Division of Endocrinology, Department of Medicine, University of Arizona, Tucson, AZ, USA; Center for Disparities in Diabetes, Obesity, and Metabolism, University of Arizona Health Science, Tucson, AZ, USA.
Division of Endocrinology, Department of Medicine, University of Arizona, Tucson, AZ, USA.
Anal Biochem. 2021 Oct 1;630:114319. doi: 10.1016/j.ab.2021.114319. Epub 2021 Jul 29.
Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmol/g wet weight muscle and 0.64 ± 0.05 nmol/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.
证据表明，人腺嘌呤核苷酸转位酶 1（ANT1）赖氨酸 23（Lys23）的乙酰化降低了 ADP 的结合。Lys23 有助于促进这种相互作用的正电荷。本研究采用一种新方法，使用少量人体骨骼肌活检组织，对 ANT1 的丰度和乙酰化进行了表征。从健康志愿者的股外侧肌针吸活检中制备了整块肌肉或同一组织线粒体的裂解物。裂解蛋白在凝胶上进行分离，包含 ANT1（周围 30Kd）的部分被切除，用胰蛋白酶消化，加入标记的未乙酰化和乙酰化合成标准肽，并通过质谱分析。对肽质量 10 倍范围内的离子强度进行自然对数转换以线性化数据。ANT1 丰度和 Lys23 乙酰化的变异系数范围为 7%至 30%。在三名志愿者中，ANT1 含量为 8.36±0.33 nmol/g 湿重肌肉和 0.64±0.05 nmol/mg 线粒体，因此线粒体含量为 13.3±2.4 mg 线粒体/克肌肉。整个肌肉和线粒体中 Lys23 的乙酰化平均值分别为 14.3±4.2%和 4.87±1.84%。该测定法使得评估乙酰化对人肌肉中 ANT1 功能的影响成为可能。
