R T Kerns | PubMed Health

PubMed1999

Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex.

The strongly anticoagulant basic phospholipase A(2) (CM-IV) from Naja nigricollis venom has previously been shown to inhibit the prothrombinase complex of the coagulation cascade by a novel nonenzymatic mechanism (S. Stefansson, R. M. Kini, and H. J. Evans Biochemistry 29, 7742-7746, 1990). That work indicated that CM-IV is a noncompetitive inhibitor and thus it interacts with either factor Va or factor Xa, or both. We further examined the interaction of CM-IV and the protein components of the prothrombinase complex. Isothermal calorimetry studies indicate that CM-IV does not bind to prothrombin or factor Va, but only to factor Xa. CM-IV has no effect on the cleavage of prothrombin by factor Xa in the absence of factor Va. However, in the presence of factor Va, CM-IV inhibits thrombin formation by factor Xa. With a constant amount of CM-IV, raising the concentration of factor Va relieved the inhibition. The phospholipase A(2) enzyme inhibits by competing with factor Va for binding to factor Xa and thus prevents formation of the normal Xa-Va complex or replaces bound factor Va from the complex. Thus factor Xa is the target protein of this anticoagulant phospholipase A(2), which exerts its anticoagulant effect by protein-protein rather than protein-phospholipid interactions.

Animals↗

PubMed1994

Three different calmodulin-encoding cDNAs isolated by a modified 5'-RACE using degenerate oligodeoxyribonucleotides.

In order to obtain the 5' ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5' RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating the annealing step from the reverse transcription reaction allowed for greater stringency by using higher temperatures than could be tolerated if the reverse transcriptase were present. Annealing was also done with lower primer concentration and was driven by a longer incubation time. After the annealing step, cDNA synthesis was initiated by diluting the annealing mixture into a 42 degrees C buffer with reverse transcriptase. The synthesized cDNA was poly(dA)-tailed to allow PCR amplification of the first-strand cDNA with an anchor-dT17 primer and the degenerate primers. The CaM cDNAs were evident after this PCR. A second PCR, with nested gene-specific primers, was used to isolate the individual CaM cDNAs from the products of the first PCR. Three distinct CaM cDNAs were cloned and sequenced. By comparison of the 5' untranslated sequences between the mouse CaM DNAs and rat CaM cDNAs, the corresponding homologs were assigned. The results suggest that application of this modified RACE method could improve the success of isolating specific cDNAs in cases where use of a nested primer is not possible or when amino-acid sequence information is available and only degenerate primers can be designed for cloning cDNAs by the 5'-RACE method.

Animals↗