Missiaen L, Wuytack F, Casteels R
Department of Obstetrics and Gynaecology, Katholieke Universiteit, Leuven, Belgium.
Biochem J. 1988 Mar 1;250(2):579-88. doi: 10.1042/bj2500579.
The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5') pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
通过连续NADH偶联酶法测定的表观Mg2+激活的ATP酶活性，在从怀孕或经17β-雌二醇预处理的大鼠子宫肌层平滑肌、牛主动脉、豚鼠结肠带、兔耳动脉和猪胃窦获得的多种微粒体制剂中进行了研究。结果表明，这种ATP酶测定法容易受到许多与组织相关的假象的影响。子宫肌层、主动脉和结肠带微粒体（微粒体组分）中的表观Mg2+-ATP酶活性在测定过程中呈非线性下降。其初始高活性在15 - 60分钟内逐渐降低，这取决于平滑肌的类型，最终达到恒定值。这种下降取决于ATP的存在，并且可以被伴刀豆球蛋白A部分阻止。兔耳动脉微粒体中的非线性有限。胃窦微粒体中的表观Mg2+-ATP酶活性实际上随时间增加，尽管增加缓慢。主动脉微粒体，尤其是怀孕大鼠子宫肌层和结肠带微粒体在冰上储存数小时后，其被ATP抑制的Mg2+-ATP酶活性成分会逐渐受到抑制。胃窦微粒体中的Mg2+-ATP酶活性保持恒定。NADH氧化酶活性占胃平滑肌微粒体中Mg2+-ATP酶活性的10%。该组织中Mg2+-ATP酶活性最初的表观非线性是由于鱼藤酮敏感的NADH氧化酶活性随时间下降所致。从腺苷酸激酶抑制剂P1,P5-二（腺苷-5′）五磷酸的作用推断，腺苷酸激酶活性分别占胃、子宫肌层和主动脉微粒体中Mg2+-ATP酶活性的45.0%、35.0%和31.0%。在兔耳动脉和结肠带微粒体中未检测到腺苷酸激酶活性。当胃平滑肌微粒体在蔗糖梯度上分离时，腺苷酸激酶和NADH氧化酶对Mg2+-ATP酶活性的贡献在高密度组分中最为明显。部分NADH氧化酶活性和Mg2+-ATP酶活性，以及大部分腺苷酸激酶活性，在224000 gmax离心30分钟后不会沉淀，因此可能以可溶性酶的形式存在。（摘要截于400字）
