Characteristics of the enzyme system responsible for melanin formation in verticillium Public Deposited

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  • This paper describes an attempt to ascertain the enzyme system responsible for melanin formation in a melanin-and-microsclerotia-forming strain of Verticillium albo-atrum Reinke and Berth. Previous work showed that near-UV radiation (3200-400 A⁰) inhibits melanin synthesis and microsclerotia development in this strain and that catechol almost wholly reverses this inhibition. During the present study, additional chemicals were tested in living cultures. Only one of these, hydroquinone, almost consistently reversed the effects of near-UV, and it did so to a much lesser extent than did catechol. Chemicals which failed to induce the pigment formation include: aniline, ascorbic acid, chlorogenic acid, p-cresol, dopa, gallic acid, phenylalanine, p-phenylenediamine, pyrogallol, resorcinol, shikimic acid and tyrosine. Attempts to demonstrate phenolase activity in extracts and whole cells revealed either weak activity or none. Manometric tests for phenolase demonstrated activity in potato and mushroom but littte phenolase activity in Verticillium. A modified purpurogallin test indicated the presence of peroxidase activity in Verticillium extracts. With this information an assay for the catechol-oxidizing-system was developed. It involved mixing 1.0 ml of 0.1 M Tris-HC1 buffer, pH 7.2, and 0.9 mI of distilled water with 0.4 mL of 1 M H₂O₂, 0.4 ml of 10⁻¹ M catechol and 0.2 ml of 10⁻³ M Mn⁺² in a test tube, bringing the reaction mixture to 30°C, then adding 0.1 mI of cell-free extract and incubating for ten minutes more. The O. D. of the dark red-brown pigrnent formed was measured at 490 mu. The system was saturated with each variable component to find the optimum concentration for the maximum reaction rate. Mg⁺² and Ca⁺² stimulated the reaction about one-half as much as Mn⁺². Other cations tested include: Co⁺², Cu⁺², Fe⁺², Ni⁺², and, Zn⁺². Substrates oxidized by the cell-free systern include: catechol, dopa, p-phenylenediarnine and pyrogallol. It is concluded that the enzyme responsible for melanin formation in Verticillium is likely a peroxidase.
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