Assays were developed to investigate the catalytic potential and apparent expression of
tyrosinase activities.
Tyrosine hydroxylase activity determined with cell lysates (in vitro), entire fixed cells (postfixation), or intact living cells (in situ), and
3,4-dihydroxyphenylalanine oxidase assayed spectrophotometrically or by
3,4-dihydroxyphenylalanine staining on
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, demonstrated the following results: 1) The in situ assay displayed reduced
tyrosine hydroxylase activity in all three
tyrosinase-positive oculocutaneous albino (OCA) lines except for
Chediak-Higashi Syndrome melanocytes, which displayed normal activity; 2) The in vitro assay had comparable activity of
tyrosinase-positive OCA melanocytes as controls, except for one
tyrosinase-positive OCA cell line, which demonstrated increased activity; 3) The postfixation assay, compared with the in situ assay, had elevated activity (ie. normalization) of
tyrosinase in OCA cells but reduced activity in controls; 4) The spectrophotometric 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 electrophoresis of melanocyte lysates either stained with
3,4-dihydroxyphenylalanine or immunoblotted with anti-
tyrosinase detected abnormal
tyrosinase bands in the
Chediak-Higashi Syndrome and one line of
tyrosinase positive OCA melanocytes, and both lines had release of
tyrosinase into the growth media. In conclusion, the selection and combination of these
tyrosinase assays would be informative for differentiation and characterization of human
albinism.