Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

Journal Article · Sun Jul 01 04:00:00 EDT 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:5926084

Blumentals, I I; Robinson, A S; Kelly, R M ^[1]

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1. Johns Hopkins Univ., Baltimore, MD (USA)

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons (kDa)) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98{degree}C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% {beta}-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N{prime},N{prime}-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.

• OSTI ID: 5926084
• Journal Information: Applied and Environmental Microbiology; (USA), Journal Name: Applied and Environmental Microbiology; (USA) Vol. 56:7; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOASSAY
CHEMICAL REACTIONS
DECOMPOSITION
ENZYME ACTIVITY
ENZYMES
HYDROLASES
IMMUNOASSAY
METABOLISM
MICROORGANISMS
PEPTIDE HYDROLASES
PROTEOLYSIS
SULFUR-OXIDIZING BACTERIA
THERMAL WATERS
THERMOPHILIC CONDITIONS