Mutagenesis of rat dopamine beta-hydroxylase: examination in cell-free system.

Abstract:

:Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) exists as membrane-bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell-free system. Translation of full-length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl-terminal, in contrast to amino-terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site-directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu41-->Leu41 and Ser43-->Thr43 yielded only the GH form with membranes, whereas mutation of Ser43-->Ala43 generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.

journal_name

J Neurochem

authors

Feng Z,Sabban EL

doi

10.1046/j.1471-4159.1995.64010025.x

subject

Has Abstract

pub_date

1995-01-01 00:00:00

pages

25-33

issue

1

eissn

0022-3042

issn

1471-4159

journal_volume

64

pub_type

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