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

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200

250

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350

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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!