Bissonnette M, Kuhn D, de Lanerolle P
Department of Medicine, Michael Reese Hospital and Medical Center, Chicago, IL 60616.
Biochem J. 1989 Mar 15;258(3):739-47. doi: 10.1042/bj2580739.
We have partially purified a protein kinase from rat pancreas that phosphorylates two light-chain subunits of pancreatic myosin, a doublet with components of 18 and 20 kDa. This protein kinase was purified approx. 1000-fold by sequential (NH4)2SO4 fractionation, gel filtration, ion-exchange and affinity chromatography on calmodulin-Sepharose 4B. The resultant enzyme preparation is free of cyclic AMP-dependent protein kinase, protein kinase C and calmodulin-dependent type I or II kinase activities. The purified protein kinase is completely dependent on Ca2+ and calmodulin, and phosphorylates a 20 kDa light-chain subunit of intact gizzard myosin, suggesting that it belongs to a class of enzymes known as myosin light-chain kinase (MLCK). The apparent Km values of the putative pancreatic MLCK for ATP (73 microM), gizzard myosin light chains (18 microM) and calmodulin (2 nM) are similar to those reported for MLCKs isolated from smooth muscle, platelet and other sources. The enzyme is half-maximally activated at a free Ca2+ concentration of 2.5 microM. A single component of the affinity-purified kinase reacts with antibodies to turkey gizzard MLCK. The apparent molecular mass of this component is 138 kDa. Immunoprecipitation of a pancreatic homogenate with these antibodies decreases calmodulin-dependent kinase activity for pancreatic myosin by over 85%. The immunoprecipitate contains a single electrophoretic band of 138 kDa. Tryptic phosphopeptide analyses of pancreatic myosin, phosphorylated by either gizzard or pancreatic MLCK, are identical. Thus the enzyme that we have purified from rat pancreas is a MLCK, as judged by (1) absolute dependence on Ca2+ and calmodulin, (2) high affinity for calmodulin, (3) narrow substrate specificity for the light-chain subunit of myosin, and (4) reactivity with antibodies to turkey gizzard MLCK. These studies establish the existence of a pancreatic MLCK which may be responsible for regulating myosin phosphorylation and enzyme secretion in situ.
我们已从大鼠胰腺中部分纯化出一种蛋白激酶，该激酶可使胰腺肌球蛋白的两个轻链亚基磷酸化，这两个轻链亚基是由18 kDa和20 kDa组分构成的双峰。通过在硫酸铵分级沉淀、凝胶过滤、离子交换以及钙调蛋白琼脂糖凝胶4B亲和层析等步骤，这种蛋白激酶被纯化了约1000倍。最终得到的酶制剂不含环磷酸腺苷依赖性蛋白激酶、蛋白激酶C以及钙调蛋白依赖性I型或II型激酶活性。纯化后的蛋白激酶完全依赖于钙离子和钙调蛋白，并且能使完整的砂囊肌球蛋白的一个20 kDa轻链亚基磷酸化，这表明它属于一类被称为肌球蛋白轻链激酶（MLCK）的酶。推测的胰腺MLCK对ATP（73 microM）、砂囊肌球蛋白轻链（18 microM）和钙调蛋白（2 nM）的表观Km值与从平滑肌、血小板和其他来源分离得到的MLCK所报道的值相似。该酶在游离钙离子浓度为2.5 microM时被激活至最大活性的一半。亲和纯化的激酶的单一成分与针对火鸡砂囊MLCK的抗体发生反应。该成分的表观分子量为138 kDa。用这些抗体对胰腺匀浆进行免疫沉淀，可使针对胰腺肌球蛋白的钙调蛋白依赖性激酶活性降低超过85%。免疫沉淀物包含一条138 kDa的单一电泳带。对由砂囊或胰腺MLCK磷酸化的胰腺肌球蛋白进行胰蛋白酶磷酸肽分析，结果是相同的。因此，根据以下几点判断，我们从大鼠胰腺中纯化出的酶是一种MLCK：（1）对钙离子和钙调蛋白的绝对依赖性；（2）对钙调蛋白的高亲和力；（3）对肌球蛋白轻链亚基的狭窄底物特异性；（4）与针对火鸡砂囊MLCK的抗体发生反应。这些研究证实了胰腺MLCK的存在，它可能负责在原位调节肌球蛋白磷酸化和酶分泌。
