Mattsson J P, Skyman C, Palokangas H, Väänänen K H, Keeling D J
Department of Cell Biology, Astra Hässle AB, Molndal, Sweden.
J Bone Miner Res. 1997 May;12(5):753-60. doi: 10.1359/jbmr.1997.12.5.753.
Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.
骨表面的酸化会导致骨吸收，这是由破骨细胞质膜特定区域（称为皱褶膜）中存在的一种液泡型H⁺-ATP酶完成的。在这类多亚基酶中，其结构和功能似乎高度保守。然而，互补DNA的克隆和测序表明，催化结构域中的一个亚基，即B亚基，至少以两种形式存在，B1和B2。几乎仅在肾脏中发现了B1信使核糖核酸，而在包括肾脏在内的所有研究组织中都发现了B2的信使核糖核酸。据推测，B1同工型可能参与靶向质膜。在本研究中，我们使用针对具有B亚基同工型特异性或保守序列的肽段的抗体，对鸡破骨细胞H⁺-ATP酶的B亚基进行了表征。对鸡破骨细胞膜囊泡和部分纯化的鸡破骨细胞H⁺-ATP酶进行了蛋白质免疫印迹分析，并与从牛肾和脑中分离的H⁺-ATP酶的类似分析进行了比较。B1特异性抗体在肾H⁺-ATP酶的免疫印迹上与一条约56kD的多肽发生反应，而在破骨细胞H⁺-ATP酶或破骨细胞膜囊泡制剂上未检测到反应。相反，针对B2特异性序列的抗体在破骨细胞H⁺-ATP酶、肾H⁺-ATP酶和网格蛋白包被囊泡H⁺-ATP酶的免疫印迹上与一条约56kD的肽段发生反应。针对B亚基保守区域的抗体没有提供任何证据表明破骨细胞中存在除B2以外的同工型。使用B2抗体对牛骨切片上的大鼠破骨细胞进行免疫细胞化学分析，结果显示，在活跃吸收的破骨细胞中，沿着面向骨表面的质膜有强烈的极化染色，而不吸收的破骨细胞在整个细胞质中呈弥漫性染色。通过共聚焦显微镜观察，B2染色位于皱褶膜水平，似乎集中在与封闭区相邻的膜的周边区域。我们得出结论，破骨细胞液泡H⁺-ATP酶含有B2同工型，并表明在吸收开始时，该泵从细胞内部转运到靠近封闭区的皱褶膜的一个特殊区域。
