Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.

中文翻译：

---

神经元NO合酶α-和β-亚型在小鼠骨骼肌中的表达

关于骨骼肌中神经元一氧化氮合酶（nNOS）的一级结构的知识仍然存在争议，需要进一步澄清。为了阐明 nNOS 亚型在 mRNA 和蛋白质水平上的表达模式，对 C57/BL6 小鼠的骨骼肌进行了系统逆转录 (RT)-PCR 和定性免疫印迹分析表位作图。 nNOS 亚型形成聚集体的能力通过天然低温聚丙烯酰胺电泳 (LT-PAGE) 进行表征。分子分析的重点是股直肌 (RF) 肌肉，这是一种 nNOS α- 和 β- 亚型比例接近平衡的骨骼肌。来自 RF 肌肉的 RT-PCR 扩增显示独有的外显子 1d mRNA 表达，无论有或没有外显子 μ。表位作图证明了 nNOS 剪接变体 α/μ、α/non-μ、β/μ 和 β/non-μ 的同时表达。此外，免疫印迹表明 nNOS α- 和 β-亚型之间的转变位于外显子 3 内。在 LT-PAGE 中，在 RF 肌肉和胫骨前肌匀浆中检测到三种蛋白质 nNOS 相关聚集体：包含 nNOS α-同种型的 320 kDa 条带，而 250 和 300 kDa 条带由 nNOS β-同种型组成，形成同二聚体或异二聚体非 nN​​OS 蛋白。