Transcript 幻灯片 1
Is Mitochondrial Nitric Oxide Synthase A New Nitric Oxide Synthase? Ciser HSU NO production and signalling ERP spectra of permeabilized mitochondria supplemented with NOS subtrates and inhibitors After 1h of incubation After 8h of incubation NO production by mitochondrial homogenates, submitochondrial particles, and intact mitochondrial ●intact mitochondria ○ homogenate ▲submitochondrial NO production by intact, coupled mitochondrial and toluenepermeabilized mitochondria Now, there are two opinions to identify the enzymatic characterization of mtNOS: 1. mtNOS is nNOS (by Guilivi. et al) 2. mtNOS is the novel type of the NOS (by Lacza. et al) 1. mtNOS is nNOS The electropharesis and immunoreactivity of mtNOS A.SDS-PAGE B. native PAGE C. Western blot I, 8,000 X g pellet II, 150,000 X g supernatant III, NADPH eluate from the 2’,5’-ADP Sepharose 4B column Primary sequence of rat mtNOS Sequence of Tryptic Digests and their Homology to NOSs Amino acid analysis of purified mtNOS Amino acid Asx His Ala Val Leu Glx Pro Met Phe Ser Thr Arg Ileu Lys Gly Experimental % 10.2 5.0 8.3 7.3 6.4 16.9 5.5 0.9 3.2 7.2 3.3 5.9 5.1 7.6 7.1 Theoretical 3.0 6.5 7.4 9.2 12.4 6.0 2.2 4.4 7.4 5.8 5.9 5.0 6.7 7.5 % 10.6 Experimental is the amino acid composition of mtNOS, and Theoretical is the composition of nNOS. From this table, the authors said that the amino acid composition of mtNOS closely matched that of nNOS. Mass spectra of mtNOS acylation products Expected m/z Experimental m/z SESIAFIEESKK 1367.70 1367.48 LRSESIAFIEESK 1508.76 not found SESIAFIEESKK 1447.67 1447.30 LRSESIAFIEESK 1588.76 1588.52 Fragment After phosphatase treatment Before phosphatase treatment Expected m/z was calculated by adding (before phosphatase) 80 units to the in silico tryptic digest of nNOS (aftere phosphatase). 2. mtNOS is the novel type Two disadvantages to prove mtNOS is the isoform of nNOS: 1. Identified both NADPA diaphorase activity and nNOS immunoreactivity in mitochondria from neuronal structures of wild-type, but not in nNOS knockout mice. 2. Failed to show any positive evidence for the presence of a nNOS protein in the preparation. A B 7 mtNOS activity (pmol/mg/30 min) 900 850 800 NOS activity (pmol/mg/30 min) 750 700 650 600 550 500 450 400 6 5 control L-NAME 4 3 2 1 0 control 350 L-NAME nNOS mtNOS 300 In panel A, it shows that mtNOS activity was not inhibited by L-NAME. In panel B, it shows the comparison between nNOS activity and mtNOS activity. 250 200 150 100 50 0 nNOS mtNOS mtNOS is eNOS? mtNOS is nNOS? mtNOS is iNOS? THANKS!