Katoh N, Raynor R L, Wise B C, Schatzman R C, Turner R S, Helfman D M, Fain J N, Kuo J F
Biochem J. 1982 Jan 15;202(1):217-24. doi: 10.1042/bj2020217.
Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.
研究了两亲性多肽蜂毒明肽对多种蛋白激酶的作用。发现蜂毒明肽抑制新鉴定出的磷脂敏感的Ca2 + 依赖性蛋白激酶（来自心脏、大脑、脾脏和中性粒细胞）以及心肌肌球蛋白轻链激酶，后者是一种钙调蛋白敏感的Ca2 + 依赖性酶。相比之下，蜂毒明肽对心肌环磷酸腺苷依赖性和环磷酸鸟苷依赖性蛋白激酶的全酶或前者的催化亚基几乎没有影响。动力学分析表明，蜂毒明肽对ATP而言非竞争性抑制磷脂敏感的Ca2 + 依赖性蛋白激酶（Ki = 1.3 μM）；尽管表现出复杂的动力学，但磷脂酰丝氨酸（一种磷脂辅因子）可克服其对该酶的抑制作用，而蛋白底物（组蛋白H1）或Ca2 + 则不能。另一方面，蜂毒明肽对ATP（Ki = 1.4 μM）或Ca2 + （Ki = 1.9 μM）而言非竞争性抑制肌球蛋白轻链激酶，对钙调蛋白而言竞争性抑制（Ki = 0.08 μM）；尽管表现出复杂的动力学，但肌球蛋白轻链（底物蛋白）可逆转其对该酶的抑制作用。目前的研究结果表明，在Ca2 + 依赖性蛋白激酶上存在功能重要的疏水或亲水位点，而在环核苷酸依赖性蛋白激酶类别上则不存在，蜂毒明肽可与这些位点相互作用。此外，动力学数据表明，蜂毒明肽通过与酶上与钙调蛋白结合位点相同或相邻的位点相互作用来抑制肌球蛋白轻链激酶，从而干扰活性酶 - 钙调蛋白 - Ca2 + 复合物的形成。
