Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide
| Autor: | K R, Gibson, P G, Vanek, W D, Kaloss, G B, Collier, J F, Connaughton, M, Angelichio, G P, Livi, P J, Fleming |
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| Rok vydání: | 1993 |
| Předmět: |
Binding Sites
Base Sequence Recombinant Fusion Proteins Blotting Western Cell Membrane Genetic Vectors Molecular Sequence Data Restriction Mapping Dopamine beta-Hydroxylase Protein Sorting Signals Transfection Recombinant Proteins Cell Line Oligodeoxyribonucleotides Animals Cattle Drosophila Amino Acid Sequence Cloning Molecular Subcellular Fractions |
| Zdroj: | The Journal of biological chemistry. 268(13) |
| ISSN: | 0021-9258 |
| Popis: | To characterize the mechanism of membrane attachment of dopamine beta-hydroxylase, an expression system producing the processed form of this enzyme has been developed. We have replaced the endogenous signal peptide of bovine dopamine beta-hydroxylase with a heterologous signal peptide which is efficiently recognized and cleaved in Drosophila Schneider 2 cells. A cDNA encoding this chimeric recombinant bovine enzyme has been stably transfected into Schneider 2 cells. The inducible expression of active dopamine beta-hydroxylase in these cells has been verified by Western blotting and enzyme activity assays. N-terminal sequence analysis of purified recombinant enzyme demonstrates complete removal of the signal peptide. Subcellular analysis shows that the recombinant enzyme exists as both a soluble and a membrane-bound form in these cells. These data demonstrate that the endogenous signal peptide is not required for the formation of the membranous dopamine beta-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence. |
| Databáze: | OpenAIRE |
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