Westhoff D, Kamp G
Institut für Zoophysiologie, Westfälische Wilhelms-Universität Münster, Germany.
J Cell Sci. 1997 Aug;110 ( Pt 15):1821-9. doi: 10.1242/jcs.110.15.1821.
Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3-phosphate dehydrogenase to be a hexamer. Rabbit polyclonal antibodies raised to purified glyceraldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3-phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label. During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis. The inverse compartmentation of the glycolytic enzyme and mitochondria in the mammalian sperm flagella suggests that ATP-production in the principal piece mainly occurs by glycolysis and in the midpiece by respiration.
有证据表明，糖酵解酶3-磷酸甘油醛脱氢酶与纤维鞘共价连接。纤维鞘是哺乳动物精子的典型结构，围绕着鞭毛主段的轴丝。通过离心使细胞解体后，超过90%的公猪精子3-磷酸甘油醛脱氢酶活性沉淀下来。去污剂、不同盐浓度或用胰凝乳蛋白酶短期孵育都不能使该酶溶解，而用胰蛋白酶或弹性蛋白酶消化则可以。用胰蛋白酶短期孵育（15分钟）甚至会导致3-磷酸甘油醛脱氢酶的激活。从凝胶电泳SDS-PAGE和天然梯度PAGE判断，在苯基琼脂糖上纯化得到了均一的3-磷酸甘油醛脱氢酶。分子量分别为41.5和238 kDa，表明天然的3-磷酸甘油醛脱氢酶是六聚体。针对纯化的3-磷酸甘油醛脱氢酶产生的兔多克隆抗体对哺乳动物精子的3-磷酸甘油醛脱氢酶具有高度特异性，而公猪精子的其他蛋白质或肌肉中的3-磷酸甘油醛脱氢酶则未被标记。在包埋后程序中进行的免疫金染色揭示了3-磷酸甘油醛脱氢酶在公猪、公牛、大鼠、种马和人类精子中沿纤维鞘的定位。其他结构如细胞膜、致密纤维、轴丝或线粒体没有标记。在精子成熟过程中，精子中段的大部分细胞质在通过附睾时作为液滴被去除。在未成熟的公猪精子和液滴中这种细胞质的标记表明，3-磷酸甘油醛脱氢酶在附睾精子成熟过程中从中段完全去除。哺乳动物精子鞭毛中糖酵解酶和线粒体的反向区室化表明，主段中的ATP产生主要通过糖酵解，中段则通过呼吸作用。
