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Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein.
- Source :
-
Archives of biochemistry and biophysics [Arch Biochem Biophys] 1986 Dec; Vol. 251 (2), pp. 543-50. - Publication Year :
- 1986
-
Abstract
- A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.
- Subjects :
- Animals
Cattle
Chromatography, Gel
Chromatography, High Pressure Liquid
Electrophoresis, Polyacrylamide Gel
Female
Hydrolysis
Peptide Hydrolases metabolism
Substrate Specificity
Cytoplasmic Granules enzymology
Hypothalamus enzymology
Peptide Hydrolases isolation & purification
Pituitary Hormone-Releasing Hormones biosynthesis
Protein Precursors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0003-9861
- Volume :
- 251
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Archives of biochemistry and biophysics
- Publication Type :
- Academic Journal
- Accession number :
- 3541788
- Full Text :
- https://doi.org/10.1016/0003-9861(86)90362-0