Bessière P, Cottin P, Balny C, Ducastaing A, Bancel F
Laboratoire de Biochimie et Technologie des Aliments (INRA UA 429), ISTAB, Université de Bordeaux I, Avenue des Facultés, 33405, Talence Cedex, France.
Biochim Biophys Acta. 1999 Mar 19;1430(2):254-61. doi: 10.1016/s0167-4838(99)00006-0.
The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.
在高达650兆帕的高静水压力下，通过荧光光谱法研究了μ-钙蛋白酶和m-钙蛋白酶的解离情况。压力增加导致无钙酶的色氨酸荧光发生红移。光谱转变的浓度依赖性与压力诱导的亚基解离一致。温度升高增加了钙蛋白酶异二聚体的稳定性，并证实了单体之间疏水相互作用的主导地位。在钙饱和时，天然或碘乙酰胺失活的钙蛋白酶均未观察到光谱转变，表明它们已被钙解离。两种同工型的反应体积约为-150毫升/摩尔，在大气压下，μ-钙蛋白酶和m-钙蛋白酶的解离常数分别约为10^-12摩尔/升和10^-15摩尔/升。这一结果表明，该同工型中存在更紧密的相互作用，其活性需要更高的钙浓度。
