Anagli J, Hofmann F, Quadroni M, Vorherr T, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
Eur J Biochem. 1995 Nov 1;233(3):701-8. doi: 10.1111/j.1432-1033.1995.701_3.x.
A domain in the inducible, macrophage nitric oxide (NO) synthase has been selected as the putative calmodulin-binding site. The domain was synthesized as a peptide of 29 residues [P29, NO synthase-(504-532)-peptide], having the accepted hydrophobic/basic composition of calmodulin-binding domains and containing, like most of them, an aromatic amino acid at its N-terminus and a long chain aliphatic residue 12 amino acids downstream of it. A 34-residue peptide from the synthase sequence [P34, NO synthase-(499-532)-peptide], consisting of peptide P29 and of the five extra N-terminal amino acids, three of them basic, was also synthesized. Both peptides bound calmodulin in the presence as well as in the absence of Ca2+ (i.e. in the presence of excess EGTA). The KD of the binding in the presence of Ca2+ was < or = 1 nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin-target enzyme, the Ca2+ pump of the plasma membrane.
诱导型巨噬细胞一氧化氮合酶中的一个结构域被选为假定的钙调蛋白结合位点。该结构域被合成为一个含29个残基的肽段[P29，一氧化氮合酶-(504 - 532)-肽段]，具有公认的钙调蛋白结合结构域的疏水/碱性组成，并且与大多数此类结构域一样，在其N端含有一个芳香族氨基酸，在其下游12个氨基酸处含有一个长链脂肪族残基。还合成了一个来自合酶序列的含34个残基的肽段[P34，一氧化氮合酶-(499 - 532)-肽段]，它由肽段P29和额外的五个N端氨基酸组成，其中三个是碱性氨基酸。在有Ca2+和无Ca2+（即存在过量乙二醇双四乙酸）的情况下，这两种肽段都能结合钙调蛋白。在有Ca2+存在时结合的解离常数KD≤1 nM。在乙二醇双四乙酸存在时，结合亲和力较低，但仍然非常高。这些肽段能对抗钙调蛋白对一种经典的钙调蛋白靶酶——质膜Ca2+泵的刺激作用。
