Morita Masaya, Takemura Akihiro, Nakajima Ayako, Okuno Makoto
Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, Nishihara, Okinawa, Japan.
Cell Motil Cytoskeleton. 2006 Aug;63(8):459-70. doi: 10.1002/cm.20137.
Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.
在浓度超过10(-6) M的Ca(2+)存在下，去膜的广盐性罗非鱼莫桑比克罗非鱼精子被重新激活。当Ca(2+)浓度从10(-6) M增加到10(-5) M时，运动特征发生变化。尽管搏动频率没有增加，但鞭毛搏动的剪切角和波幅增加，这表明轴丝中微管的滑动速度（代表动力蛋白活性）随着Ca(2+)的增加而上升。因此，由于动力蛋白活性增强，Ca(2+)可能与鞭毛蛋白结合以激活鞭毛运动。通过(45)Ca覆盖分析和免疫方法检测到一种Ca(2+)结合蛋白（分子量18 kDa，pI 4.0），即钙调蛋白（CaM）。CaM拮抗剂W-7抑制了重新激活率和游泳速度，这表明18 kDa的Ca(2+)结合蛋白是CaM，并且CaM调节鞭毛运动。通过免疫方法在轴丝低离子提取物部分和轴丝残余物中均检测到CaMKIV为单一的48 kDa条带，这表明CaMKIV结合在轴丝的不同位置。CaMKIV有可能以Ca(2+)/CaM依赖的方式使轴丝蛋白磷酸化，从而调节动力蛋白活性。轴丝中的(32)P摄取表明，48、75、120、200、250、380和400 kDa的蛋白以Ca(2+)/CaM激酶依赖的方式被磷酸化。在10(-5) M Ca(2+)存在下，380 kDa的蛋白被磷酸化。Ca(2+)的增加可能诱导Ca(2+)/CaM激酶依赖的调节，包括蛋白磷酸化，以激活/调节鞭毛轴丝中动力蛋白的活性。
