Iraburu M J, López-Zabalza M J, Santiago E
Departamento de Bioquímica Universidad de Navarra, Pamplona, Spain.
Rev Esp Fisiol. 1994 Mar;50(1):55-62.
The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.
胰蛋白酶激活的CF1的Ca(2+)-ATP酶活性呈现单相模式，表明该酶的活性中心以相同的动力学特性起作用。对阴离子氰酸盐（OCN-）和硫氰酸盐（SCN-）对ATP酶活性影响的研究表明存在阳离子调节位点，这些位点能够以竞争性方式结合这些调节剂，从而导致ATP酶活性受到抑制。高浓度的核苷酸ADP和ATP是底物Ca(2+)-ATP的竞争性抑制剂。低浓度的ATP呈现激活作用。对低浓度ATP和SCN-对ATP酶活性的联合作用的研究揭示了阴离子和核苷酸之间存在非竞争性关系。用异硫氰酸荧光素（一种与核苷酸结合中心的氨基结合的特异性试剂）对CF1进行修饰，无论是对胰蛋白酶处理的酶还是未处理的酶，都得到摩尔比FITC/CF1 = 4。这种特异性掺入发生在CF1的α和β亚基上，并导致ATP酶活性降低约30%。这些结果与CF1上存在三个催化位点和三个调节位点或四个催化位点和两个调节位点一致。
