Wieben E D
J Cell Biol. 1981 Mar;88(3):492-8. doi: 10.1083/jcb.88.3.492.
Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell-free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.
当在源自兔网织红细胞的无细胞系统中进行翻译时，小鼠睾丸总RNA可指导乳酸脱氢酶-X（LDH-X）精子特异性C亚基的合成。新合成的C亚基通过用针对该同工酶的抗体进行免疫沉淀来分离，并通过在SDS聚丙烯酰胺凝胶上进行电泳进行定量。掺入酶亚基的放射性量与添加到翻译系统中的睾丸RNA量直接成正比，从而提供了一种评估相对LDH-X mRNA活性的灵敏且可靠的方法。蔗糖梯度离心和寡聚（dT）-纤维素层析相结合，使得LDH-X mRNA相对于睾丸总细胞质RNA得到了23倍的纯化。对发育中睾丸的LDH-X mRNA活性分析表明，功能性LDH-X mRNA活性的出现与产后14天LDH-X催化活性的出现相一致。通过离心淘析制备的分离睾丸细胞群体中LDH-X mRNA水平的测量表明，在富含粗线期精母细胞和圆形精子细胞的组分中，LDH-X mRNA占总功能性mRNA活性的0.17 - 0.18%，但在长形精子细胞的翻译产物中仅占0.09 - 0.10%。
