| pubmed-article:9325309 | rdf:type | pubmed:Citation | lld:pubmed |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C0205245 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C0596988 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C0600499 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C1514562 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C1883221 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C1883204 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C1880389 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C0871161 | lld:lifeskim |
| pubmed-article:9325309 | lifeskim:mentions | umls-concept:C0006785 | lld:lifeskim |
| pubmed-article:9325309 | pubmed:issue | 41 | lld:pubmed |
| pubmed-article:9325309 | pubmed:dateCreated | 1997-11-13 | lld:pubmed |
| pubmed-article:9325309 | 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. | lld:pubmed |
| pubmed-article:9325309 | pubmed:language | eng | lld:pubmed |
| pubmed-article:9325309 | pubmed:journal | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9325309 | pubmed:citationSubset | IM | lld:pubmed |
| pubmed-article:9325309 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9325309 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9325309 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9325309 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9325309 | pubmed:status | MEDLINE | lld:pubmed |
| pubmed-article:9325309 | pubmed:month | Oct | lld:pubmed |
| pubmed-article:9325309 | pubmed:issn | 0021-9258 | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:CarafoliEE | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:MakiMM | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:HitomiKK | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:BerardiSS | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:CalderaroEE | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:AnagliJJ | lld:pubmed |
| pubmed-article:9325309 | pubmed:author | pubmed-author:VileiE MEM | lld:pubmed |
| pubmed-article:9325309 | pubmed:issnType | Print | lld:pubmed |
| pubmed-article:9325309 | pubmed:day | 10 | lld:pubmed |
| pubmed-article:9325309 | pubmed:volume | 272 | lld:pubmed |
| pubmed-article:9325309 | pubmed:owner | NLM | lld:pubmed |
| pubmed-article:9325309 | pubmed:authorsComplete | Y | lld:pubmed |
| pubmed-article:9325309 | pubmed:pagination | 25802-8 | lld:pubmed |
| pubmed-article:9325309 | pubmed:dateRevised | 2006-11-15 | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:meshHeading | pubmed-meshheading:9325309-... | lld:pubmed |
| pubmed-article:9325309 | pubmed:year | 1997 | lld:pubmed |
| pubmed-article:9325309 | pubmed:articleTitle | Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit. | lld:pubmed |
| pubmed-article:9325309 | pubmed:affiliation | Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland. | lld:pubmed |
| pubmed-article:9325309 | pubmed:publicationType | Journal Article | lld:pubmed |
| pubmed-article:9325309 | pubmed:publicationType | Research Support, Non-U.S. Gov't | lld:pubmed |
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| http://linkedlifedata.com/r... | pubmed:referesTo | pubmed-article:9325309 | lld:pubmed |
