Rhyner J A, Durussel I, Cox J A, Ilg E C, Schäfer B W, Heizmann C W
Department of Pediatrics, University of Zurich, Switzerland.
Biochim Biophys Acta. 1996 Oct 11;1313(3):179-86. doi: 10.1016/0167-4889(96)00087-0.
Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet. of 4.9 x 10(3) M-1. The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet. of 3.4 to 4.6 x 10(3) M-1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively. The K'Mg.compet values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human alpha-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.
表达并纯化了人重组α-原肌球蛋白（PVwt）以及九种突变蛋白，这些突变蛋白在CD Ca(2+)结合位点（PVE62V、PVD51A、PVD51A,62V）、EF位点（PVE101V、PVD90A、PVD90A,E101V）或两者（PVE62V,E101V、PVD51A,D90A、PVD51A,E62V,D90A,E101V）的Ca2+结合关键位置含有失活取代。流动透析显示，PVwt以相等的K'Ca（2.3×10(7) M-1）结合2个Ca2+，并且Mg2+以K'Mg.compet（4.9×10(3) M-1）进行竞争。CD位点失活的三种突变体以2.0至2.3×10(7) M-1的K'Ca结合1个Ca2+，K'Mg.compet为3.4至4.6×10(3) M-1，即与PVwt的非常相似。EF位点失活的突变体对于PVD91A、PVE102V和PVE101V,D91A分别以7.9×10(6)、4.5×10(6)和3.6×10(6) M-1的K'Ca值结合1个Ca2+。这些突变体的K'Mg.compet值比PVwt低约4倍。两个位点均失活的三种突变体既不结合Ca2+也不结合Mg2+。在259 nm激发后，不含酪氨酸和色氨酸的人PV在283 nm处显示最大荧光发射。Ca2+或Mg2+与PVwt或EF位点失活的突变体结合导致荧光强度降低1.8倍，而CD失活的突变体在结合Ca2+或Mg2+时仅显示非常轻微的降低。在兔中制备了针对人α-原肌球蛋白的特异性抗体。测试了它们与突变蛋白的反应性，并研究了它们在定位和功能研究中的潜在价值。
