ABSTRACT
To characterize the mechanism of membrane attachment of dopamine beta-hydroxylase, an expression system producing the processed form of this enzyme has been developed. We have replaced the endogenous signal peptide of bovine dopamine beta-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 beta-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 beta-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence.
