Baystate Medical Center, Tufts University School of Medicine, Springfield, Massachusetts 01199, USA.
Endocrinology. 2011 Aug;152(8):3082-92. doi: 10.1210/en.2011-0137. Epub 2011 May 31.
RT-PCR shows that mouse skeletal muscle contains type-2 iodothyronine deiodinase (D2) mRNA. However, the D2 activity has been hard to measure. Except for newborn mice, muscle homogenates have no detectable activity. However, we have reported D2 activity in mouse muscle microsomes. As the mRNA, activity is higher in slow- than in fast-twitch muscle. We addressed here the major problems in measuring D2 activity in muscle by: homogenizing muscle in high salt to improve yield of membranous structures; separating postmitochondrial supernatant between 38 and 50% sucrose, to eliminate lighter membranes lacking D2; washing these with 0.1 M Na(2)CO(3) to eliminate additional contaminating proteins; pretreating all buffers with Chelex, to eliminate catalytic metals; and eliminating the EDTA from the assay, as this can bind iron that enhances dithiothreitol oxidation and promotes peroxidation reactions. Maximum velocity of T(3) generation by postgradient microsomes from red muscles was approximately 1100 fmol/(h · mg) protein with a Michaelis-Menten constant for T(4) of 1.5 nM. D2-specific activity of Na(2)CO(3)-washed microsomes was 6-10 times higher. The enrichment in D2 activity increased in parallel with the capacity of microsomes to load (sarco/endoplasmic reticulum Ca(2+)-ATPase) and bind Ca(2+) (calsequestrin), indicating that D2 resides in the inner sarcoplasmic reticulum, close to the nuclei. The presence of D3 in the sarcolemma suggests that the most of D2-generated T(3) acts locally. Estimates from maximum velocity, Michaelis-Menten constant, and muscle T(4) content suggest that mouse red, type-1, aerobic mouse muscle fibers can generate physiologically relevant amounts of T(3) and, further, that muscle D2 plays an important role in thyroid hormone-dependent muscle thermogenesis.
实时聚合酶链反应显示，鼠骨骼肌含有Ⅱ型碘甲状腺原氨酸脱碘酶（D2）mRNA。然而，D2 活性很难测量。除了新生鼠，肌肉匀浆没有可检测的活性。然而，我们已经报道了鼠肌肉微粒体中的 D2 活性。因为 mRNA，活性在慢肌比在快肌中更高。我们在这里通过以下方法解决了在肌肉中测量 D2 活性的主要问题：在高盐中匀浆肌肉以提高膜结构的产量；在 38%和 50%蔗糖之间分离线粒体后上清液，以消除缺乏 D2 的较轻的膜；用 0.1 M Na2CO3 洗涤这些膜，以消除额外的污染蛋白；用 Chelex 预处理所有缓冲液，以消除催化金属；并从测定中去除 EDTA，因为它可以结合铁，铁增强二硫苏糖醇氧化并促进过氧化物反应。红肌后梯度微粒体生成 T3 的最大速度约为 1100 fmol/（h·mg）蛋白，T4 的米氏常数为 1.5 nM。用 Na2CO3 洗涤的微粒体的 D2 比活度高 6-10 倍。D2 活性的富集与微粒体装载（肌浆/内质网 Ca2+-ATP 酶）和结合 Ca2+（钙结合蛋白）的能力平行增加，表明 D2 位于肌浆内质网内，靠近核。肌膜中的 D3 的存在表明，大部分由 D2 生成的 T3 局部作用。最大速度、米氏常数和肌肉 T4 含量的估计表明，鼠红肌、I 型、需氧鼠肌纤维可以产生生理相关量的 T3，而且，肌肉 D2 在甲状腺激素依赖性肌肉产热中发挥重要作用。
