2P102 Analyses of caspase-independent apoptosis caused by the expression of a candidate human tumor suppression gene, 101F6(03. Membrane proteins,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Abstract

We have developed a new method to analyze structure of substrate, which is bound to enzyme, by detecting its resonance Raman (RR) spectra. We have applied the new methods to indoleamine 2,3-dioxygenase (IDO) which catalyses direct incorporation of two oxygen atoms into tryptophan (Trp) and whose reaction mechanism is not fully understood due to the lack of structural information. We have successfully obtained UVRR spectra of the bound-substrate in IDO and found some spectral change in Trp bands by binding to IDO; 10 cm -1 down-shift of W3 mode and 10 cm -1 up-shift of W18 mode. These results clearly show that the bound Trp to IDO as substrate has a specific conformation as compared to Trp in solution. We developed a real-time analysis system for biological membranes in the whole-cell recording configuration: two micro-fabricated flow channels connected with a micro aperture (2 μm) where a large-sized liposome is captured. To validate the system, we assessed the proton pump activity of complex I (CI) of the respiratory chain as follows. First, a large-sized protoplast from E. coli (10 μm) was trapped and perforated at the aperture with an electrical pulse. Then, one of the channels was infused with NADH, a substrate for CI. As NADH diffused into the E. coli via the aperture, pH fluorescent indicator started to become bright at another channel. It suggests proton was transferred across the membrane by CI, as observed in bulk biochemical assays. 2P099 ブタ心臓由来ミトコンドリア調製法の確立と活性測定Establishment of a new process for preparation of porcine heart mitochondria, and their activity measurementsWe have been developing a novel real time monitoring device for membrane protein activities. As a membrane sample, we chose the mitochondrial inner membrane (IM) prepared from porcine hearts. Porcine heart, which is not required any assessment of BSE, is extremely fresher than bovine heart, because the hearts can be obtained immediately after the animals are slaughtered.We confirmed the proton pumping activity of the IM proteins in bulk experiment by using a fluorescent pH indicator. The respiratory complexes were selectively activated depending on various substrates, which is consistent with bovine IM assay. By fusing the IM, we also successfully prepared a giant vesicle (>10 μm), which will allow us to image membrane protein activities using our device. Ras is one of small G-protein known as a molecular switch mediating cellular signalling. We performed basic study to control the function of Ras reversibly using photochromic molecules, 4-phenylazophenyl maleimide (PAM) upon visible (VIS) light and ultra-violet (UV) light irradiation. We have prepared the Ras mutants which have a single cysteine at functional sites and modified with PAM stoichiometrically. The GTPase activities of PAM-Ras were reversibly altered upon VIS and UV light irradiations. In order to monitor the effect on GTPase kinetic pathway by the photoisomerization of PAM, We synthesized a novel fluorescent GTP analogue, NBD-GTP. The Kinetic studies suggested that initial ...