A mass spectrometry-based unique fragment approach for the identification of microcystins

Both qualitative and quantitative methods for the analysis of microcystin variants have been established.

A fragment of antithrombin that binds both heparin and thrombin

In order to identify the regions of antithrombin that interact with heparin and thrombin, it was degraded with CNBr and the activities of the isolated products were investigated. These fragments did not exhibit direct thrombin-neutralizing activity; however, one unique fragment was found to bind to heparin-Sepharose and also to interfere with the inhibition of thrombin by intact antithrombin. This fragment was identified as the one consisting of three disulphide-linked polypeptide chains containing residues 1-17, 104-251 and 424-432. At a concentration of 46 nM, this product decreased the heparin-enhanced thrombin-inhibitory activity of antithrombin by half, and completely abolished this inhibition when above 300 nM. In the absence of heparin, the action of antithrombin was not completely nullified by the fragment, even when present at relatively high concentrations. At a given fragment concentration, the extent of inhibition was independent of antithrombin concentration over the range tested. It was found that the fragment decreased the second-order rate constant for the antithrombin-thrombin reaction. Reduction and alkylation of the fragment showed that the above properties reside primarily in the peptide with residues 104-251. It is concluded that this peptide possesses portions of the antithrombin molecule that bind to heparin as well as to a site on thrombin.

Comparative study of the U1, U4, and U7 strains of tobacco mosaic virus

Experiments were undertaken to compare three strains of tobacco mosaic virus (TMV) belonging to the tobacco group 1 of the tobamoviruses. Although the U1, U4, and U7 strains of TMV differ significantly in virulence, it was shown that these strains are very closely related. No difference was detected in the major spots of the coat protein tryptic maps and the virions from the three strains shared the same electrophoretic mobility in agarose gels with different buffer systems. The RNA fingerprints of U1 and U7 were indistinguishable and the largest T1 omega fragments of the three strains were identical in size. However, the avirulent U4 RNA fingerprint exhibited a single spot mobility difference from the U1 and U7 RNA fingerprints. This spot corresponded to the T1 phi fragment. This unique fragment is located in the so-called l2 gene between nucleotides 976 and 992 from the 3′ end. This suggests and extends the proposal that the l2 gene is most probably implicated as a factor in the control of virulence (symptomatology).