AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

准噶尔盆地侏罗系层序格架和油气成藏条件研究

Jurassic is an important hydrocarbon-bearing formation in Junggar Basin. Analyzing in strata sequence stratigraphy and hydrocarbon formation has both theoretical and practical values. First of all, strata sequence stratigraphy of continental facies is a new development and supplement in the theory of stratigraphic geology. Stratum of continental facies, unlike sea facies, has rich sup-plements, rapid facies changes, and was influenced slightly by sea level changes. The structural background and sedimentary environment of the basin in west China are greatly different from those of the basins in east China. So it is important to build the patterns of strata sequence stratigraphy in west China basins. Secondly, it is also of significance to find out all kinds of traps, for the dominant types are structural ones so far. After 50 years exploration, the stratigraphic or litholigic traps have become the main concern. This desertation is mainly focused on establishing the isochronal strtaum frame for Junggar Basin to show the evolvement characters of the basin sediment system and the regionalstuctrue background. By analyzing the conditions and patterns of the regional oil and gas bearing formations with typical cross-sections, we have established the patterns of sedimentary conditions for different reserviors. By authur's study, several fruitful results have been obtained in the following: Strata sequence frame and evolvement characteristics of Jurassic: By studing strata sequence, Jurassic has been divided into 2 second rank strata sequences and 3 third rank strata sequences based on the interface unconformities. Only 2 fourth rank strata sequences were grouped in BaDaoWan group. Also different seismic facies and sediment units have been recognized with the establishment of the of sediment system model. The oil-gas system characteristics in Jurassic: We conclude that hydrocar bon resources have the best oil potential. Potential of coal, carbonaceous and dark mudstone were reduced in turn. In this thesis we have made the evaluation of three hydrocarbon sources and the distribution oil-gas resource, and studied the potentials of hydrocarbon and evolvement for each kind of micro-component of the two main resource rocks. Prediction of paleo-temperature: In Junggar basin the evolvement of paleo-ground temperature can be divided into three stages. From Carboniferous to early Permian grads of ancient ground temperature was 8-5 ℃/100m, 5-3 ℃/100m from later period of Permian to end Trias, 3-2 ℃/100m from Jurassic to early Tertiary. Patterns of Jurassic hydrocarbon-bearing reserviors: There were two kinds of hydrocarbon source of Permian and Jurassic. They form different hydrocarbon - bearing systems. Six fundamental hydrocarbon - bearing trap modeS have been established. Directions for later exploration: There were two kinds of regional belts in Jurassic, One is structural belt caused by Yanshan and Ximalaya process, and the other was the stratum one caused by paleostructural rises.