Immunochemical studies on tyrosinase induction in Neurospora.

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An immunoassay for tyrosinase, using the modified bacteriophage technique, was developed: Tyrosinase of Neurospora was conjugated to bacteriophage T4 using glutaraldehyde as a cross-linking agent. The conjugated phage that survived the coupling process could be inactivated by antiserum raised in rabbits against pure tyrosinase, but not by normal serum. This inactivation was specifically inhibited by pure Neurospora tyrosinase, and the degree of inhibition was proportional to the concentration of tyrosinase within the range of 30-150 ng/ml. Crude mycelial extract possessing tyrosinase activity could similarly inhibit the inactivation of the conjugated phage by the antiserum. To evaluate the tyrosinase content of crude extracts their inhibitory capacity was compared to that of known amounts of pure tyrosinase, and the amounts thus calculated agreed with those predicted from an enzymatic assay. The tyrosinase-bacteriophage immunoassay was used for the quantitation of tyrosinase-antigen in crude extracts of Neurospora cultures that had been induced to form tyrosinase by the addition of ethionine. Enzymatic activity appeared after a lag of several hours, increased for 2-3days and then declined. Immunological assays of these cultures showed: (a) serologically reactive protein started to accumulate upon culture starvation and was evident during the lag period; (b) specific activity (units per mg antigen) was constant throughout induction; (c) at the phase of decrease in mycelial enzyme content, increasing amounts of serologically reactive protein were detected in the medium, indicating that some enzyme was eventually excreted. These results show that the lag is not a qualitatively distinct period, and support the previously forwarded notion that tyrosinase is synthesized de novo upon induction.