Immunochemical Studies on Tyrosinase Induction in Neurospora

Abstract

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 extracts 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 2-3 days 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

An EPR study of Neurospora tyrosinase

“…The pure enzyme had an extinction coefficient AXl%cm of 22 at 280 nm [4]. The determination of the specific activity was carried out according to Fling et al [2] and the obtained value, 1100 units per mg protein, is slightly higher than those reported by others [2,3,9].…”

Neurospora tyrosinase: Molecular weight, copper content and spectral properties

“…Freshly isolated enzyme contained about 30% oxytyrosinase as has been reported by Gutterridge and Robb [5] using a different purification method. The specific activity of the purified enzyme measured by the procedure of Fling et al [4] was slightly higher than the ones reported by Gutteridge and Robb [5], Katan et al [9], and Fling et al [4]. The enzyme was stored in 10 mM sodium phosphate buffer, pH 7.2, + 0.5 M NaC1 at 4°C.…”

Amino acid sequence of tyrosinase from Neurospora crassa.

“…Sci. USA 75 (1978) indications (8,22) that methionine is the sole sulfur-containing amino acid in Neurospora tyrosinase, the enzyme was found to contain also a single chemically modified cysteine residue. Digestion of in dvo labeled [asSityrosinase with pepsin resulted, in addition to the three methionine peptides, in a fourth radioactive peptide with the amino acid composition 0.95 Thr, 2.00 Gly, 0.91 Tyr, and an unknown ninhydrin-positive, radioactive species "X" eluting just after aspartic acid.…”