The purification and characterisation of m-calpain from ostrich brain.
Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. The purpose of the present study was to investigate the physico-chemical and kinetic properties of ostrich brain m-calpain. m-Calpain was purified by successive chromatographic steps on Toyopearl-Super Q 650s and Pharmacia Mono Q HR 5/5 columns. A Ca(2+) concentration of 5mM and a casein concentration of 5mg/ml were found to be necessary for optimum calpain activity. Ostrich m-calpain exhibited a M(r) of 84K using SDS-PAGE and a M(min) of 79.3K from amino acid analysis. The pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. The amino acid composition of m-calpain revealed 700 residues. The N-terminal sequence of m-calpain showed sequence identity with chicken (27%), human (23%) and rabbit (18%) and Schistoma mansoni (9%).
Mkwetshana
Noxolo
N
Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa.
Naudé
Ryno J
RJ
Oelofsen
Willem
W
Muramoto
Koji
K
Naganuma
Takako
T
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020310:Struthioniformes / Q000033:anatomy & histology; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
D002364:Caseins; D004791:Enzyme Inhibitors; D006454:Hemoglobins; C026018:azocasein; D002154:Calpain; C449000:m-calpain; D002118:Calcium