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Mutagenesis of Rat Dopamine β‐Hydroxylase: Examination in Cell‐Free System
Author(s) -
Feng Zhehui,
Sabban Esther L.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64010025.x
Subject(s) - biochemistry , signal peptide , cleavage (geology) , membrane , mutant , microsome , biology , glycosylation , endoglycosidase h , chemistry , microbiology and biotechnology , endoplasmic reticulum , enzyme , peptide sequence , golgi apparatus , gene , paleontology , fracture (geology)
Dopamine β‐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 Glu 41 → Leu 41 and Ser 43 → Thr 43 yielded only the GH form with membranes, whereas mutation of Ser 43 → Ala 43 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.