Mutagenesis of Rat Dopamine β-Hydroxylase: Examination in Cell-Free System |
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Authors: | Zhehui Feng Esther L. Sabban |
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Affiliation: | Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, U.S.A. |
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Abstract: | Abstract: 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 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. |
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Keywords: | Dopamine β-hydroxylase Norepinephrine mRNA mutagenesis Glycosylation Translation |
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