Role of nonmuscle myosins in cardiac development

人类非肌肉肌球蛋白重链（NMHC）II-A中保守氨基酸Arg 702突变为Cys（R702 C）导致肾脏、血小板、白细胞、内耳和眼睛的异常，并且N93 K突变导致血小板和白细胞的缺陷。小鼠中NMHC II-B中同源残基（R709 C）的突变导致心脏和大脑的缺陷。为了更好地理解导致这些缺陷的机制，我们使用杆状病毒表达系统表达了具有R709 C突变的非肌肉HMM II-B以及具有N97 K突变的第二同源HMM。我们比较了肌动蛋白激活的MgATP酶活性和结果，从体外肌动蛋白滑丝测定与野生型非肌肉HMM II-B获得的值。磷酸化的野生型HMM II-B被肌动蛋白激活（Vmax = 0.17+0.04s-1）比N97 K突变体激活（Vmax = 0.12+0.04s-1）稍多，但比R709 C突变体HMM激活（Vmax = 0.05+0.003s-1）多得多。N97 K突变HMM移动肌动蛋白丝的速率降低（野生型，0.18 ± 0.03 μ m/s vs 0.05 ± 0.005 μ m/s），但R709 C突变HMM完全不能移位肌动蛋白丝，但确实可逆地结合肌动蛋白丝。在一组单独的实验中，我们表征了杆状病毒表达的HMM的非插入非肌肉HMM II-C和HMM II-C与ATP结合区附近的8个氨基酸插入。与平滑肌HMM的结果相似，插入的NMHC II-C HMM具有更高的肌动蛋白激活的MgATP酶活性（Vmax = 0.85 ± 0.07s-1 vs 0.29 ± 0.04s-1）;并且与未插入的非肌肉HMM II-C（0.03 ± 0.005 um/s）相比，肌动蛋白丝移位更快（0.08 ± 0.003 um/s）。这些值不仅有助于表征两种肌球蛋白的生物学特性，而且有助于理解我们正在产生的突变小鼠的表型。

Mutation of the conserved amino acid Arg 702 to Cys (R702C) in nonmuscle myosin heavy chain (NMHC) II-A in humans leads to abnormalities in the kidney, platelets, leukocytes, inner ear and eye and mutation of N93K results in defects in the platelets and leukocytes. Mutation of the homologous residue (R709C) in NMHC II-B in mice results in defects in the heart and brain. To better understand the mechanism leading to these defects, we expressed nonmuscle HMM II-B with the R709C mutation as well as a second homologous HMM with the N97K mutation, using the baculovirus expression system. We compared the actin-activated MgATPase activity and results from the in vitro actin gliding filament assay with values obtained for wild-type nonmuscle HMM II-B. Phosphorylated wild-type HMM II-B was activated by actin (Vmax = 0.17+0.04s-1) somewhat more than the N97K mutant (Vmax = 0.12+0.04s-1), but considerably more than the R709C mutant HMM (Vmax = 0.05+0.003s-1). The N97K mutant HMM moved actin filaments at a decreased rate (wild-type, 0.18+0.03 um/s vs 0.05+0.005 um/s), but the R709C mutant HMM could not translocate actin filaments at all, but did bind reversibly to actin filaments. In a separate set of experiments, we characterized baculovirus-expressed HMM for both non-inserted nonmuscle HMM II-C and HMM II-C with an 8-amino acid insert near the ATP binding region. Similar to the findings for smooth muscle HMM, inserted NMHC II-C HMM had a higher actin-activated MgATPase activity (Vmax = 0.85+0.07s-1 vs 0.29+0.04s-1); and translocated actin filaments more rapidly (0.08+0.003 um/s) than non-inserted nonmuscle HMM II-C (0.03+0.005 um/s). These values should not only help in characterization of the biological properties of the two myosins, but also help in understanding the phenotypes of the mutant mice we are generating.