Expression, partial purification and functional properties of themuscle-specific calpain isoform p94.
The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.
Branca
D
D
Department of Biological Chemistry, University of Padova, Italy.
Gugliucci
A
A
Bano
D
D
Brini
M
M
Carafoli
E
E
D000818:Animals; D016367:Baculoviridae; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry* / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009124:Muscle Proteins / Q000737:chemistry; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera
D007527:Isoenzymes; D009124:Muscle Proteins; D011994:Recombinant Proteins; D002154:Calpain; C078134:calpain p94; D002118:Calcium