at a much slower rate. The bleomycin-reductase complex was capable of demethylating dimethylaniline and aminopyrine but failed to oxidize aniline.
Animals↗