Distinguishing between the catalytic potential and apparent expression of tyrosinase activities.
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.
Zhao
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Department of Dermatology, University of Cincinnati College of Medicine, Ohio.
Boissy
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D016115:Albinism, Oculocutaneous / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D002609:Chediak-Higashi Syndrome / Q000201:enzymology / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008543:Melanins / Q000096:biosynthesis; D008544:Melanocytes / Q000201:enzymology / Q000378:metabolism; D008562:Membrane Glycoproteins; D014442:Monophenol Monooxygenase / Q000032:analysis / Q000378:metabolism*; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism; D012880:Skin Pigmentation / Q000502:physiology; D013053:Spectrophotometry; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D008543:Melanins; D008562:Membrane Glycoproteins; D011506:Proteins; D004295:Dihydroxyphenylalanine; D010088:Oxidoreductases; D014446:Tyrosine 3-Monooxygenase; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase