Molecular cloning and biochemical characterization of the skin tyrosinase from Rana esculenta L.
Amphibian tyrosinases display unique and poorly understood properties such as seasonal activity variations, different activities in dorsal and ventral skin and the occurrence as inactive forms requiring proteolytic activation. For the first time we have sequenced and characterized Rana esculenta L. tyrosinase by functional expression of the cloned cDNA, and compared it with frog skin extracts. R. esculenta tyrosinase ORF is well conserved compared with tyrosinases of various sources. The amino acid similarities between the tyrosinases from R. esculenta and other amphibia range from 85% to 98%. Homology remains high with mammalian tyrosinases (65% identity with Homo sapiens, and 63% with Mus musculus) and with bird orthologues (66% identity with Gallus gallus). Tyrosinase was expressed in HEK293T cells as an active enzyme. Activity staining on non reducing SDS-PAGE revealed two bands around 63 and 68 kDa. R. esculenta skin extracts were mildly active and reached maximal activity upon protease treatment, revealing a high molecular weight dopa-positive band in the 200 kDa range and one of higher MW, after nagarse treatment, in activity stainings. The different behaviour of recombinant tyrosinase compared to skin extracts suggests formation in vivo of a multimeric complex.
Zanna
Paola T
PT
Dipartimento di Biochimica Medica, Biologia Medica e Fisica Medica, Facoltà di Medicina e Chirurgia, Università degli Studi di Bari, Policlinico-Piazza Giulio Cesare, Bari, Italy.
Maida
Immacolata
I
Arciuli
Marcella
M
Jimenez-Cervantes
Celia
C
Garcia-Borron
José C
JC
Cicero
Rosa
R
Guida
Gabriella
G
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism*; D011893:Rana esculenta / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012867:Skin / Q000201:enzymology* / Q000378:metabolism; D013045:Species Specificity
D014442:Monophenol Monooxygenase