Huang Shu-Gui
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, D-80336 Munich, Germany.
Arch Biochem Biophys. 2003 Dec 1;420(1):40-5. doi: 10.1016/j.abb.2003.07.005.
Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.
对来自仓鼠棕色脂肪组织线粒体的解偶联蛋白-1（UCP1）进行了有限的蛋白水解消化研究。在最佳条件下，胰蛋白酶和糜蛋白酶分别在Lys-292和Phe-102处切割，产生主要产物31 kDa的T1和22 kDa的Ch1。与UCP1一样，T1和Ch1均保持二聚体形式。使用荧光核苷酸衍生物2'-O-丹磺酰鸟苷三磷酸（2'-O-dansyl GTP）表明，T1保留核苷酸结合亲和力（丹磺酰鸟苷三磷酸的K(D)=1 microM），而Ch1不结合核苷酸。先前的动力学结合和H(+)转运研究[《生物化学》35(1996)7846]表明，UCP1与核苷酸及其衍生物在不同程度上形成紧密复合物。核苷酸对胰蛋白酶消化有很强的保护作用，但对糜蛋白酶消化的保护作用较小，因为糜蛋白酶产物Ch1不结合核苷酸。核苷酸及其衍生物在防止胰蛋白酶消化和糜蛋白酶消化方面表现出相同的效力特征，这表明UCP1在核苷酸结合后经历了重大的构象变化，从最初的松散复合物转变为紧密复合物，其中切割位点被掩盖而免受蛋白水解作用。
