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Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide

1993; Elsevier BV; Volume: 268; Issue: 13 Linguagem: Inglês

10.1016/s0021-9258(18)98377-8

1083-351X

Keisha Gibson, Philip G. Vanek, William D. Kaloss, Galen Collier, John F. Connaughton, Monica Angelichio, G P Livi, Patrick Fleming,

Nerve injury and regeneration

To characterize the mechanism of membrane attachment of dopamine @-hydroxylase, an expression system producing the processed form of this enzyme has been developed.We have replaced the endogenous signal peptide of bovine dopamine @-hydroxylase with a heterologous signal peptide which is efficiently recognized and cleaved in Drosophila Schneider 2 cells.A cDNA encoding this chimeric recombinant bovine enzyme has been stably transfected into Schneider 2 cells.The inducible expression of active dopamine @-hydroxylase in these cells has been verified by Western blotting and enzyme activity assays.N-terminal sequence analysis of purified recombinant enzyme demonstrates complete removal of the signal peptide.Subcellular analysis shows that the recombinant enzyme exists as both a soluble and a membrane-bound form in these cells.These data demonstrate that the endogenous signal peptide is not required for the formation of the membranous dopamine @-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence.