Match!

Heterogeneity of proteinases from the hyperthermophilic archaeobacterium Pyrococcus furiosus

Published on May 1, 1991in Microbiology1.027
· DOI :10.1099/00221287-137-5-1193
Helen Connaris1
Estimated H-index: 1
,
Don A. Cowan51
Estimated H-index: 51
,
Richard J. Sharp9
Estimated H-index: 9
Abstract
Intracellular and extracellular samples from the extremely thermophilic archaeobacterium Pyrococcus furiosus showed the presence of multiple active proteinases. Using gelatin-containing SDS-PAGE, up to 13 activity bands were visualized with apparent molecular masses of between 66 and 135 kDa. Characterization studies revealed these bands to be due to discrete polypeptides, and not artefacts. Results from gel permeation chromatography, sucrose density gradient centrifugation and non-denaturing PAGE suggested that some of these proteohtic polypeptides may exist as active aggregates either in vivo or in vitro before being dissociated by SDS to active monomers.
  • References (11)
  • Citations (40)
📖 Papers frequently viewed together
78 Citations
90 Citations
684 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
References11
Newest
#1Rik I. L. EggenH-Index: 41
#2Ans C. M. GeerlingH-Index: 6
Last. W.M. de VosH-Index: 136
view all 4 authors...
78 CitationsSource
Abstract A thermostable, acid proteolytic activity has been found to be associated with the cells and in the culture medium of Sulfolobus acidocaldarius, an archaebacterium. This acid protease, which has been named thermopsin, was purified to homogeneity from the culture medium by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, monoQ (fast protein liquid chromatography), and gel filtration (high pressure liquid chromatograp...
74 Citations
#1Angelo Fontana (UNIPD: University of Padua)H-Index: 49
Abstract The molecular mechanisms responsible for the unusual stability of enzymes isolated from thermophilic microorganisms are much more complex and subtle than was originally thought. In particular, a general mechanism cannot be proposed, since individual enzymes can be stabilized by specific molecular interactions and forces. The results of studies on thermophilic enzymes obtained in recent years in our laboratory will be summarized, with particular emphasis being placed on those obtained wi...
81 CitationsSource
#1Don A. Cowan (University of Waikato)H-Index: 10
#2K.A. Smolenski (University of Waikato)H-Index: 1
Last. Hugh W. Morgan (University of Waikato)H-Index: 37
view all 4 authors...
86 CitationsSource
#1Noboru Horie (UCSF: University of California, San Francisco)H-Index: 1
#2Kimie Fukuyama (UCSF: University of California, San Francisco)H-Index: 32
Last. William L. Epstein (UCSF: University of California, San Francisco)H-Index: 40
view all 4 authors...
Abstract 1. 1. Electrophoresis of cornified cell extracts of 2-day-old rats, using SDS polyacrylamide gels copolymerized with α-casein or gelatin with or without plasminogen, was performed. 2. 2. Both Tris-HCl buffer soluble protein and KSCN solubilized protein contained a number of hydrolases for α-casein and/or gelatin, but PA (mol. wts 57 and 50K) was found only in the KSCN extract. 3. 3. The pH dependency, substrate specificity and mol. wt of plasminogen-independent proteinases were comparat...
19 CitationsSource
: The thermophilic enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase, decarboxylating, EC 1.1.1.44) from Bacillus stearothermophilus was much more resistant to inactivation under different conditions of temperature, pH, guanidine-hydrochloride, and organic solvents (dioxane, dimethylformamide, acetone) than its mesophilic counterpart from yeast. In addition, the thermophilic enzyme largely withstands proteolysis with trypsin, chymotrypsin, and elastase when compa...
19 Citations
#1Don A. Cowan (University of Waikato)H-Index: 10
#2Roy M. Daniel (University of Waikato)H-Index: 46
Abstract An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus -like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D.phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21000, a pli optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and i...
71 CitationsSource
#1Jeff T. Barach (NCSU: North Carolina State University)H-Index: 1
#2Daniel M. Adams (NCSU: North Carolina State University)H-Index: 2
Thermal inactivation at 110–150°C of thermolysin (EC 3.4.24.4), produced by the thermophile Bacillus thermoproteolyticus, and the extracellular protease of Pseudomonas sp. MC60 a psychrotroph, were investigated at 130°C, both enzymes had approximately the same ΔH∗ (22 kcal/mol) and ΔS∗ (−13.5 cal/mol per degree) values. Both enzymes contain zinc and calcium. The amino acid compositions of the enzymes were similar except that MC60 protease exhibited a more typical tyrosine content. Comparable hea...
33 CitationsSource
#1Teruo AbeH-Index: 2
#2Kenji TakahashiH-Index: 4
Last. Toshio AndoH-Index: 47
view all 3 authors...
12 CitationsSource
#1Ulrich K. Laemmli (LMB: Laboratory of Molecular Biology)H-Index: 58
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
199k CitationsSource
Cited By40
Newest
#1Julie A. Maupin-Furlow (UF: University of Florida)H-Index: 31
Archaea are phylogenetically distinct from bacteria, and some of their proteolytic systems reflect this distinction. Here, the current knowledge of archaeal proteolysis is reviewed as it relates to protein metabolism, protein homeostasis, and cellular regulation including targeted proteolysis by proteasomes associated with AAA-ATPase networks and ubiquitin-like modification. Proteases and peptidases that facilitate the recycling of peptides to amino acids as well as membrane-associated and integ...
Source
#1Xiaowei Gao (WHU: Wuhan University)H-Index: 2
#2Jing Zeng (WHU: Wuhan University)H-Index: 2
Last. Xiao-Feng Tang (WHU: Wuhan University)H-Index: 10
view all 6 authors...
: Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS14...
1 CitationsSource
#1Jing Zeng (WHU: Wuhan University)H-Index: 2
#2Xiaowei Gao (WHU: Wuhan University)H-Index: 2
Last. Xiao-Feng Tang (WHU: Wuhan University)H-Index: 2
view all 5 authors...
Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin a...
17 CitationsSource
#1Zheng Dai (WHU: Wuhan University)H-Index: 3
#2Heting Fu (WHU: Wuhan University)H-Index: 1
Last. Xiao-Feng Tang (WHU: Wuhan University)H-Index: 2
view all 6 authors...
ABSTRACT Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both...
9 CitationsSource
#1Robert M. Kelly (NCSU: North Carolina State University)H-Index: 55
#2Tonya L. Peeples (NCSU: North Carolina State University)H-Index: 1
Last. Guy D. Duffaud (NCSU: North Carolina State University)H-Index: 5
view all 5 authors...
12 CitationsSource
#1Semra Kocabiyik (METU: Middle East Technical University)H-Index: 9
#2Bilge Erdem (METU: Middle East Technical University)H-Index: 1
Abstract In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60–70 °C and showed a pH optimum over a range of pH 7.0–8.5. ...
22 CitationsSource
#1Deborah S. Kelley (UW: University of Washington)H-Index: 40
#2John A. Baross (UW: University of Washington)H-Index: 48
Last. John R. Delaney (UW: University of Washington)H-Index: 37
view all 3 authors...
▪ Abstract The recent recognition of a potentially vast, unexplored hot microbial biosphere associated with active volcanism along the global mid-ocean ridge network has fundamentally shifted concepts of how planets and life coevolve. Many processes intrinsic to the dynamics of the spreading center volcanic system provide partial or complete nutritional fluxes that support diverse microbial communities that thrive under extreme conditions on and beneath the seafloor. Mantle melting, volcanism, a...
313 CitationsSource
#1Donald WardH-Index: 11
#2Keith R. ShockleyH-Index: 14
Last. Robert M. KellyH-Index: 55
view all 7 authors...
Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus ...
37 CitationsSource
#1Jerry Eichler (BGU: Ben-Gurion University of the Negev)H-Index: 35
Archaea have developed a variety of molecular strategies to survive the often harsh environments in which they exist. Although the rules that allow archaeal enzymes to fulfill their catalytic functions under extremes of salinity, temperature or pressure are not completely understood, the stability of these extremophilic enzymes, or extremozymes, in the face of adverse conditions has led to their use in a variety of biotechnological applications in which such tolerances are advantageous. In the f...
144 CitationsSource
#1James F. Holden (UW: University of Washington)H-Index: 22
#2Ken Takai (UW: University of Washington)H-Index: 1
Last. John A. Baross (UW: University of Washington)H-Index: 48
view all 6 authors...
Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9°50′N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and roc...
75 CitationsSource