Lamport-Vrana S J, Vrana K E, Fedan J S
Department of Pharmacology and Toxicology, West Virginia University, Morgantown.
J Pharmacol Exp Ther. 1991 Jul 1;258(1):339-48.
Extracellular ATP (greater than 3 x 10(-5) M) elicits a transient, biphasic contraction of the vas deferens. The specific P2x-purinoceptor photoaffinity label antagonist, arylazido aminopropionyl ATP (ANAPP3), inhibits the first contractile phase. The second phase, which is increased for adenosine 5'-O-(3-thio-triphosphate)(ATP gamma S) and attenuated for 5'-anhydride-substituted ATP analogs, has been hypothesized to be initiated by phosphate chain cleavage, and is inhibited irreversibly by periodate-oxidized ATP (P-ATP), an affinity label. We examined whether phosphorylation occurs during contraction to ATP gamma S and ATP, and whether the irreversible inhibition of the second phase of contraction by P-ATP results from its covalent incorporation. After incubation of intact tissues with [35S]ATP gamma S (3 mM), homogenization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, 35SPO2(3-) (35S) was incorporated predominantly into a single, 19 to 21 kD protein. The incorporation was transient and paralleled the time course of the transient contraction to ATP gamma S (peak by 5 to 10 sec, then rapid decline during 60 sec). Treatment with P-ATP (10 mM; 5 min) or ANAPP3 (10(-4) M) inhibited 35S incorporation at 60 sec. 32PO4(3-) (32P) from [gamma-32P] ATP (3 mM) also was incorporated rapidly into the protein; removal of 32P, however, was faster than removal of 35S. [3H]P-ATP was incorporated into several proteins, but not into the 19 to 21 kD protein nor the ones labeled by photolyzed ANAPP3. Incorporation of 35S from [35S]ATP gamma S, and [3H]P-ATP, were not affected by incubation in the presence of histamine or norepinephrine (10(-4)-10(-3) M), whereas ATP (10(-2) M) reduced incorporation of [3H]P-ATP (10(-5)-10(-2) M). The results support the hypothesis that the second phase of contraction to ATP involves a transductive phosphoryl transfer involving an ecto-kinase, which is inhibited by P-ATP.
细胞外ATP（大于3×10⁻⁵ M）可引起输精管的短暂双相收缩。特异性P2x嘌呤受体光亲和标记拮抗剂芳基叠氮基氨丙酰基ATP（ANAPP3）可抑制第一收缩期。第二收缩期对腺苷5'-O-（3-硫代三磷酸）（ATPγS）增强，对5'-酸酐取代的ATP类似物减弱，据推测是由磷酸链裂解引发，且被亲和标记物高碘酸盐氧化的ATP（P-ATP）不可逆抑制。我们研究了在对ATPγS和ATP收缩过程中是否发生磷酸化，以及P-ATP对收缩第二阶段的不可逆抑制是否源于其共价掺入。完整组织与[³⁵S]ATPγS（3 mM）孵育后，进行匀浆、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影，³⁵SPO₂(³⁻)（³⁵S）主要掺入一种单一的19至21 kD蛋白质中。掺入是短暂的，且与对ATPγS的短暂收缩时间进程平行（5至10秒达到峰值，然后在60秒内迅速下降）。用P-ATP（10 mM；5分钟）或ANAPP3（10⁻⁴ M）处理在60秒时抑制³⁵S掺入。来自[γ-³²P]ATP（3 mM）的³²PO₄(³⁻)（³²P）也迅速掺入该蛋白质中；然而，³²P的去除比³⁵S的去除更快。[³H]P-ATP掺入几种蛋白质中，但未掺入19至21 kD蛋白质或被光解的ANAPP3标记的蛋白质中。在组胺或去甲肾上腺素（10⁻⁴ - 10⁻³ M）存在下孵育，[³⁵S]ATPγS的³⁵S掺入和[³H]P-ATP的掺入不受影响，而ATP（10⁻² M）降低[³H]P-ATP（10⁻⁵ - 10⁻² M）的掺入。结果支持这样的假设，即对ATP收缩的第二阶段涉及一种涉及胞外激酶的转导性磷酰转移，该激酶被P-ATP抑制。
