Gheber L, Priel Z, Aflalo C, Shoshan-Barmatz V
Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva, Israel.
J Membr Biol. 1995 Sep;147(1):83-93. doi: 10.1007/BF00235399.
3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.
3'-O-(4-苯甲酰基)苯甲酰基ATP（BzATP）被用作ATP的光亲和类似物，以标记纤毛细胞中的潜在ATP受体。与ATP一样，在没有光激活的情况下，BzATP可将组织培养中的纤毛搏动频率刺激高达三倍。在[α-32P]BzATP存在下对完整细胞进行照射，然后进行SDS-PAGE和放射自显影，结果显示出两种分子量分别为46 kDa和96 kDa的标记蛋白（p46和p96）。两种蛋白的光标记都易受胰蛋白酶消化的影响，这意味着标记蛋白至少部分暴露在质膜的细胞外表面。两种蛋白中32P掺入对[α-32P]BzATP浓度的依赖性相似。p46而非p96的标记需要Ca2+或Mg2+。各种核苷酸刺激纤毛频率，并抑制p46和p96的光标记。对p46的表观亲和力的排序为：ATP≈ADP＞GTPγS＞ADPβS、UTP、2MeSATP、AMP-PNP＞AMP-PCP＞AMP＞腺苷；对p96而言则为：ADP≈ADPβS≥ATP＞＞AMP-PCP、AMP-PNP＞GTPγS≥AMP＞2MeSATP、UTP、腺苷。纤毛搏动频率刺激的排序为：ADPβS、UTP≥2MeSATP、GTPγS、AMP-PNP、ATP≥ADP＞AMP-PCP＞腺苷＞AMP。这些结果表明p46作为一种P2嘌呤受体参与了细胞外ATP对纤毛搏动的刺激作用。另一方面，p96可能代表一种P2嘌呤受体或一种胞外核苷酸酶。
