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Is Mitochondrial Nitric Oxide Synthase A New Nitric Oxide Synthase?

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sylvester-haynes
20 diapositives · 428 Views · Oct 27, 2014

Is Mitochondrial Nitric Oxide Synthase A New Nitric Oxide Synthase?. Ciser HSU. NO production and signalling. After 1h of incubation. After 8h of incubation. ERP spectra of permeabilized mitochondria supplemented with NOS subtrates and inhibitors.

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Is Mitochondrial Nitric Oxide Synthase A New Nitric Oxide Synthase?
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  1. Is Mitochondrial Nitric Oxide Synthase A New Nitric Oxide Synthase? Ciser HSU

  2. NO production and signalling

  3. After 1h of incubation After 8h of incubation ERP spectra of permeabilized mitochondria supplemented with NOS subtrates and inhibitors

  4. NO production by mitochondrial homogenates, submitochondrial particles, and intact mitochondrial ●intact mitochondria ○ homogenate ▲submitochondrial

  5. NO production by intact, coupled mitochondrial and toluene-permeabilized mitochondria

  6. 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)

  7. 1. mtNOS is nNOS

  8. 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

  9. Primary sequence of rat mtNOS

  10. Sequence of Tryptic Digests and their Homology to NOSs

  11. Amino acid analysis of purified mtNOS 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.

  12. Mass spectra of mtNOS acylation products

  13. Expected m/z was calculated by adding (before phosphatase) 80 units to the in silico tryptic digest of nNOS (aftere phosphatase).

  14. 2. mtNOS is the novel type

  15. 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.

  16. 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.

  17. mtNOS is eNOS?

  18. mtNOS is nNOS?

  19. mtNOS is iNOS?

  20. THANKS!