The purification and characterization of mu-calpain and calpastatin from ostrich brain.
Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.
Mkwetshana
Noxolo
N
Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, 6000, Port Elizabeth, South Africa.
Naudé
Ryno J
RJ
Muramoto
Koji
K
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D001921:Brain / Q000201:enzymology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013045:Species Specificity; D020310:Struthioniformes / Q000378:metabolism*; D013552:Swine; D013696:Temperature
D002135:Calcium-Binding Proteins; C033668:calpastatin; D002154:Calpain; C449002:mu-calpain