EFFECT OF ENVIRONMENTAL CONDITIONS UPON TYROSINASE ACTIVITY IN GLOMERELLA CINGULATA

Abstract

PREVIOUS EXPERIMENTS with Glomerella cingulata have established that tyrosinase activity first appears in plate culture about 120 hr. after inoculation and that its formation is probably adaptive in nature (Sussman and Markert, 1953). On the other hand, cytochrome oxidase activity is present at all times in the organism and in roughly equivalent quantities. In an earlier investigation (Markert, 1950) it was noted that the amount of tyrosinase in the mycelium of Glomerella varies with the conditions of nutrition, aeration, and temperature under which the mycelium is grown. More recently, other workers have called attention to the importance of temperature (Horowitz and Shen, 1952) in the formation of tyrosinase in Neurospora, and in the formation of other fungous enzymes (Fries, 1953). The following experiments were designed, therefore, to explore the effects of these factors and others upon tyrosinase formation in Glomerella. METHODS.-The organism used in these experiments was the standard type (A1B13) of Glomerella cingulata which is a conidiating double mutant from the wild type. Surface cultures were grown from mass conidial inoculum, usually on 30 ml. of agar medium in Petri dishes, and incubated at 200C. until harvested. Where divergence from these techniques occurred, mention will be made before the results are presented. The complete Neurosporac medium (Ryan, 1950) was used, except where otherwise noted. Mycelia were harvested and handled as described by Sussman and Markert (1953). Enzyme extracts were made by grinding the weighed mycelial mats in 1/15 M phosphate buffer at pH 7.2 with a glass homogenizer. The brei was centrifuged at 100 X g for 15 min. after which the supernatant fluid was collected and used. Tyrosinase activity was determined colorimetrically by the method of Markert (1950) as modified (Sussman and Markert, 1953) to employ a Beckman spectrophotometer. In this investigation the model DU was used in place of the model B. The reaction mixture consisted of 1 ml. of 0.02 M DL-dihydroxyphenylalanine (dopa), 0.1 ml. of the enzyme preparation, and 1.5 ml of 1/15 M phosphate buffer at pH 6.5. In certain instances, less enzyme was used because of the preparation's high activity; when this was done, enough buffer was added to bring the total volume to 3.0 ml. The substrate was added at zero time and readings were made at 480 mln at 10-sec. intervals. The change in