A low-molecular-weight cytotoxic protein has been purified from Pyrularia pubera Michx. (Santalaceae). By comparison with the behavior of proteins of known molecular weight during Sephadex G-75 gel filtration and denaturing electrophoresis, a molecular weight of somewhat less than 6000 is indicated. Purification involves ammonium sulfate fractionation followed by either gel filtration on Sephadex G-75 or separation on a carboxymethyl cellulose CM52 column. At concentrations of 0.04 mg/ml the protein causes visible disruption of cultured mouse B16 melanoma cells. The complete amino acid sequence has been determined. The toxin contains 47 amino acids arranged as follows:Lys-Ser-Cys-Cys-Arg-Asn-Thr-Trp-Ala-Arg-Asn-C ys-Tyr-Asn-Val-Cys-Arg-Leu-Pro-Gly-Thr-Ile-Ser-Arg-Glu-Ile-Cys-Ala-Lys- Lys-Cys-Asp-Cys-Lys-Ile-Ile-Ser-Gly-Thr-Thr-Cys-Pro-Ser-Asp-Tyr-Pro-Ly s-OH. The protein is clearly a thionin, as shown by its close resemblance to the thionins from wheat and barley, to the viscotoxins from mistletoes, and to crambin.
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A minimalist design approach to antimicrobial agents based on a thionin template.
J Med Chem
January 2006
Department of Experimental and Health Sciences, Pompeu Fabra University, E-08003 Barcelona, Spain.
Numerous studies have been devoted to the stabilization of secondary structure elements to improve receptor-ligand recognition. We report a novel application of this principle to create new antimicrobial agents using the highly folded thionin from Pyrularia puberaas a template. Non-native disulfide bonds have been used to induce two short linear segments of the protein into an amphipathic helix.
View Article and Find Full Text PDFCharacterization and structural role of disulfide bonds in a highly knotted thionin from Pyrularia pubera.
Biopolymers
December 2005
Department of Experimental and Health Sciences, Pompeu Fabra University, Dr. Aiguader, 80, E-08003 Barcelona, Spain.
Disulfide bonds play a crucial role in the stabilization of the amphipathic folding of the diverse families of cysteine-rich antimicrobial peptides. The determination of cysteine pairings in these peptides has largely depended on sequence homology criteria, since the classical methods of disulfide bond characterization, which usually require proteolysis as a first step, encounter serious drawbacks derived from the tight folding and the presence of vicinal cysteines. We have chosen the Pyrularia pubera thionin, a 47-residue peptide with four internal disulfides and a remarkable resistance to most proteases, as a representative member of this type of cysteine-rich peptides and have shown that a combination of partial reduction and cyanylation readily allows the determination of its disulfide bonds.
View Article and Find Full Text PDFStimulation of prothrombinase activity by the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, a segment of a plant thionin.
Peptides
April 2003
Escola Superior Agrária de Castelo Branco, Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal.
Pyrularia thionin (PT) is a basic 47 amino acid peptide isolated from the nuts of Pyrularia pubera. Its structure and properties have been studied in some detail. Its receptor site is a domain of membrane phosphatidyl serine (PS), where it binds with a relatively high specificity.
View Article and Find Full Text PDFSynthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against Gram-negative bacteria.
FEBS Lett
February 2003
Department of Experimental and Health Sciences, Pompeu Fabra University, Dr. Aiguader 80, E-08003 Barcelona, Spain.
The thionin from Pyrularia pubera (Pp-TH), a 47-residue peptide with four internal disulfide bonds, was efficiently produced by chemical synthesis. Its antimicrobial activity in vitro against several representative pathogens (EC(50)=0.3-3.
View Article and Find Full Text PDFBinding to and hemolysis of human erythrocytes by pyrularia thionin and Naja naja kaouthia cardiotoxin: inhibition by prothrombin.
J Nat Toxins
August 2001
Escola Superior Agraria, Instituto Politecnico de Castelo Branco, Portugal.
Pyrularia thionin and snake venom cardiotoxin are strongly basic proteins which bind to and induce hemolysis of erythrocytes, cause depolarization of muscle cells, and influence the order and properties of phospholipids in cellular membranes. Earlier studies showed a competition between the thionin and cardiotoxin for a common binding site on erythrocytes, and the present study extends these studies to show a similar competition between prothrombin and both basic proteins. The competition between the thionin and prothrombin for binding sites on erythrocytes was shown by direct binding experiments using radiolabeled thionin.
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