Abstract
An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V max of 92 nmol arginine per 15 min/mg protein and a K m of 2.1×103 nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.
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Abbreviations
- CGP:
-
Cyanophycin granule polypeptide
- CAP:
-
chloramphenicol
- MSO:
-
L-methionine-DL-sulphoximine
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Dedication: This manuscript is dedicated to the memory of Roger Y. Stanier whose excitement, enthusiasm, and vision for the study of all aspects of the microbial world led to a stimulating diversity of interests and achievements in his laboratory. The senior author is particularly indebted to him for his inspiring and articulate teaching and for his influence on her scientific development
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Allen, M.M., Morris, R. & Zimmerman, W. Cyanophycin granule polypeptide protease in a unicellular cyanobacterium. Arch. Microbiol. 138, 119–123 (1984). https://doi.org/10.1007/BF00413011
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DOI: https://doi.org/10.1007/BF00413011