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Division of Science and Mathematics, Bethune-Cookman College, Daytona Beach, Florida. 32114, U.S.A.. Received November 20, 1997. Abstract.
Vol. 45, No. 6, September 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL PQges ! 081-1087

Constitutive Nitric Oxide Synthase in Saccharomyces cerevisiae R.N. Kanadia, W.N. Kuo*, M. Mcnabb, and A. Botchway Division of Science and Mathematics, Bethune-Cookman College, Daytona Beach, Florida 32114, U.S.A. Received November20, 1997 Abstract After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive-band at 60kDa of constitutive nitric oxide synthase (cNOS) from Saccharomyces cerevisiae was detected by Western blot using mouse monoclonal anti-neuronal NOS (cNOS). The activity of yeast cNOS, which was prepared by either histone-agarose chromatography or anti-neuronal NOS immunoprecipitation, was monitored by the formation of citrulline. Yeast cNOS was activated in the presence of calmodulin and arginine, whereas it was inhibited by L-NAME, a mammalian NOS inhibitor. Moreover, actinomycin-D decreased the extracellular and the intracellular levels of nitrate and nitrite which had been converted from NO. The results suggest that cNOS occurs in unicellular eukaryotes and the enzyme activity can be regulated. Key words:

Nitric oxide synthase, Western blot, Nitrate/Nitrite, Citrulline, Yeast. Introduction

Nitric oxide synthase (NOS), an important enzyme catalyzing the conversion of LArginine to nitric oxide and citrulline is found in various tissues in mammals. Nitric oxide (NO) is not only a critical regulator in the nervous, the immune and the cardiovascular systems (1), but also an important mediator in certain pathophysiologic conditions (2). In addition, NO binds to post synaptic density -95 (PSD-95) which is a receptor protein found in postsynaptic membranes of the central nervous system (3), and it activates soluble guanylate cyclase which catalyzes the formation of cyclic GMP (4). Moreover, there are two kinds of NOS, constitutive NOS (cNOS) and inducible NOS (iNOS). Constitutive NOS is a membrane-bound protein (20) found in brain and endothelium, which requires Ca 2+, calmodulin, FAD, FMN, and BH4 as cofactors (5). In comparison, inducible NOS found in macrophage is a cytosolic enzyme (21), which has a tightly bound calmodulin prosthetic group, and is calcium/calmodulin independent (6). Mammalian NOS has been extensively studied, however, very little is kalown about NOS in unicellular organisms, such as yeast. Hence, a current report of the presence of NO-activated soluble

*Address Correspondenceto: W.N. Kuo, Division of Science and Mathematics,DaytonaBeach FL 32114, U.S.A. 1039-9712/98/121081~07505.00/0 108 |

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guanylate cyclase in yeast (22, 13) along with the elevated levels of cyclic GMP in exponentially growing Saccharomyces cerevisiae lead this investigation to explore the presence of cNOS in yeast. Materials and Methods Mouse monoclonal anti-neuronal nitric oxide synthase (cNOS) and mouse monoclonal antimacrophage nitric oxide synthase (iNOS) were obtained from Transduction Laboratories, Lexington, U.S.A. Sodium dodecyl sulfate (SDS), acrylamide, and mouse IgG were provided by Sigma Chemical Company, St. Louis, U.S.A. Western blotting reagents, nitrocellulose membranes, alkaline phosphatase (AP)-conjugated secondary antibody, and goat anti-mouse immunoglobulin G (IgG) were obtained from Bio-Rad Laboratories, Hercules, U.S.A. Saccharomyces cerevisiae (Fleishmann's active dry yeast) was supplied by Burn's Philip Food Inc., San Francisco, U.S.A. Nitrate/Nitrite Colorimetric Assay Kit was obtained from Cayman Chemical Company, MI, U.S.A. Crude extracts of yeast were prepared according to previously described methods (7,8). After the removal of nonspecific immunoreactivities from crude extracts by affinity chromatography (9,10), appropriate amount of samples were subjected to sodium dodecyl sulfate polyacrylamide gel (15%) electrophoresis (SDS-PAGE) (1 l). Then, Western blot analysis (12,15-19 SGS paper) was performed by incubating the experimental membranes with mouse monoclonal anti-neuronal NOS (cNOS) or mouse monoclonal anti-iNOS (9,12). Finally, antimouse IgG-AP conjugate was used for color development. Constitutive NOS activity was measured by citrulline formation. Yeast cells were suspended in extraction buffer containing Tris buffer (pH 7.4), lmM EGTA, and lmM dithiothreitol along with the addition of Triton X-100 (0.25 %), and were homogenized with a French pressure cell (SLM). After centrifugation at 45,000 rpm for 10 minutes in a Beckman SW50.1 rotor, the supernatant was repeatedly passed through a histone-agarose column (3 x 0.5 cm) for 5 times, thus removing most of the endogenous calmodulin. The resultant eluate was used for cNOS assay. Alternatively, 2 ml of the above mentioned supernatant was immunoprecipitated by incubating 100~tg of monoclonal anti-neuronal NOS (cNOS) with gentle stirring for 30 minutes at 4~ followed by an additional 30-minute incubation with 1.0 ml of protein G-Sepharose. The mixture was centrifuged at 2000 rpm for 10 minutes, and the pellet, which contained partially purified enzyme was suspended in 3.0 ml extraction buffer for cNOS assay. In each cNOS assay, aliquots of 701al of sample obtained from histone column or 140p.l of immunoprecipitated sample was used as enzyme source. The incubation was performed at 25~ for 7.5 minutes in final volume of 160p.1 of solution containing 1mM NADPH, 1~tM FAD, 10txM BH4, and 2mM CaC12 (5). After reaction was terminated by the addition of 60 gl of 100% trichloroacetic acid, the yeast cNOS activity was monitored by the formation of citrulline, which was subsequently converted into a colored product. Finally, the turbidity of the colored product was removed by using 0.2 ~m PVDF filter media, and the filtrate was spectrophotometrically analyzed at 540 nm (19). Regulation of NO formation catalyzed by cNOS in yeast cell was monitored by the changes of intracellular and extracellular nitrate and nitrite levels. Intact yeast cells were incubated for 30 minutes in phosphate buffer saline (PBS) containing 0.9% dextrose, and with or without 1 jaM actinomycin-D. The supernatant, collected from the yeast sample centrifuged at 3000 rpm with a Beckman JS7.5 rotor, was used for measuring extracellular nitrate and nitrite.

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Thereafter, the precipitated yeast cells were immediately washed with deionized H20 by centrifugation, followed by resuspension in PBS for homogenization with a French pressure cell (SLM instrument). The supernatant, collected by centrifuging yeast homogenate at 45,000 rpm in a Beckman SW50.1 rotor, was used for measuring intracellular nitrate and nitrite level. In addition, prior to determining the extracellular and the intracellular nitrate and nitrite levels by nitrate and nitrite colorimetric assay kit (technical note Cayman Chemical Company), the yeast samples were passed through 0.45btm and 0.2btm PVDF filter media. Result and Discussion Positive immunoreactivity of the yeast sample tested against rabbit polyclonal antineuronal NOS (cNOS) was previosly reported (9). A similar immunoreactive-band at 60 kDa was shown in yeast samples tested against mouse monoclonal anti-neuronal NOS (cNOS) (Figure lb, lanes 2 and 3), with a more intense band in 210 p.g sample (Figure lb, lane 3) than that of 150btg (lane 2). It has been previously demonstrated that yeast soluble guanylate cyclase (22) is the target enzyme of NO (13), further strengthening the possible occurrence of cNOS in yeast. The cross-kingdom/phylum immunoreactivity suggests an evolutionary conservation with a higher degree of homology between lnamnaalian cNOS and the yeast enzyme. Unlike the mammalian central nervous system, PSD-95 a NO receptor protein was not detected in the yeast (data not shown), suggesting evolutionary divergence. In addition, the membrane incubated with mouse monoclonal anti-macrophageNOS (iNOS) did not exhibit any immunoreactivities (Figure la, lanes 1 and 2), except for one sharp immunoreactive-band for the control (macrophage lysate) at 150 kDa (Figure 1b, lane 3), suggesting that under this experimental condition yeast may not have inducible NOS. However, it remains to be elucidated whether yeast NOS can be induced under different physiological conditions. Brain NOS (cNOS) is 50% homologous to inducible macrophage NOS (20, 21). This possible homology was confirmed by the traceable immunoreactivc-band observed at 150kDa (Figure 1b, lane 1), when macrophage lysate, a positive control for iNOS, was incubated with monoclonal anti-neuronal NOS. Citrulline and NO are two stoichiometric equivalent products of the constitutive NOS catalyzed conversion of L-arginine. Hence, the amount of NO produced, can be indirectly monitored by the formation of citrulline, which can be converted into a colorimetrically stable product (Table 1). For the sample prepared by histonc-agarose chromatography, maximal activation of yeast NOS as indicated by 1071plnol ofcitrulline formed in the presence of arginine and cahnodulin was observed, compared to 95 pmol of citrulline fbrmed with arginine. These results indicate that yeast NOS,.just as mammalian cNOS, is calciuln/calmodulindependent. Likewise, the anti-neuronal NOS (cNOS) immunoprecipitated sample also displayed

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Figure 1: Western blot analysis using 1btg/ml (a) mouse monoclonal anti-macrophage NOS (iNOS) and (b) mouse monoclonal anti-neuronal NOS (cNOS). The yeast crude extract was loaded onto an anti-mouse lgG-agarose and mouse IgG-agarose columns. Aliquot of eluate, containing 155 to 210 btg proteins, was separated by SDS-PAGE. The antibody buffer contained 500mM NaC1. Anti-mouse IgG-AP conjugate was used as secondary antibody. Figure la: lane 1, 150 Jag ofmacrophage lysate; lane 2,155 gg yeast eluate; lane 3,210 btg yeast eluate. Figure lb: lane 1,155 btg yeast eluate; lane 2,210 btg yeast eluate; lane 3, 150 j,tg macrophage lysate.

calmodulin-dependent cNOS activity, as indicated by the increase from 312 pmol for arginine alone to 644 pmol for the additon of colmodulin. However, for the sample prepared by histoneagarose chromatography, L-NAME, an arginine analogue that competes with arginine (14), reduced cNOS activity by 63%. Such inhibition is similar to that observed in mammalian cNOS (14). In addition, L-NAME reduced cNOS activity in the sample prepared from anti-neuronal NOS (cNOS) immunoprecipitation (22) by 87.4%. Similar to the mammalian enzyme, yeast cNOS is also a membrane bound protein because the enzyme activity was much lower by omitting Triton-X 100 in the extraction buffer (data not shown). Despite the above-mentioned positive result, the citrulline cNOS assay has some limitations. These limitations are: endogenous citrulline that resulted in high basal values, turbidity that interfered with absorbency at 540nm, susceptibility of the yeast enzyme to proteolysis, and the extremely unstable nature of the enzyme. Furthermore, in mammalian tissue, NO has only 5-10 seconds of half-life (25), resulting in quick conversion to nitrate and nitrite (26). Such formation of nitrate and nitrite may also occur in the yeast, interestingly, after 30- minute incubation, microscopic examination revealed tightly clustered yeast cells and rapidly fermented actinomycin-D treated yeast. Thus, it is highly

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Table 1 Constitutive NOS Activity

Addition

Citrullinc Formation (pmol) Histone-agarose Anti-neuronal NOS Chromatography immunoprecipitation

None (control)

95

197

Arginine (100j_tM)

95

312

Cahnodulin (300~tM)

0

0

Arginine + Cahnodulin

1071

644

L-NAME (20raM) + Arginine + Calmodulin

393

81

Each value shown is the mean from three samples and is corrected from basal value. Samples were either prepared by histone-agarose chromatography or anti-neuronal NOS (cNOS) immunoprecipitation as described under "Materials and Methods".

desirable to investigate whether there is any change in nitrate/nitrite level after 30-minute incubation. Accordingly, indirect evidence for the presence of cNOS in yeast was obtained by measuring nitrate and nitrite levels. Also, regulation of the yeast enzyme can be indirectly confirmed in vitro by measuring the changes of the levels of nitrate and nitrite. Indeed, the intracetlular nitrate and nitrite levels for the yeast treated with actinomycin-D, was reduced from 421pmol (control) to 337 pmol (Table 2), thus exhibiting 20% inhibition. In addition, for actinomycin-D treated yeast, the extracetlular nitrate and nitrite level was inhibited by 25%, as indicated by reduction from 253 pmol (control) to 191 pmol (Table 2). In mammals, actinomycin-D has been shown to inhibit transcription by binding between guanosine and cytosine residues (23), and to cause DNA-damage in long-term effect (27). After 30-minute incubation of yeast with actinomycin-D, the observed decrease in intracellular and extracellular level of nitrate and nitrite may be due to the indirect inhibitory effect of actinomycin-D. However, an exact inhibitory mechanism of actinomycin-D on yeast eNOS and a time-dependent effect of actionmycin-D are yet to be elucidated. The results suggest that NO may play a role in

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Table 2 Effect of Actinomycin-D on Nitrate and Nitrite tbrmation

Addition

Nitrate and Nitrite (pmol) Intracellular

Extracellular

None (control)

421

253

Actinomycin-D (1 pM)

337

191

Each value shown is the mean from three samples. Incubation of intact yeast cells, and determination of nitrate and nitrite level was performed as described under "Materials and Methods".

intracellular and intercellular communication and regulation while under external chemical stress. The findings of yeast soluble guanylate cyclase in previous study (22) and yeast cNOS in this investigation imply the biological significance of NO/cyclic GMP cascade in unicellular eukaryotes. Acknowledgements This work was supported by grant GM08119 from the National Institutes of Health, U.S.A. The first three authors contributed equally in this investigation.

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