Nakanishi S, Yamada K, Kase H, Nakamura S, Nonomura Y
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.
J Biol Chem. 1988 May 5;263(13):6215-9.
Effects of K-252a, (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, purified from the culture broth of Nocardiopsis sp., on the activity of myosin light chain kinase were investigated. 1) K-252a (1 x 10(-5) M) affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca2+-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca2+-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10(-6) M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10(-4) M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lower in the presence of 100 microM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [gama-32P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP (Ki = 20 nM). These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase. The direct action of the compound on the enzyme would explain the multivarious inhibition of myosin ATPase, of superprecipitation, and of the contractile response of smooth muscle.
研究了从诺卡氏菌属（Nocardiopsis sp.）培养液中纯化得到的K-252a，即(8R*, 9S*, 11S*)-(-)-9-羟基-9-甲氧基羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b,11a-三氮杂二苯并[a,g]环辛[cde]茚-1-酮，对肌球蛋白轻链激酶活性的影响。1）K-252a（1×10⁻⁵ M）对鸡砂囊肌球蛋白-B（天然肌动球蛋白）的三个特征性质有相似程度的影响：ATP酶的Ca²⁺依赖性活性、超沉淀以及肌球蛋白轻链的磷酸化。2）K-252a抑制纯化的肌球蛋白轻链激酶以及通过戊二醛交联肌球蛋白轻链激酶和钙调蛋白构建的一种不依赖Ca²⁺的酶形式的活性。3×10⁻⁶ M K-252a对纯化酶和交联复合物的抑制程度分别为对照活性的69%和48%。钙调蛋白拮抗剂氯丙嗪（3×10⁻⁴ M）抑制天然酶，但不抑制交联酶。这些结果表明，K-252a通过与酶直接相互作用抑制肌球蛋白轻链激酶，而氯丙嗪通过与钙调蛋白相互作用抑制酶的激活。3）K-252a对交联激酶的抑制受磷酸供体ATP浓度的影响。在100 μM ATP存在下导致50%抑制的浓度比在2 mM ATP存在下低两个数量级。4）使用[γ-³²P]ATP进行的动力学分析表明，K-252a的抑制模式相对于ATP是竞争性的（Ki = 20 nM）。这些结果表明，K-252a在肌球蛋白轻链激酶的ATP结合结构域相互作用。该化合物对酶的直接作用可以解释对肌球蛋白ATP酶、超沉淀和平滑肌收缩反应的多种抑制作用。
