The membrane-binding segment of dopamine β-hydroxylase is not an uncleaved signal sequence

Abstract

Dopamine beta-hydroxylase exists in bovine adrenal medulla chromaffin granules in both soluble and membrane-bound forms. The mechanism by which membranous dopamine beta-hydroxylase is bound to granule membranes has been elusive. Recently, evidence that covalently attached phosphatidylinositol does not serve as an anchor for membranous dopamine beta-hydroxylase was reported (Stewart, L. C., and Klinman, J. P. (1988) J. Biol. Chem. 263, 12183-12186). It was suggested that an uncleaved signal sequence could serve as a mode of attachment for the membrane-bound hydroxylase. Amino-terminal sequence analysis of purified bovine membranous dopamine beta-hydroxylase demonstrates that this form of the enzyme possesses an amino-terminal sequence similar to the soluble enzyme. Additionally, the 75- and 72-kDa bands of membranous dopamine beta-hydroxylase were electrophoretically eluted from a preparative sodium dodecyl sulfate-polyacrylamide gel and sequenced. Both bands had the amino-terminal sequence characteristic of the soluble bovine enzyme. These sequence results eliminate the possibility that an uncleaved signal sequence serves as the membrane anchor.