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Many proteins containing domains of a cysteine-rich repeated motif, such as epidermal growth factor (EGF), have been reported. Here we report strong similarity between the amino acid sequence of a plant toxin--i.e., purothionin and its homologues--and with those of a domain found in mammalian pore-forming cytoplasmic proteins: components of complement and perforin of cytotoxic T-lymphocytes or natural killer-like cytotoxic cells. These similar sequences were found to be identical to the so-called EGF-like cysteine-rich repeated motif itself. Electron-microscopic observations indicated that, like complement and perforin, purothionin forms pores in the cytoplasmic membrane of target cells, resulting in their death within a few hours. On the basis of these sequence comparisons and physiological functions, we propose a scheme for the evolution of proteins containing modules of the cysteine-rich repeat motif.

A purothionin homolog was isolated from barley flour and purified by CM-52 column chromatography. It showed potent lethal activity towards brewer's yeast and its complete amino acid sequence was determined to be as follows. Lys-Ser-Cys-Cys-Arg-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Val-Arg-Gly-Ala-Gln-Lys-Leu-Cys-Ala-Gly-Val-Cys-Arg-Cys-Lys-Leu-Thr-Ser-Ser-Gly-Lys-Cys-Pro-Thr-Gly-Phe-Pro-Lys. It thus consists of 45 amino acid residues with 8 cysteines. The number of amino acid residues and the positions of the 8 cysteines are identical with those of wheat purothionins. There is a high degree of homology in the primary structures of these proteins.

The effect of purothionin, a peptide isolated from wheat flour, on adrenal medullary cells was examined in perfused bovine adrenal glands. Addition of purothionin (4 microgram/ml) to the perfusion fluid caused the release of cytoplasmic glutamic oxaloacetic transaminase (c-GOT), DOPA decarboxylase (DDC) and magnesium ions, which are present in the cytoplasmic fraction of adrenal medulla, but not the release of dopamine-beta-hydroxylase (DBH) or mitochondrial glutamic oxaloacetic transaminase (m-GOT), which are located in the particulate fraction. These results suggest that purothionin makes small pores in the cytoplasmic membranes of bovine adrenal medullary cells, resulting in the leakage of the cytoplasmic proteins and ions from the cells.

Purothionin from wheat flour was chemically modified by acetic or succinic anhydride under specific conditions. The complete modification of all amino groups of purothionin caused a large change in the net charge of the molecule, leading to the loss of the toxicity to mice and yeast. The sole tyrosyl residue in purothionin was nitrated by tetranitromethane at neutral pH or iodinated by the lactoperoxidase method. The nitro- and diiodo-derivatives of purothionin showed considerably reduced toxicity. Based on these modification studies we conclude that the positive charges of lysyl residues have an important role in the interaction with the negatively charged cell surface, and that the emergence of the toxicity of purothionin depends on a certain state of the tyrosyl residue.

Purothionin isolated from commercial wheat flour contained several components and two of them (A-I and A-II) were isolated in pure form by CM-52 column chromatography. Each component contained 45 amino acid residues with a 4 disulfide bonds. Purothionin A-II was digested with trypsin and thermolysin to isolate cystine peptides. These were separated and purified by chromatography on an SP-Sephadex column, and paper electrophoresis and chromatography. A peptide containing a -Cys-Cys- sequence was hydrolyzed with 10 N sulfuric acid. Amino acid compositions and partial sequence studies of the cystine peptides and their performic acid-oxidized peptides revealed the positions of all 4 disulfide bonds in purothionin A-II. They were formed between residues 3 and 39, 4 and 31, 12 and 29, and 16 and 25. The results of a partial study of purothionin A-I are also presented.