DISTINGUISHING BETWEEN THE CATALYTIC POTENTIAL AND APPARENT EXPRESSION OF TYROSINASE ACTIVITIES

Assays were developed to investigate the catalytic potential and appar ent expression of tyrosinase activities. Tyrosine hydroxylase activity determined with cell lysates (in vitro), entire fixed cells (postfixa tion), or intact living cells (in situ), and 3,4-dihydroxyphenylalanin e oxidase assayed spectrophotometrically or by 3,4-dihydroxyphenylalan ine staining on sodium dodecyl sulfate-polyacrylamide gel electrophore sis, demonstrated the following results: 1) The in situ assay displaye d reduced tyrosine hydroxylase activity in all three tyrosinase-positi ve oculocutaneous albino (OCA) lines except for Chediak-Higashi Syndro me melanocytes, which displayed normal activity; 2) The in vitro assay had comparable activity of tyrosinase-positive OCA melanocytes as con trols, except for one tyrosinase-positive OCA cell line, which demonst rated increased activity; 3) The postfixation assay, compared with the in situ assay, had elevated activity (ie, normalization) of tyrosinas e in OCA cells but reduced activity in controls; 4) The spectrophotome tric assay for 3,4-dihydroxyphenylalanine oxidase activity correlated very well with the tyrosine hydroxylase activity determined by the in vitro assay; 5) sodium dodecyl sulfate-polyacrylamide gel electrophore sis of melanocyte lysates either stained with 3,4-dihydroxyphenylalani ne or immunoblotted with anti-tyrosinase detected abnormal tyrosinase bands in the Chediak-Higashi Syndrome and one line of tyrosinase posit ive OCA melanocytes, and both lines had release of tyrosinase into the growth media. In conclusion, the selection and combination of these t yrosinase assays would be informative for differentiation and characte rization of human albinism.