To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.

To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues.

Previously, several studies have demonstrated that autophosphorylation of the C-terminal tyrosine residues (Tyr 1316 and Tyr 1322 ) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C-terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD-Y2F) the two C-terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD-Y2F kinase (Tyr 1146 , Tyr 1150 , and Tyr 1151 ) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the K m values for exogenous substrates. However, the mutation in IRKD-Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD-Y2F led to an increase in the apparent K m values for ATP, suggesting a cross-talk of the C-terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C-terminal tyrosines. These data suggest a regulatory role of the two C-terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor. ß