Pyrularia
thionin, isolated from nuts of Pyrularia pubera, is a strongly basic
peptide of 47
amino acids. The amino acid sequence and configuration of its four
disulfide bonds place this plant
peptide, known to be hemolytic, cytotoxic, and neurotoxic, among the
thionins. We report and compare several cellular responses mediated by Pyrularia
thionin:
hemolysis of human erythrocytes, activation of an endogenous
phospholipase A2 in Swiss 3T3 cells, cytotoxicity toward HeLa and mouse
B16 melanoma cells in culture, viability of rat hepatocytes and lymphocytes measured by
trypan blue exclusion, and lethality in mice. Cellular responses related to ion movement include a toxin-mediated influx of Ca2+ into mouse P388 cells measured by
Fura-2 fluorescence, depolarization of mouse P388 plasma membrane measured by fluorescence of bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol), and depolarization of frog (Rana pipiens) sartorius muscle determined by direct measurement of membrane potential. Graded iodination of Pyrularia
thionin leads to a related loss of activity for
hemolysis,
phospholipase A2 activation, cytotoxicity, and lethality in mice. The mediated Ca2+ influx into and depolarization of P388 cells require Ca2+ in the external medium and are inhibited by 100 microM Ni2+. Depolarization of sartorius muscle by Pyrularia
thionin also requires a functional Ca2+ channel, as shown by
verapamil inhibition. This muscle depolarization also involves
phospholipase A2 activation because
dexamethasone and quinacrin, but not
indomethacin, protect against depolarization. The IC50 values for viability of rat hepatocytes and splenic lymphocytes measured by
trypan blue exclusion were 0.17 and 40 microM, respectively. The general response of cells to Pyrularia
thionin involves a membrane alteration leading to depolarization and a channel-mediated influx of Ca2+. There is a related activation of
phospholipase A2 that results in loss of membrane integrity,
hemolysis in the case of erythrocytes, and eventually cell death. Iodination of Pyrularia
thionin leads to a corresponding inhibition of all three cellular responses, which indicates an essential role for
tyrosine in either maintenance of
peptide structure or interaction of the
peptide with cellular membranes.