Purification and Characterization of Two Functional Forms of Intracellular Protease PfpI from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Applied and Environmental Microbiology
S B HalioRobert M Kelly

Abstract

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 (plusmn) 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 (plusmn) 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was (symbl)85(deg)C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of...Continue Reading

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Citations

Mar 7, 2003·Proceedings of the National Academy of Sciences of the United States of America·Paulene M QuigleyWim G J Hol
Nov 26, 2008·Journal of Bacteriology·Alexandro Rodríguez-Rojas, Jesús Blázquez
Apr 26, 2011·Extremophiles : Life Under Extreme Conditions·Sung-Ho YunSeung Il Kim
Aug 29, 2014·World Journal of Microbiology & Biotechnology·Liuqing WangMeide Liao
Jan 10, 2012·Molecules and Cells·Hoi Jong JungSun-Shin Cha
Jul 9, 2013·FEBS Letters·Hitomi MitsugiHiroyoshi Ariga
Feb 12, 2014·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Dongling ZhanWeiwei Han
Apr 10, 2015·Marine Drugs·Gabriel Zamith Leal DalmasoAlane Beatriz Vermelho
Sep 7, 2017·Acta Crystallographica. Section D, Structural Biology·Steven B Larson, Alexander McPherson
Jun 27, 2018·FEMS Microbiology Reviews·Christopher T StraubRobert M Kelly
Dec 1, 2018·Emerging Topics in Life Sciences·Julie A Maupin-Furlow
Apr 6, 2021·Archives of Biochemistry and Biophysics·Esha SircarRajib Sengupta
Mar 1, 1997·Applied and Environmental Microbiology·M W BauerR M Kelly

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