Biochemistry of organometallic compounds

Recent studies have demonstrated that organometallics can be substrates of enzymes and may undergo biotransformations in the presence of microorganisms. Latest achievements in organometallic biosynthesis, applications of organometallics in biosensor systems and the properties and models of novel enzymes which attack the core of organometallic compounds, i.e., the metal-carbon bond, are discussed.     

Lipoxins: study of various biosynthesis pathways

Lipoxins A4 and B4 were obtained by using soybean lipoxygenase and blood cells as a source of enzymatic activity. The conditions facilitating lipoxin biosynthesis from arachidonic acid catalyzed by soybean 15-lipoxygenase were selected. A comparative analysis of lipoxin biosynthesis with the use of cell suspensions containing only granulocytes and of mixed suspensions (platelets + granulocytes and platelets + total fraction of blood leucocytes) was carried out.     

Characterization of urokinase type plasminogen activator modified by phenylglyoxal

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in the scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4-12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the scu-PA globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the double chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

Kinetic model of a multienzyme system of blood prostanoid synthesis. II. Dynamic responses to pharmacological agents--inhibitors of prostaglandin H synthetases

The dynamic replies of the multienzyme system of blood prostanoid synthesis to the introduction of an irreversible inhibitor of prostaglandin H synthetase (PGH synthetase) have been analysed by using kinetic modelling. The alterations of arachidonic acid and PGH synthetase concentrations in platelets and endothelium and the concentrations of thromboxane and prostacyclin have been demonstrated. Particularities of kinetic behaviour of the system probably providing the therapeutic effect of non-steroidal anti-inflammatory drugs have been shown. Namely, the kinetic wave of free arachidonic acid and prostacyclin concentration with respect to thromboxane concentration appears after introduction of the drugs.     

Effect of opiates on Ca2+ transport in synaptosomes

Potassium-stimulated uptake of Ca2+ by nerve-ending fractions from rat brain (synaptosomes) is inhibited by morphine and [D-Ala2, D-Leu5]enkephalin. This effect develops significantly within 1 minute. The opiates do not affect the Ca2+ efflux from the synaptosomes. Naloxone, the opiate antagonist, does not reverse the effect of morphine on synaptosomal Ca2+ uptake, and in this respect itself acts similarly to morphine).