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Predicate | Object |
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
41
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pubmed:dateCreated |
1997-11-13
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pubmed:abstractText |
The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Oct
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pubmed:issn |
0021-9258
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
10
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pubmed:volume |
272
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
25802-8
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:9325309-Animals,
pubmed-meshheading:9325309-Binding Sites,
pubmed-meshheading:9325309-Calcium,
pubmed-meshheading:9325309-Calpain,
pubmed-meshheading:9325309-Catalysis,
pubmed-meshheading:9325309-DNA, Complementary,
pubmed-meshheading:9325309-Humans,
pubmed-meshheading:9325309-Kinetics,
pubmed-meshheading:9325309-Mutagenesis, Site-Directed,
pubmed-meshheading:9325309-Protein Conformation,
pubmed-meshheading:9325309-Recombinant Proteins,
pubmed-meshheading:9325309-Spodoptera
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pubmed:year |
1997
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pubmed:articleTitle |
Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.
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pubmed:affiliation |
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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