Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.
A small basic peptide with an unusual amino acid composition has been isolated from the seeds of pumpkin, Cucurbita maxima. Amino acid analysis and sequence data show the protein to be about 36 residues in length, with an approximate composition Lys,, Arg,4, Asp3, (Glu + Gln),s, Gly,, Pro,, Trp,. On the basis of composition, the molecular weight is approximately 5000 daltons and the nitrogen content by weight is 20A%. Twelve amino acids are entirely lacking. The peptide is slightly toxic to mouse B-16 melanoma cells, but its in vivo function is unknown. It does not appear to be derived from cucurbitin, the pumpkin storage globulin; however, it could be a storage peptide involved in nitrogen mobilization during the early stages of germination.A major component of plant seeds is frequently a globulin, highly enriched in Gln, Asn, and Arg, which is thought to serve primarily as a nitrogen reserve (11). In a number of seeds, however, there are also small proteins whose biological function is unclear, although several are cytotoxic (4,13,18,21,22 granular appearance, or when the membrane was visibly fragmented. Three plus was given when 75 to 99% of the cells were altered, 2+ when 50 to 74% were altered, 1+ when 5 to 50% were altered, and 0 when all of the cells resembled those in the control wells lacking extract. Denaturing polyacrylamide electrophoresis (SDS-PAGE) as described by Laemmli (14) was used with slight modifications. Low range mol wt standard proteins from Sigma Chemical Co., as well as Cyt c and PT2 were used as standard mol wt marker proteins.Using liquid nitrogen as a grinding medium, 250 g of seeds were ground into a fine flour in a Waring Blendor at high speed. Five hundred ml ofextraction buffer A (0.1 M sodium phosphate, 0.05 M EDTA, 0.5% proteinase inhibitor PMSF at pH 7.4) prechilled to 0°C were added to 250 g of frozen flour and ground for 30 s, producing a thick homogenate which was allowed to stand for 20 min at 0°C. To test for possible proteolysis during extraction, an aliquot was allowed to stand for 6 h and analyzed at intervals by PAGE. The homogenate was centrifuged at 27,000g for 20 min at 4°C. The floating lipid layer and the sedimented pellet were discarded. The pH of the supemate was adjusted to pH 8.5 by addition of 6 N NaOH, and after standing 20 min at 0°C the suspension was centrifuged as before. Ammonium sulfate was added to 35% (w/v). After standing 20 min at 0°C the suspension was centrifuged as before. Additional ammonium sulfate precipitation steps were performed by bringing the ammonium sulfate concentration to 45% (w/v) and finally to saturation. The ammonium sulfate steps were performed at 0°C and centrifugation was as before.The pellet obtained at saturating ammonium sulfate was resuspended in buffer B (1.0 M NaCl, 0.1 M sodium phosphate, 0.05 M EDTA at pH 7.4) and purified on G-50 Sephadex using the same buffer at a flow rate of 0.5 mL/min collected in 2 mL fractions. Cytotoxicity to mouse B-16 melanoma cells identified the active material.Following lyophiliza...
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