Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties.
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.
Rhyner
J A
JA
Department of Pediatrics, University of Zurich, Switzerland.
Durussel
I
I
Cox
J A
JA
Ilg
E C
EC
Schäfer
B W
BW
Heizmann
C W
CW
D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002457:Cell Extracts; D002531:Cerebellum; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera; D007526:Isoelectric Point; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002457:Cell Extracts; D007106:Immune Sera; D010320:Parvalbumins; D011994:Recombinant Proteins; D008274:Magnesium; D002118:Calcium