影响人血小板裂解物中鸟苷酸环化酶活性的因素。

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

作者信息

Adams A F, Haslam R J

出版信息

Biochem J. 1978 Jul 15;174(1):23-35. doi: 10.1042/bj1740023.

DOI:10.1042/bj1740023

PMID:29607

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1185881/

Abstract

Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

摘要

在最佳离子条件下（4 mM - MnCl₂），新鲜血小板裂解物中鸟苷酸环化酶的比活性在30℃时约为每毫克蛋白质每20分钟形成10 nmol环鸟苷酸。在10 mM - MgCl₂存在下活性为最佳活性的15%，而在4 mM - CaCl₂存在下活性可忽略不计。当[MnCl₂]≤[GPT]时，观察到MnCl₂与MgCl₂或CaCl₂之间存在协同作用。2. 在含有大量血小板裂解物的测定中获得的比活性低于最佳值，这是由于存在可通过超滤去除的抑制因子。腺嘌呤核苷酸占抑制活性的不到50%。3. 将裂解物在30℃预孵育1小时可使血小板鸟苷酸环化酶的比活性提高约2倍。4. 十二烷基聚氧乙烯醚PX（1%，w/v）在预孵育前可使鸟苷酸环化酶活性提高3 - 5倍，预孵育后提高约2倍。Triton X - 100的效果要差得多。5. 二硫苏糖醇抑制未处理、预孵育和经十二烷基聚氧乙烯醚PX处理的裂解物的鸟苷酸环化酶活性，并阻止预孵育激活，前提是预先加入。6. 油酸可使鸟苷酸环化酶活性提高3 - 4倍，花生四烯酸提高2 - 3倍，而棕榈酸几乎无活性。用吲哚美辛预处理裂解物并不抑制花生四烯酸的这种作用。油酸和花生四烯酸在预孵育的裂解物中可显著刺激鸟苷酸环化酶，但在经十二烷基聚氧乙烯醚PX处理的裂解物中抑制该酶。7. 叠氮化钠（10 mM）可使鸟苷酸环化酶活性提高多达七倍；这种作用具有时间和温度依赖性。叠氮化钠在经十二烷基聚氧乙烯醚PX处理的裂解物中不会进一步激活该酶。8. 结果表明，预孵育、十二烷基聚氧乙烯醚PX、脂肪酸和叠氮化钠通过不同机制激活血小板鸟苷酸环化酶。9. 血小板颗粒部分无论有无十二烷基聚氧乙烯醚PX均未检测到鸟苷酸环化酶活性，其活性无法用污染的可溶性酶来解释，这表明生理聚集剂可能通过中间因子的作用增加完整血小板中的环鸟苷酸。本文所述酶的激活和抑制状态可能与这些因子的作用有关。