Is Mitochondrial Nitric Is Mitochondrial Nitric Oxide Synthase A New Oxide Synthase A New Nitric Oxide Synthase?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 toluene-permeabilized 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-PAGEB. native PAGEC. Western blot
I, 8,000 X g pelletII, 150,000 X g supernatantIII, 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
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.
Amino acid
Asx HisAl
aVa
lLeu Glx
Pro
MetPhe
SerThr 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
%10.
6
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
Mass spectra of mtNOS acylation products
FragmentExpected
m/zExperimental
m/z
After phosphatase treatment
SESIAFIEESKK 1367.70 1367.48
LRSESIAFIEESK 1508.76 not found
Before phosphatase treatment
SESIAFIEESKK 1447.67 1447.30
LRSESIAFIEESK 1588.76 1588.52
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 nNOSimmunoreactivity in mitochondria from neuronal structures of wild-type, but not in nNOS knockout mice.
2. Failed to show any positive evidence for the presence ofa nNOS protein in the preparation.
nNOS mtNOS0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
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900
nNOSmtNOS
B
NO
S a
ctiv
ity (p
mol
/mg/
30 m
in)
control L-NAME0
1
2
3
4
5
6
7
controlL-NAME
A
mtN
OS
act
ivity
(pm
ol/m
g/3
0 m
in)
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.
mtNOS is eNOS?
mtNOS is nNOS?
mtNOS is iNOS?
THANKS!