[Effect of apparatus plasmapheresis on the bowel barrier and motility function in patients with acute necrotizing pancreatitis].

Influence of therapeutic plasmapheresis on bowel barrier function and evacuation was investigated in 83 patients with severe acute necrotizing pancreatitis. Except standard therapy patient obtained therapeutic plasmapheresis using "Haemonetics" PCS 2 system. Complex treatment of patients with acute necrotizing pancreatitis and dynamic ileus using plasmapheresis increases contractive and propulsive function of stomach and duodenum and prolongs period of activity of these organs on 32%. Intestinal barrier function associates with restoration of bowel evacuation. Addition of plasmapheresis to standard therapy of necrotizing pancreatitis can be effective prevention of dynamic ileus.

[Hemocoagulation disorders in patients with acute pancreatitis].

62 patients with acute pancreatitis (AP) were examined in order to determine hemocoagulation disorders. The obtained results showed that the patients developed the consumptive coagulopathy with high levels of D-dimer, activation and exhaustion of antithrombin III (AT III). The development of hemocoagulation disturbances in patients with severe AP was confirmed through decrease of activity of AT III up to 68% and high level of D-dimer>693 ng/ml.

[The interaction of organophosphorus amino acid analogs with cytochrome P-450 2B4]. / Vzaimodeistvie fosfororganicheskikh analogov aminokislot s tsitokhromom P450 2B4.

Interaction between phosphorus amino acids analogs, 1-aminoalkylphosphonous and 1-aminoalkylthiophosphonic acids, and microsomes from the liver of phenobarbital-induced rabbits was studied. The phosphorus amino acids analogs cause type I and reverse type I spectral changes, respectively. A new reaction in the microsomal monooxygenase system was revealed. In the presence of NADPH, 1-aminoalkylphosphonous acids can be transformed to the corresponding 1-aminoalkylphosphonic acids by the reaction P-H-->P-OH. The reaction was monitored by 1H-NMR spectroscopy. 1-Aminoalkylphosphonous acids also serve as substrates for the NADPH-dependent monooxygenase system. 31P-NMR has shown that the oxidative desulfuration produces 1-aminoalkylphosphonous acids according to the reaction P=S-->P=O. Neither 1-aminoalkylphosphonous nor 1-aminoalkylphosphonic acids are deaminated in the NADPH-dependent monooxygenase system as follows from 1H-NMR spectroscopy.

[Surface of cytochrome P-450 2B4: structure and function]. / Poverkhnost' tsitokhroma P-4502B4: struktura i funktsii.

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.

[The role of heme in formation of the native structure of cytochrome P-450 LM2]. / Rol' gema v formirovanii nativnoi struktury tsitokhroma P-450 LM2.

The role of heme in the formation of cytochrome P-450 native structure was investigated. It was shown that treatment of purified and membrane-bound hemoproteins with H2O2 results in the total destruction of heme. After incubation with hemine the apoprotein thus obtained forms a catalytically active cytochrome P-450. The efficiency of this process depends on the enzyme microenvironment. The membrane-bound apoprotein may be reconstituted by 70-80%, whereas the soluble one--by 50%. It is concluded that the observed differences may be accounted for by a greater stability of the membrane-bound protein structure.

[Structural organization and localization of cytochromes P450 in the membrane]. / Strukturnaia organizatsiia i raspolozhenie tsitokhromov P450 v membrane.

The secondary structure prediction of 19 microsomal cytochrome P-450s from two different families was made based on their amino acid sequences. It was shown that there is a structural similarity between the heme-binding sites of these enzymes and the bacterial P-450cam. An average predicted secondary structure of cytochrome P-450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-strands, 9% beta-turns and 33% random coil. In the region of the 35-120 residues in microsomal P-450s two adjacent beta alpha beta-units (the Rossmann domain) were recognized, which may interact with the NADPH-cytochrome P-450 reductase. Using the procedure of identification of hydrophobic and membrane-associated alpha-helical segments of 23 cytochromes, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site perhaps includes surface-bound helix. A model of vertebrate microsomal P-450s is proposed. That is an amphypathic membrane protein located on the cytoplasmic face of the endoplasmic reticulum, their active center lies out/on the bilayer border.

[The effect of microenvironment and formation of intra-molecular links on the tertiary structure of cytochrome P-450 LM2]. / Vliianie mikrookruzheniia i obrazovaniia vnutrimolekuliarnykh shivok na formirovanie tretichnoi struktury tsitokhroma LM2.

The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation. Whereas the complete inhibition of oxidation of type II substrate turnover needs two intramolecular cross-links, that of type I substrates necessitates at least seven cross-links. The incorporation of modified and native enzymes into the membrane lipid bilayer at temperatures above the phase transition point results in the enzyme activation. However, in case of the preimmobilized enzyme the activation does not reach the maximal values. Both stabilized and liposome-incorporated cytochrome P-450 can fully be reactivated via the cross-link disulfide bond reduction. No such effect is observed at temperatures below the phase transition point.

[ESR-spectroscopy of cytochrome P-450 LM2 in the soluble and membrane-bound state]. / EPR-spektroskopiia spin-mechenogo tsitokhroma P-450 LM2 v izolirovannom i membranno-sviazannom sostoianiiakh.

Influence of microenvironment on the structure of spin-labeled cytochrome P-450 LM2 was studied. The distance between the spin-label which is covalently bound to cysteine-152 and heme ferrum in soluble protein is 17 A and 23 A in the membrane-bound one. The spin-label was exposed in water solution in both cases. It was shown that solubilized cytochrome P-450 LM2 in water solution forms the aggregates consisting of 4-6 monomers.

[Effect of lipids on CO recombination with ferrocytochrome P-450]. / Vliianie lipidov na rekombinatsiiu CO s ferrotsitokhromom P-450.

Using the flash photolysis technique the kinetics of recombination of carbon monoxide with ferrocytochrome P-450 LM-2 was investigated. Ferrocytochrome P-450 was incorporated into liposomes prepared from different lipids: from microsomal lipids, phosphatidylcholine from egg yolk, dipalmitoylphosphatidylcholine. It was found that the activity and structure of ferrocytochrome P-450 conformers is affected by the lipid microenvironment. The kinetics of the CO-binding is affected also by the nature of lipids.

[Effect of lipids and substrates on the kinetics of binding of ferrocytochrome P-450 to CO]. / Vliianie lipidov i substratov na kinetiku kompleksoobrazovaniia ferrotsitokhroma P-450 s CO.

Using the flash photolysis technique, it was found that the kinetics of recombination of carbon monoxide with ferrocytochrome P-450 LM-2 can be approximated by the sum of three exponents. Incorporation of cytochrome P-450 into liposomes prepared from microsomal lipids leads to the reduction of the number of steps to two as well as to essential changes in rate constants. Addition of type I substrates (Triton N-101, albumin) cause similar changes in the reaction kinetics. NADPH-cytochrome P-450 reductase has no effect on this process. The multistep kinetics of CO recombination with cytochrome P-450 LM-2 may be accounted for by the presence of some protein conformers. The experimental results suggest that the activity and structure of cytochrome P-450 conformers is affected by the lipid microenvironment, type I substrates and Triton N-101.

[Effect of cytochrome b5 on the functional activity and conformational state of cytochrome P-450]. / Vliianie tsitokhroma b5 na funktsional'nuiu aktivnost' i konformatsionnoe sostoianie tsitokhroma P-450.

Microsomal cytochromes P-450 and b5 were shown to form mixed complexes with the association constant of 0.24 microM in water solution. Such complex formation stabilizes cytochrome P-450 in the catalytically active conformational state characterized by increased conformational rigidity and temperature stability. This stabilization results in acceleration of the cumene hydroperoxide-dependent oxidation of p-nitroanisol catalyzed by cytochrome P-450. The thermodynamic parameters of O-demethylation of p-nitroanisol catalyzed by cytochrome P-450 and mixed haemoprotein complexes measured in water solution and in a membrane-bound state were found to be different.

[Conformational state and functional activity of NADPH-dependent cytochrome P-450 reductase isolated and incorporated into phospholipid bilayers]. / Konformatsionnoe sostoianie i funktsional'naia aktivnost' izolirovannoi i vstroennoi v fosfolipidnyi bisloi NADPH-zavisimoi tsitokhrom P-450 reduktazy.

It was shown that the alpha-helix content in both isolated and incorporated into phospholipid bilayer NADPH-dependent cytochrome P-450 reductase is 20%. NADPH or dithionite reduction of the flavoprotein is not followed by conformational changes. The incorporation of the NADPH-dependent cytochrome P-450 reductase molecule into the phospholipid bilayer does not affect its catalytic properties. It was found that the protein does not interact with the phospholipid bilayer of phosphatidyl choline liposomes but incorporates readily into the liposomal membrane from a microsomal phospholipid mixture with a binding constant of 17.4 microM.

[Conformation of cytochrome b5 in soluble form and incorporated into artificial membranes]. / Konformatsionnoe sostoianie rastvorimogo i vstroennogov iskusstvennuiu membranu tsitokhroma b5.

It was shown that 35% of the peptide groups, which form the right alpha-helices, are presented in the soluble fraction of cytochrome b5. The incorporation of cytochrome b5 into egg lecithine liposomes increases the number of alpha-helices up to 51%. On the contrary, the cytochrome incorporation into microsomal lipid liposomes slightly increases the degree of spiralization. The reduction of cytochrome b5 haem slightly decreases alpha-helices in the soluble haemoprotein as well as in the one incorporated into the artificial membrane irrespective of its phospholipid content.

[Conformation and thermal stability of soluble and liposomal forms of cytochrome P-450]. / Konformatsiia i teplovaia stabil'nost' rastvorimogo i vstroennogo v membranu liposom tsitokhroma P-450.

This secondary structure of soluble cytochrome P-450 and the one incorporated into liposomes from egg lecithin and microsomal lipids has been studied. Using circular dichroism and infrared spectroscopy, it was shown that about 60% of alpha-helices are presented in the structure of haemoprotein and the rest 40% have the structure of statistical coil. The binding of haemoprotein with the type II substrates--octylamine and diaminooctan, slightly increases alpha-helices in soluble cytochrome P-450. The type I non-polar substrates--hexane and cyclohexane--do not change the conformation of isolated enzyme. Cytochrome P-450 incorporated into the artificial membranes of phosphatidyl choline and microsomal phospholipid has almost identical secondary structure as does the soluble one. Data from circular dichroism suggest that the binding of the types I and II substrates to cytochrome P-450 incorporated into lecithin liposomes and microsomal lipid liposomes does not change the conformation of the polypeptide chain. The reduction of cytochrome P-450 haem increases the degree of alpha-spiralization by 10% for soluble haemoprotein and by 5% for the membrane-bound enzyme. The thermal stability of soluble and liposomal forms of cytochrome P-450 was investigated by circular dichroism technique. The effective values of enthalpy and the temperature transition of soluble cytochrome P-450 at pH 6.9, 7,6 and 7,9 are 78, 80 and 78 kcal/mol and 47,7 degrees, 45,2 degrees and 42,4 degrees, respectively. The enzyme incorporated into the phospholipid vesicles is much more stable. The cooperative transition of soluble cytochrome is clearly expressed in contrast to the one of the membrane-bound enzyme.