"Prohormone thiol protease" (PTP) processing of recombinant proenkephalin.
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The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed. Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E. coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC. Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing. In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing. Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo [Liston, D. L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J. (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D. L. (1983) Arch. Biochem. Biophys. 221, 309-314]. Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.
Adrenal medulla; Amino Acid Sequence; Amino Acids/analysis; Animals; Base Sequence; Cattle; Chromaffin cells; Chromaffin Granules/enzymology; Chromatography; High Pressure Liquid; Cysteine Endopeptidases/metabolism; Electrophoresis; Polyacrylamide Gel; Enkephalins/chemistry; Enkephalins/genetics; Enkephalins/metabolism; Escherichia coli/genetics; Gene Expression; Kinetics; Molecular Sequence Data; Peptide hormones; Protein Precursors/chemistry; Protein Precursors/genetics; Protein Precursors/metabolism; Proteinase; Recombinant proteins; Recombinant Proteins/metabolism; Sequence Analysis
Biochemistry | Life Sciences | Molecular Biology | Neuroscience and Neurobiology
Schiller, M. R.,
Mende-Mueller, L. M.,
Miller, K. W.,
Hook, V. Y.
"Prohormone thiol protease" (PTP) processing of recombinant proenkephalin..