Mutagenesis of rat dopamine beta-hydroxylase: examination in cell-free system.
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.
Feng
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Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Sabban
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004285:Dogs; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D006026:Glycoside Hydrolases / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D014176:Protein Biosynthesis; D051381:Rats
D018076:DNA, Complementary; D004299:Dopamine beta-Hydroxylase; D006026:Glycoside Hydrolases; D008274:Magnesium