Characteristics of Tyrosinase in B16 Melanoma

Abstract

Tyrosine hydroxylase, dopa oxidase, and peroxidase activities were studied in soluble fractions of B16 melanoma tumor homogenates by polyacrylamide gel disc electrophoresis. Stained gels were scanned photometrically and gel slices were assayed radiometrically. In these preparations, the two bands of tyrosine hydroxylating activity were completely separated from the peroxidase activity but coincided with two major bands of dopa oxidase activity. The third dopa oxidase band coincided with the single band of peroxidase activity. The soluble fraction of cultured cell homogenates had no peroxidase activity, but the two tyrosine hydroxylase bands coincided exactly with the two dopa oxidase bands. Therefore, in the soluble fraction of the murine melanoma bifunctional tyrosinase does exist as two electrophoretically separable forms which are independent of peroxidase.

Kinetic and Sensitive Analysis of Tyrosinase Activity Using Electron Transfer Complexes: In Vitro and Intracellular Study

“…The detailed and successful investigation of in vitro tyrosinase analysis using QD‐Tyr conjugates encouraged us to detect the tyrosinase in live cells. We chose B16 cells as experimental model due to its high tyrosinase expression quantity . We used HeLa cells with low tyrosinase expression as negative control.…”

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design, Synthesis, and Application in Living Cells and Zebrafish

“…Owing to its high specificity for tyrosinase,p robe 1 is anticipated to be capable of accurately detecting the enzyme activity in living cells or organisms.T od emonstrate this potential, murine melanoma B16 cells were used as amodel, because they overexpress tyrosinase. [13] As depicted in Figure 3A,B 16 cells themselves show an extremely low background fluorescence (image a), which benefits from the NIR excitation wavelength of the probe.H owever,t he B16 cells treated with probe 1 exhibit strong fluorescence (image b), suggesting ag ood cell-permeability for probe 1 and its possible reaction with tyrosinase in the cells.T ov erify that the fluorescence change was caused by tyrosinase,k ojic acid (inhibitor) was used to pretreat the cells,a nd the pretreated cells generated am arkedly lower fluorescence (image c), in which the relative pixel intensity decreases by about 50 % (compare the intensity values of ba nd ci nF igure 3B). This indicates that the fluorescence enhancement in B16 cells does result from the endogenous tyrosinase.I mportantly,b oth siRNA-transfected B16 and HeLa cells with the inhibited expression of tyrosinase,asfurther evidenced by the enzymelinked immunosorbent assay (ELISA), showed ag reatly decreased intracellular fluorescence (Supporting Information, Figure S25), clearly indicating that this intracellular fluorescence change reflects the alteration of the relative tyrosinase activity.I nterestingly,n oo bvious fluorescence increase was observed outside the cells ( Figure 4A), and no mass peak of HXPI (Supporting Information, Figure S26) was…”

Peroxidase-Mediated Mechanisms Are Involved in the Melanocytotoxic and Melanogenesis-Inhibiting Effects of Chemical Agents

“…Therefore, it was suggested that 1) peroxidase, and not tyrosinase, is responsible for converting tyrosine to dopa, and 2) peroxidase and tyrosinase (aerobic dopa oxidase) then act synergistically to oxidize dopa to dopaquinone [13]. The hypothesis of Okun and associates was objected to by several studies supporting the tyrosinase theory of mammalian melanogenesis by presenting evidence on the ability of tyrosinase to hydroxylate tyrosine and/or the inability of horseradish peroxidase or melanocyte peroxidase to mediate this reaction [14, 15, 16]. The peroxidase hypothesis was therefore considered to be ruled out.…”