Cunningham H B, Yazaki P J, Domingo R C, Oades K V, Bohlen H, Sabbadini R A, Dahms A S
Department of Chemistry, San Diego State University, California.
Arch Biochem Biophys. 1993 May 15;303(1):32-43. doi: 10.1006/abbi.1993.1252.
Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M(r) 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M(r) 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P1- and P2-purinoceptors. It is also proposed that the ecto-MgATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-MgATPase may modulate the neurotransmitter role of ATP.
纯化的鸡骨骼肌横管（T管，TT）膜制剂含有一种活性很强的钙或镁ATP酶（EC 3.6.1.3），以前认为它是T系统特异性标记酶。镁ATP酶的功能尚未确定，尽管其在连接复合体中的显著活性和浓度支持其在兴奋-收缩循环中可能发挥的作用。镁ATP酶的一个重要成分已被鉴定为一种分子量为85,000的糖蛋白（85k-GP）。针对TT 85k-GP产生的多克隆抗体具有特异性，在免疫印迹上与其他骨骼肌蛋白无交叉反应。利用这种抗85k糖蛋白IgG，我们研究了其他鸡组织以确定85k-GP的组织分布。在砂囊平滑肌、脑、心脏、脾脏和肺组织中发现了分子量为85,000的抗体反应性多肽。进一步纯化脑和平滑肌膜蛋白，并对与85k-GP相关的镁ATP酶活性进行了表征。就一系列激活剂和抑制剂、氨基末端氨基酸序列以及去糖基化的影响而言，脑和平滑肌酶表现出与骨骼肌TT特异性镁ATP酶无法区分的特性。所有三种组织中的酶都被二酰基甘油激酶抑制剂R 59022抑制。在砂囊平滑肌中鉴定出TT镁ATP酶后，就可以利用分离的完整平滑肌细胞研究镁ATP酶的膜拓扑结构。数据支持平滑肌细胞酶的外向定位。尽管脑和骨骼肌酶的定位尚未最终确定，但所有三种酶几乎相同的特性也表明这些酶的活性位点也是外向定位的。这三种酶对调节性脂质的反应性表明，外向镁ATP酶可能作为一个主开关，控制细胞外ATP浓度以及配体与P1和P2嘌呤受体的可及性。还提出外向镁ATP酶可能调节各种组织中细胞外蛋白激酶对ATP的可及性，并且在脑中，外向镁ATP酶可能调节ATP的神经递质作用。
