0022-3565/9&2761-0030$03.

0O/O
THE Jotmxu. OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 276, No. 1
Copyright 0 1996 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 276:30-33, 1996

Determination of Km for Oxygen of Nitric Oxide Synthase

Isoforms1

APPAVOO RENGASAMY and ROGER A. JOHNS

Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville, Virginia
Accepted for publication September 1 , 1995

ABSTRACT
Biosynthesis of nitric oxide
requires L-arginine(NO) and molec- cultured bovine aortic endothelial cells and RAW 264.7 macro-
ular oxygen. Although the apparent
Km values for L-arginine of phages. The apparent Km values for oxygen were 23.2 ± 2.8,
NO synthase isoforms have been
reported, the apparent Km 7.7 ± 1 .6 and 6.3 ± 0.9 M for the brain, endothelial and
values for oxygen are unknown. Low oxygen tension has been macrophage NO synthases, respectively. This suggests that
shown to attenuate NO synthase activity and NO-dependent pathophysiological conditions involving a decrease in tissue
vascular relaxation. We investigated the effect of different con- oxygen concentration may attenuate NO production.
centrations of oxygen on NO synthase activity of bovine brain,

NO is a ubiquitous cellular messenger implicated in di- part due to a decrease in NO production caused by depletion of
verse cellular functions (Griffith and Stuehr, 1995; Nathan, oxygen. Although the Km values for L-arginine and NADPH of
1992). NO is synthesized by the oxidation of a guanidino NO synthases are well characterized, the Km value for oxygen
nitrogen atom of L-argimne by NO synthase (Griffith and has not been reported. In this study, we investigated the effect
Stuehr, 1995; Nathan, 1992). It has been shown that the of different concentrations of oxygen on isolated NO synthase
oxygen atom in NO and citrulline is derived from molecular activity ofbovine brain, cultured bovine aortic endothelial cells
oxygen (Kwon et at. , 1990; Leone et at. , 1991; Marletta, 1993). and RAW 264.7 macrophages.
Initially, L-arginine is oxidized to N”-hydroxy-L-arginine,
which utilizes one
equivalent of oxygen and NADPH. Addi-
tional oxygen NADPH
and are required for the subsequent Materials and Methods
oxidation ofN”-hydroxy-L-arginine to NO and citrulline. The Preparation ofNO syntheses. Crude bovine brain NO synthase
mechanisms of NO synthesis by constitutive and inducible was prepared as described previously (Rengasamy and Johns, 1991).
isoforms of NO synthase appear to involve similar synthetic Endothelial NO synthase was prepared from cultured bovine aortic
pathways (Leone et at. , 1991). endothelial cells as reported (Pollock et al. , 1991). The enzyme
Before the discovery of EDRFINO, De Mey and Vanhoutte was obtained by solubilization of the particulate fraction with 3-[(3-
(1978, 1980) observed inhibition by anoxia of ACh-induced cholamidopropyl)-dimethylammoniol-1-propanesulfonate (CHAPS).
endothelium-dependent relaxation in canine femoral arterial Macrophage NO synthase was isolated from cultured W 264.7
rings. Subsequent studies confirmed this finding in rabbit tho- macrophages exposed to 1 .tg/ml lipopolysaccharide for 24 hr. The
racic aortic rings (Furchgott and Zawadzki, 1980; Furchgott et cells were homogenized and centrifuged at 25,000 X g for 1 hr. The
at., 1981). Studies from our laboratory (Johns et at., 1989) and supernatant was used as the enzyme source.
Determination of NO synthase activity. NO synthase activity
several others (Rodman et at. , 1990; Warren et al. , 1989) sug- was determined by measuring the conversion of L-[3H]arginine to
gest that hypoxia causes attenuation of the EDRF/NO compo- L-[3H]citrulline. The brain NO synthase reaction was performed in a
nent ofendothelium-dependent vascular relaxation. A previous 300-il volume mixture containing 50 mM Tris-HC1, pH 7.4, 1 mM
study from our laboratory showed that the activity of isolated EDTA, 1.25 mM CaCl2, 1 mM dithiothreitol, 1 mM NADPH, 100 nM
brain NO synthase was attenuated by hypoxic conditions calmodulin, 20 M tetrahydrobiopterin and 25 .il enzyme extract at
(R.engasamy and Johns, 1991). This suggested that hypoxia- 37#{176}C.
The activity of endothelial NO synthase
fol- was determined
induced attenuation of vascular relaxation may be at least in lowing the method described for the brain NO synthase with 0.1 mM
EDTA, 0.1 mM EGTA and 2 mM CaCl2 instead of 1 mM EDTA and
1.25 mM CaCl2. The macrophage NO synthase reaction mixture was
Received for publication June 6, 1995. the same as the endothelial NO synthase assay mixture without
This study
1 was supported by National Institutes of Health Grants ROl
GM 49111 and ROl HL39706 (R.A.J.) and by a Grant-in-Aid from the Amer- exogenous calcium or calmodulin. The reaction was terminated at 3
ican Heart Association-Virginia Affiliate (A.R.). mm, and L-[3H]citrulline production was measured as described pre-

ABBREVIATIONS: NO, nitric oxide; P, inorganic phosphate; EDRF, endothelium-derived relaxing factor.

30
1996 Km Value for Oxygen of NO Synthases 31

viously (Rengasamy and Johns, 1991). To determine the Km value for top of which was covered with a styrofoam block. The oxygen elec-
oxygen, we measured NO synthase activity in duplicate for each trode wire, the solution injection tubing and the gas inlet and outlet
concentration of oxygen in each experiment and used the average. tubings passed through the styrofoam block. Measurement of NO
Each experiment was repeated at least three times. synthase activity at different concentrations of oxygen was per-
Determination of oxygen concentration. Gas mixtures con- formed as follows: The vial containing the reaction mixture without
taming oxygen and nitrogen in different ratios were passed through the enzyme was placed in the thermostatted block, stirred and equil-
the reaction mixture to yield a desired concentration of oxygen. The ibrated with varying concentrations ofoxygen by passing humidified
oxygen concentration of the reaction mixture was continuously mea- gas mixtures at a flow rate of 40 rnL/min. After 10 mm, the enzyme
sured using a Clark-type polarographic electrode (Model 125/05 elec- (30 j.il) that had been pre-equilibrated with the respective gas mix-
trode, Instech Laboratories, Inc., Plymouth Meeting, PA) connected ture and prewarmed at 37#{176}Cfor 5 mm was injected into the vial. The
to a YSI Model 5300 Biological Oxygen Monitor (Yellow Springs oxygen concentration in the reaction mixture was continuously re-
Instruments Co., Inc., Yellow Springs, OH). The signal from the corded. The reaction was stopped at 3 mm by injecting 100 j.il ice-cold
oxygen monitor was recorded continuously using a chart recorder. stopping solution containing 120 mM HEPES and 10 mM EDTA. The
Experimental set-up. NO synthase activity and oxygen concen- vial was removed from the thermostated block, and the reaction
tration were measured in each reaction mixture in a custom-de- mixture was transferred to a 5-ml plastic tube and kept on ice. The
signed chamber-electrode assembly (Instech Laboratories Inc., fig. electrode was washed twice with 0.75 ml ice-cold distilled water. The
1), which consisted of a thermostatted aluminum block, an oxygen reaction mixture and the washes were pooled and passed through a
electrode, a 1-mi polystyrene reaction vial, a plastic thermal jacket 1-ml Dowex AG5O-X8 column. L-[3H]citrulline was eluted with 1.5 ml
and a miniature precision stirring system. The temperature of the distilled water.
reaction mixture was kept at 37#{176}C by circulating water from a
temperature-controlled water bath internal to the thermostatted
aluminum block. The oxygen electrode was threaded through the cap
Results
of the reaction vial. The cap and the reaction vial were sealed by an The effect of different concentrations of oxygen on the
0-ring. The presence ofan inlet and an outlet facilitated the passage activity of isolated NO synthase preparations from bovine
of gas mixtures containing varying concentrations of oxygen into the
brain, cultured bovine aortic endothelial cells and cultured
reaction vial. A separate inlet on the cap permitted the addition of
RAW 264.7 macrophages was determined by measuring the
solutions to the reaction vial. The reaction vial was placed in a
conversion of L-[3H]arginine to L-[3H]citrulline. The oxygen
tapered mating hole in the thermostatted aluminum block, and a
miniature precision stirrer was positioned right below the reaction concentration of the reaction mixture was continuously mon-
vial. The reaction mixture in the vial was continuously stirred with itored using a Clark-type polarographic electrode. The appar-
a 1.5/8-mm stirring bar that could be seen through a narrow view ent Km values for oxygen were found to be 17.7 (23.2 ± 2.8),
port in the thermostatted block. The reaction vial was enclosed by a 9.6 (7.7 ± 1.6) and 5.2 (6.3 ± 0.9) .tM (the numbers in
tubular acrylic thermaljacket seated on the thermostatted block, the parentheses represent the mean ± S.E.M. of three experi-
ments) for the brain, endothelial and macrophage NO syn-
Injection Port thases, respectively (figs. 2A, B and C), which corresponded
to partial pressures ofoxygen of 17, 6 and 5 ton’, respectively.
Styrofoam Block The Vmax values for the brain, endothelial and macrophage
NO synthases were 616 (705 ± 45), 530 (435 ± 43) and 2637
(2081 ± 235) pmollmg/3 mm (the values in parentheses rep-
Gas Inlet Tubing resent the mean ± S.E.M. of three experiments), respec-
Gas Outlet Tubing tively.

Discussion
Thermal Jacket The apparent Km values for oxygen of NO synthases ob-
served in this study are close to the Km values of enzymes
Kel-F Cap such as cytochrome P-450 and cytochrome oxidase that uti-
. Oxygen Electrode
lize oxygen as a substrate (Brown, 1992; Jones, 1986). Iso-
. 0- Ring lated preparations including rat liver microsome cytochrome
Reaction Vial P-450 and cytochrome oxidase have been shown to have Km
values ranging from 1 to 9 p.M (Brown, 1992; Jones, 1986).
. Stirrer Bar Further studies on cytochrome P-450 and cytochrome oxidase
have revealed that the Km value for oxygen in intact cellular
Water Outlet systems is higher than that observed in isolated preparations
. View Port (Jones, 1986). The increase in Km for oxygen in intact tissues
appears to be due to the presence of an oxygen gradient. The
magnitude ofthe oxygen gradient depends mainly on the rate
. Aluminum Block
of oxygen consumption and on the effective diffusion coeffi-
cients in extracellular and intracellular spaces. The oxygen

Inlet
gradient from the extracellular medium to the endoplasmic
reticulum has been shown to be 0.6 to 2 M when the extra-
Fig. I . Measurement of oxygen concentration. The oxygen concentra- cellular medium is 6 to 10 tM (Ericson et at. 1982, Jones,
,
tion of the NO synthase reaction mixture was determined using a
Clark-type oxygen electrode (lnstech Laboratories Inc., Model 125/05 1986). Studies on glycolate oxidase in perfused liver and
electrode) in a custom-designed chamber-electrode assembly as de- isolated peroxysomes and on 4-hydroxyphenyl pyruvate di-
scribed in “Materials and Methods.” oxygenase and homogentisate 1,2-dioxygenase in hepato-
32 Rengasamy and Johns Vol. 276

intracellular oxygen gradient exists (Jones and Mason,

A
1978b). Endothelial NO synthase appears to be associated
C with intracellular organelles including Golgi bodies (O’Brien
a,
0
et at. , 1995) and endoplasmic reticulum (Loesch et at. , 1994),
0. whereas brain (Hecker et at. , 1993) and macrophage
0)
E (Vodovotz et at. , 1995) NO synthases are cytosolic and mem-

r’)
C

0
77/7/770
Km 17.7 AM
brane-bound.
types
ditions
oxygen
is subjected
from
of NO
It is unclear

the
synthase
to an
extracellular
oxygen
whether

is likely
gradient
medium.
NO synthase

to be higher
under
The Km
in these
hypoxic
value
if there
for
is an
cell
con-

E
C Vmax 616 pmol/3 mm/mg
oxygen gradient under in vivo conditions.
>
The oxygen concentration in intact tissues is in the range
0 of 1 to 90 j.tM with a median of 35 .tM (Brown, 1992). NO
0 25 50 75 100 125 synthases in intact tissue are exposed to saturating concen-
i/[sJ (mM’) trations of oxygen under normoxic conditions, as expected
from the Km values for oxygen. However, pathophysiological
B6
conditions including hypoxia are likely to cause a consider-
able decrease in oxygen concentration in intact tissue, result-
C
a, ing in an attenuation of NO production.
0
0.4 Several studies demonstrate that hypoxia attenuates en-
0) dothelium-dependent vascular relaxation, the mechanism of
E
which has not been completely elucidated. Decreased NO

r)
C

E activity
hibition
appears
of vascular
to be
relaxation
a principal
(Johns
mechanism
et at. , 1989;
of hypoxic
Rodman
in-
et
0
E
C

>
Km
Vmax =9.6 M
530 pmol/3 mmn/mg
at.
showed
1990;
, Warren
that moderate
both EDRFINO-dependent
et at. , 1989).
hypoxia
In previous studies,
(35 ± 5 mm Hg) attenuated
vasodilation (Johns
we

et at. , 1989)
and the activity of isolated NO synthase (Rengasamy and
0
0 50 100 150 200
Johns, 1991), which suggests that hypoxia-induced decrease
i/[s] (mM’)
in NO synthesis may be a result of limitation of availability
of oxygen substrate for enzyme activity. The Km values for
oxygen determined in the current study suggest that oxygen
0.75
substrate limitation is indeed the major mechanism of the
inhibition of EDRFINO-dependent vasodilation that we ob-
served.
0.50 Brain NO synthase showed a higher Km value for oxygen
than did the endothelial and macrophage NO synthase iso-
forms, which suggests that the brain NO synthase is more
sensitive to oxygen concentration. This result may have im-
0.25
Km
Vmax =5.2 M
2637 pmol/3 mmn/mg
portant
cluding
implications
ischemia-induced
The Km value
modulated
under

for oxygen
in the intact
pathophysiological
neuronal

system
reported
injury.

by several
in this study
conditions

factors,
may be
including
in-

0.00 the redox state of NO synthase, the availability of NADPH

0 100 200
and the interaction of other ligands with oxygen binding to
i/[s} (mM’)
NO synthase. Wilson et al. (1977) investigated the effect of
Fig. 2. Determination of Km value for oxygen of NO synthases. The decreasing oxygen tension on cellular energetics in cultured
initial rate of NO synthase activity at varying oxygen concentrations was kidney cells; they observed a decrease in respiratory rate and
determined as described in “Materials and Methods.” The data were a progressive reduction in cytochrome c with relatively con-
analyzed by Uneweaver and Burk plot. The Km values for oxygen were
stant ATP levels. They suggest that the mitochondrial respi-
1 7.7 (23.2 ± 2.8). 9.6 (7.7 ± 1 .6) and 5.2 (6.3 ± 0.9) M for the bovine
brain (A), bovine aortic endothelial (B) and RAW 264.7 macrophage (C) ratory chain between NAB couple and cytochrome c remains
NO synthases, respectively. The numbers in parentheses represent the at or near equilibrium even under low oxygen concentration
mean ± S.E.M. of three experiments. because of compensatory adjustments in the [ATPV[ADPI x
[P] ratio. Although the Km value for oxygen of cytochrome c
cytes have revealed that the oxygen gradient from the extra- oxidase is 0.8 .tM, the metabolic effects of limited oxygen
cellular space to the cytoplasm is small (Jones and Mason, supply occur at oxygen concentrations greater than 3 tM,
1978a). Several studies suggest that the plasma membrane is whereas the effects on respiration are masked by compensa-
a minimal barrier to oxygen diffusion and that the maximal tory mechanisms. In another study, synaptosomes isolated
gradient due to an unstirred layer surrounding the cell is 1 to from the brains of nonanesthetized rats subjected to 30 mm
2 j.tM (Kreuzer and Yahr, 1960, Huxley and Kutchai, 1981). of hypoxia exhibited lower rates of oxygen uptake and a
However, studies on mitochondrial function in isolated hepa- decrease in the steady-state reduction levels of cytochrome c
tocytes and isolated mitochondria show that a substantial compared with normoxic animals (Rafalowska, 1995). This
1996 Km Value for Oxygen of NO Synthases 33

suggests that damage that occurred to the synaptosomal or HECKER, M., MULSCH, A. s.rm BUSSE, R.: Subcellular localization and character-
ization of neuronal nitric oxide synthase. J. Neurochem. 62: 1524-1529,
mitochondrial membrane during hypoxia has modified sub-
1993.
strate oxidation in mitochondria and decreased the availabil- Huxizy, V. H. AND Kurs.&, H.: The effect of red cell membrane and a diffusion
ity of reducing equivalents for the respiratory chain. boundary layer on the rate of oxygen uptake by human J. erythrocytes.
Physiol. (Lond.) 316: 75-83, 1981.
Although previous studies show that NO binds tightly to
JOHNS, R. A., LINDEN, J. M. AND PEACH, M. J.: Endothelium-dependent relax-
the reduced form ofcytochrome oxidase (Brudvig et at. , 1980), ation and cyclic GMP accumulation in rabbit pulmonary artery are selec-
the complex is unstable in the presence of oxygen (Wilson tively impaired by hypoxia. Circ. Res. 65: 1508-1515, 1989.
and Erecinska, 1978). Recently, Brown and Cooper (1994) JONES, D. P.: Intracellular diffusion gradients of 0 and ATP. Am. J. Physiol.
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have shown that nanomolar concentrations of NO reversibly Jor.izs, D. P. AND MASON, H. S.: Metabolic hypoxia: Accumulation of tyrosine
inhibited oxygen consumption by brain synaptosomes. This metabolites in hepatocytes at low P02. Biochem. Biophys. Res. Commmun.
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JoNEs, D. P. AND MASON, H. S.: Gradients ofO2 concentration in hepatocytes. J.
cytochrome oxidase. NO competes with oxygen to bind to
Biol. Chem. 253: 4874-4880, 1978b.
cytochrome oxidase and significantly attenuates oxygen con- KREUZER, F. AND YAHR, W. Z.: Influence of red cell membrane on diffusion of
sumption. This has been suggested to increase the Km value oxygen. J. Appi. Physiol. 15: 1117-1122, 1960.
KWON, N. S., NATHAN, C. F., GIucsR, C., Gawrim, 0. W., MATHEWS, D. E. AND
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O’BRIEN, A. J., YOUNG, H. M., Povsx, J. M. n FURNESS, J. B.: Nitric oxide
that pathophysiological conditions causing a decrease in tis- synthase is localized predominantly in the Golgi apparatus and cytoplasmic
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POLLOCK, J. S., Foantius.’, U., MITCHELL, J. A., WARNER, T. D., SCHMIDT, H. H.
H. W., NAXANE, M. m Mi.mu, F.: Purification and characterization of
Acknowledgments
particulate endothelium-derived relaxing factor synthase from cultured and
We thank Ms. Patty Jenkins for her secretarial assistance. native bovine aortic endothelial cells. Proc. NatI. Acad. Sci. U.S.A. 88:
10480-10484, 1991.
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