Indian Journal of Experimental Biology Vol. 43, October 2005, pp. 873-879

Effect of sodium nitroprusside on H+ -ATPase activity and ATP concentration in Candida albicans

Mohammad Mahfuzul Haque, Pooja, Nikhat Manzoor, Luqman A Khan & Seemi Farhat Basir*

Enzyme Kinetics and Molecular Physiology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India

Received 15 September 2004; revised 5 July 2005

ATP hydrolysis by plasma membrane W-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells . Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type A TPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+ -ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+ - ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain . It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+ - ATPase of plasma membrane by changing its conformation resulting in decreased activity.

Keywords: ATP, Candida albicans, W -ATPase, Nitric oxide, Sodium nitroprusside, Nutrients

Candida albicans is a common opportunistic pathogen in immuno-compromised patients and its dimorphic property is generally considered to be a virulent factor . Phagocytosis by macrophages linked with release of nitric oxide (NO) is probably the most important mechanism in protecting the non­compromised host against candidiasis2

. NO has been identified as a potent effector molecule3

, released mainly by phagocytes performing vital roles in vascular cell signaling and immune system4

•

It is well known that C. albicans and other yeasts possess W -ATPase that nurtures intracellular pH and generates an electrochemical gradient of protons necessary for secondary transport systems. It uses free energy of A TP hydrolysis to translocate protons from the cell interior to the medium. Earlier, we have reported that C. albicans exhibits a strong stimulation of W -extrusion in presence of nutrients and NO affects thiss. The plasma membrane W-ATPase is an important new target of therapeutic intervention6

• It is

Correspondent author: Phone: 09810597159 Email: seemifb@rediffmail.com

one of the few antifungal targets that have been shown to be essential7

• In addition to its role in cell growth, W-ATPase has been implicated in fungal pathogenicity through its effect on dimorphism, nutrient uptake and medium acidification8

. Complete inhibition of this proton pump will certainly be lethal, partial inhibition can also be lethal depending on the environment of the cell. There are number of reports about the effect of NO on activity of other P-type ATPases. However, less attention has been paid to W-ATPase from yeast plasma membrane. Therefore, in the present study we have measured the concentration of ATP and activity of H+-ATPase of Candida in presence of various nutrients and NO. Sodium nitroprusside (SNP) was used as a potent source of N09

, a compound widely used by several workers for in vitro studies. Effect of NO on activity of W -ATPase from Candida plasma membrane has been studied for the first time.

Materials and Methods All biochemicals and enzymes were obtained from

Sigma Chemicals, USA, whereas all inorganic chemicals were of analytical grade and procured from E.Merck (India).

874 INDIAN J EXP BIOL. OCTOBER 2005

The Candida cells were grown in YEPD medium as described by Haque et al 5. Mid-log cells from YEPD medium were harvested and washed twice with sterile distilled water. The washed cells (1 g wet wt.) were suspended in 2 ml of homogenizing buffer (250 mM. sucrose; 1 mM, PMSF; 10 mM, Tris-CI; pH 7.5). To this suspension of cells, 2 g of glass beads (0.45-0.5 J.lm size) were added. The cells were then mechanically disrupted in a COrcooled homogenizer. The suspension was agitated for a total of nine cycles, each 5sec with an interval of 3 sec, at 4,000 rpm. The homogenate was centrifuged at 4,OOOx g for 5 min at 4 ° C to remove unbroken cells and glass beads. The pellet was washed once with the same homogenizer buffer. The combined supernatants were then centrifuged at 15,OOOx g for 45 min at 4°C, and the crude membrane pellet was resuspended in suspension buffer (1 mM, Tris-CI; pH 7.5). The preparation obtained routinely contained 4-6 mg/ml protein.

The concentration of ATP was estimated by using ATP estimation kit obtained from Sigma-Aldrich Chemical Company.

A TPase assay-- W -ATPase activity was assayed with 50-100 J.lg of membrane protein at room temperature accordin¥ to the protocol of Gupta et al lO

and Manzoor et al 1 with some modifications. The assay medium (500 J.lI final volume) contained 25 mM, MES-KOH; 10 mM, MgCh; 10 mM, NaN3 (to inhibit mitochondrial ATPase) and 10 mM, ATP (sodium salt). After 8 min of incubation (optimal time for ATP hydrolysis) of enzyme with substrate in assay medium, the reaction was stopped by addition of 2 ml of ice-cold stop solution (2%, H2S04; 0.5%, ammonium molybdate; and 0.5%, SDS) with 20 J.lI of 10% ascorbic acid. The tubes were incubated for 15 min at 37°C to allow formation of Pi-molybdate complex. After 15 min, stable color developed, and absorbance at 750nm was determined. Each assay was performed in presence or absence of 1 mM of vanadate, and the difference between the two measurements was used to estimate ATPase activity. NaN3 was used as mitochondrial ATPase inhibitor and sodium nitroprusside (SNP) was used as NO donor. W-ATPase was incubated with SNP for 30 min for in vitro studies.

Results PM-ATPase or W-ATPase of Candida uses the

free energy of ATP hydrolysis to translocate protons

from cell interior to the medium. In the present study, we have estimated the concentration of ATP and activity of W -ATPase in presence of various nutrients and effect of SNP (NO donor) has been monitored.

ATP concentration in presence of SNP and nutrients- It has been reported that NO acts as an inhibitor of mitochondrial electron transport chain. Cells of Candida were given exposure to NO by treating them with SNP (20 mM). The concentration of ATP was about 87.7 nmole/mg of Candida cells in the control, but once the cells were exposed to SNP for 30 min, the concentration of ATP dramatically decreased by nearly 62% (Table 1). In the presence of glucose (5mM), the concentration of ATP was around 64 nmole/mg cells, whereas cells with glvcose and SNP showed only 45 nmole of ATP/mg of cells ie, there was a decrease of around 30% in A TP concentration in SNP treated cells. To confirm that decrease in ATP concentration was due to secondary active transport of glucose, two analogues of glucose namely, xylose and 2-deoxy-D-glucose were used. Interestingly, we found similar results with them in presence and absence of SNP (Table 1). Amino acids being important molecules of cellular machinery, we have also tried to study the effect of some of them on C. albicans. One neutral (proline), one acidic (glutamic acid) and one basic (arginine) amino acids were selected to study the roles of amino acids. In the presence of proline the concentration of A TP was observed to be 66.5 nmole/mg of cells, whereas in SNP treated cells the concentration was 33.15 nmole/mg cells only. The concentration of ATP in

Table 1 - Effect of SNP and different nutrients on ATP concentrations in Candida albicans

Treatment

Cells (control) Cells + SNP Cells + glucose Cells + glucose + SNP Cells + xylose Cells + xylose + SNP Cells + 2-deoxy-D-glucose Cells + 2-deoxy-D-glucose + SNP Cells + Proline Cells + Proline + SNP Cells + Glutamic acid. Cells + Glutamic acid + SNP Cells + Arginine Cells + Arginine + SNP

[ATP) (nmole/mg of cells)

87.7 32.76 63.9 45.2

73.12 35.8 78.1 42.5 66.5

33.15 56.55 32.76 70.2 52.55

Concentration of amino acids, glucose and analogues was 5mM and SNf'was 20mM for various treatments.

HAQUE et al.: EFFECT OF SODIUM NITROPRUSSIDE ON W -ATPase ACTIVITY 875

presence of proline was almost 50% in SNP treated cells as compared to untreated cells (Table 1). Similar observations were seen in the presence of glutamic acid and arginine (Table 1). Our study showed that there was a decrease in ATP concentration in the presence of SNP irrespective of the nature of amino acids. This decrease in ATP concentration may be attributed to the effect of NO generated by SNP as reported earlier5

, which blocks the electron transport chain leading to inhibition of ATP synthesis.

H+ -A TPase activity in presence of SNP and nutrients- In the present study, we have also measured the activity of H+-ATPase in presence of SNP, nutrients (sugars and amino acids) and an inhibitor, vanadate. For in vivo experiment, Candida cells were grown in the medium (YPD) with SNP. For in vitro experiment, H+ -ATPase was isolated and exposed to SNP for 30 min. In control, H+ - ATPase activity was found to be 3.78 /lmole/min/mg of proteins, whereas in presence of vanadate (a P-type H+- ATPase inhibitor) the activity was only 0.64. The mixture of crude membrane (eM) and SNP (20mM) showed an activity of 1.12 /lmole/min/mg of protein. In the presence of SNP, it was observed that the activity was decreased by 70% (Table 2). When eM was treated with SNP in presence of vanadate the activity was found to be 0.98 /lmole/min/mg of protein, which was almost equal to the activity in vanadate absent condition. Present results indicated that vanadate did not have any independent or cumulative effect in presence of SNP. Decrease in activity in presence of SNP was almost equal to the value of vanadate present condition. It was quite possible that NO might be affecting W - ATPase directly by binding with its tyrosyl residues. Table 2 gives the value of W-ATPase activity in the presence of glucose and its structural analogue, 2-deoxy-D­glucose and other monosaccharide xylose. In the presence of glucose the H+- ATPase activity was around 120% of the control, indicating increased W­extrusion rate. Earlier, it has been reported from our lab that C. albicans exhibits a strong stimulation of W -extrusion in the presence of nutrients, but the activity of W-ATPase is inhibited by around 78% in presence of glucose and vanadate. Activity in the presence of SNP and glucose was only 26% of its control. It reinforced that NO might be involved in change of conformation of W- ATPase. We observed that the activity of PM ATPase in presence of 2-deoxy-D-glucose and xylose were 112 and 105% of

control, respectively (Table 2). Results of vanadate and SNP were almost similar to those of glucose where eM was incubated with 2-deoxy-D-glucose and SNP (Table 2).

Experiments were also conducted to study the effect of amino acids (proline, glutamic acid and arginine) on W-ATPase activity in the presence or absence of SNP and Vanadate. It was observed that proline showed similar change in activity as seen with sugars. Proline showed an increase in activity from

Table 2 - Effect of SNP (in vitro) and vanadate on specific activity of H+-ATPase isolated from plasma membrane of Candida albicans in the presence of different sugars

Treatment Sp. Activity (J.lmole/minlmg of

protein) ± SD

CM + ATP 3.78 ± 0.Q25 CM + ATP + 0.64 ± 0.Q2 Van CM + SNP 1.12 ± 0.030 CM + SNP+ 0.98 ± 0.Q15 Van CM + ATP + 4.64 ± 0.030 glucose CM + ATP + 0.86 ± 0.036 glucose + Van CM + ATP + 1.23 ± 0.021 glucose + SNP CM + ATP + 1.09 ± 0.025 glucose + SNP + Van CM + ATP + 4.24 ± 0.040 2-deoxy-D-glucose CM + ATP + 0.97 ± 0.030 2-deoxy-D-glucose + Van CM + ATP + 1.20 ± 0.015 2-deoxy-D-glucose + SNP CM + ATP + 0.97 ±0.026 2-deoxy-D-glucose + SNP + Van CM + ATP + 4.0 ± 0.015 xylose eM + ATP + 0.94 ± 0.015 xylose + Van CM + ATP + 1.08 ±0.02 xylose + SNP CM + ATP + 0.93 ± 0.036 xylose + SNP + Van

% increase/decrease in activity

(-) 83.0

(-) 70.0 (-)74.0

23.0

(-)77.0

(-) 67.0

(-)71.0

12.0

(-)74.0

(-)68.0

(-)74.0

6.0

(-) 75.0

(-) 71.0

(-)75.0

(-) sign indicates decrease in activity; CM = Crude membrane Concentration of glucose and analogues was 5mM and SNP was 20 mM. Vanadate was used at 1 mM concentration for various treatments.

876 INDIAN J EXP BIOL, OCTOBER 2005

3.78 to 4.16 /lmole/min/mg of protein ie, 110% of control, whereas in presence of SNP the activity decreased to 23% only. Like proline, in the presence of glutamic acid and arginine the activity was increased to 106 and 111 % of control, respectively (Table 3). In the presence of SNP and glutamic acid the activity of PM ATPase was only 0.92 /lmole which was almost equal to the value of vanadate present condition. Similar results were observed with arginine. (Table 3). Throughout the study, it was seen that in the presence of SNP, vanadate did not have any independent or cumulative effect.

To confirm the effect of NO on If"- ATPase from plasma membrane of C. albicans, cells were grown in SNP (NO donor). It was seen that few cells survived when they were grown in presence of SNP (Table 4). It was observed that the proton ATPase activity in control was only 1.02 /lmole/min/mg of proteins in SNP treated cells. This value was almost equal to the value of in vitro treatment ie, 1.12 /lmole/min/mg of protein.

Activity of PM ATPase was also studied in presence of glucose and its structural analogues. There was almost no increase in the activity in

Table 3- Effect of SNP (in vitro) and vanadate on specific activity of H'-ATPase isolated from plasma membrane of Candida albicans in the presence of different amino acids

Treatment Sp. Activity % (ILmole/minlmg increase/ of protein) ± SD decrease

in activity

CM +ATP 3.78 ± 0.025 CM + ATP+ Van 0.64 ±0.02 (-) 83.0 CM +SNP 1.12 ± 0.030 (-) 70.0 CM + SNP + Van 0.98 ±0.Dl5 (-) 74.0 CM + ATP+ Pro 4.16±0.D41 10.0 CM + A TP + Pro + Van 0.88 ± 0.Dl5 (-) 77.0 CM + ATP+ Pro + SNP 0.98 ±0.030 (-)74.0 CM + A TP + Pro + SNP + 0.87 ± 0.030 (-) 77.0 Van CM +ATP+Glu 4.02 ±0.021 6.0 CM + ATP+ Glu + Van O.92±0.017 (-) 76.0 CM + A TP + Glu + SNP 0.92 ± 0.Dl5 (-)76.0 CM + ATP + Glu + SNP+ 0.91 ±0.015 (-) 76.0 Van CM + ATP+ Arg 4.20 ± 0.030 11.0 CM+ATP+Arg+Van 0.78 ± 0.025 (-) 79.0 CM + A TP + Arg + SNP 0.98 ± 0.035 (-) 74.0 CM + A TP + Arg + SNP + 1.0l ± 0.020 (-) 73.0 Van

(-) sign indicates decrease in activity; CM = Crude membrane Concentration of the amino acids Pro, Glu and Arg was 5mM and that of SNP was 20 mM Vanadate was used at 1 mM concentration for various treatments

presence of glucose, when cells are grown with SNP (Table 4A). Vanadate did not show any effect on the activity of PM ATPase. Similar results were observed with 2-deoxy-D-glucose and xylose. Table 4B shows the activity of W-ATPase in presence of amino acids (proline, glutamic acid and arginine). Similar results as those of sugars were observed in presence of amino acids in SNP treated cells (Table 4B). From the results (Tables 3 and 4), it appeared that different isozymes of H+-ATPase were involved, in which one had greater affinity for ATP than the other. But the concentration of A TP used during the study was enough to saturate the binding site of both high and low affinity ATPases, and the contribution would be from both types of isoforms.

Table 4 - Effect of SNP and vanadate on specific activity of H+ -ATPase isolated from plasma membrane of Candida albicans in the presence of nutrients. Cells were given in vivo treatment of SNP

Treatment

A Sugars CM +ATP CM+ATP+ Van CM+ATP+ glucose CM +ATP+ glucose Van CM + ATP+ 2-deoxy-D-glucose CM +ATP+ 2-deoxy-D-glucose + Van CM + ATP+ xylose CM+ ATP+ xylose + Van

B Amino acids CM +ATP+ Pro CM+ ATP+ Pro + Van CM +ATP+ Glu CM+ATP+ Glu + Van CM +ATP+ Arg CM+ATP+ Arg + Van

Sp. Activity (ILmole/minlmg of protein) ± SD

1.02 ± 0025 0.95 ± 0.015

1.20 ± 0.02

0.97 ±0.025

1.09 ± 0.021

0.99 ±0.02

1.10 ± 0.036

1.00 ±0.027

1.12 ±0.035

1.02 ± 0.031

1.10 ± 0.026

0.98 ± 0.036

1.08 ± 0.02

0.99 ±0.OO6

% increase/decrease in activity

(-) 4.5

17

(-) .5.0

6.0

(-) 3.0

8.0

(-) 2.0

10.0

8.0

(-) 4.0

6.0

(-) 3.0

(-) sign indicates decrease in activity; CM = Crude membrane Concentration of glucose and its analogues and all amino acid was 5mM, while that of vandate was 1 mM

HAQUE et al.: EFFECT OF SODIUM NITROPRUSSIDE ON W-ATPase ACTIVITY 877

Discussion Nitric oxide production has been proposed as one of

the major antimicrobial mechanisms of murine macrophages l2

, which are active against different kinds of pathogens, such as viruses, bacteria, fungi, protozoa and helminthes. It is a very labile molecule and can pass easily through the cell membrane. Role of NO in anti Candida activities is reported to be well established u, but its effect on plasma membrane W-ATPase is not known. In view of this, we conducted this study to see the role of NO on ATP concentration and W-ATPase activity in presence of various nutrients. We also tried to find out whether vanadate and NO act on same site or at different sites. In our study we observed that there was a decrease in ATP concentration in SNP exposed cells in both absence and presence of nutrients. In presence of nutrients, the decrease in A TP concentration was more. Our results may be correlated to earlier reports that prolonged exposure to NO inhibits activity of a number of enzymes such as aconitase and cytochrome c oxidase by triggering the production of peroxynitrite (ONOO) in mitochondria, leading to the depletion in ATP concentration 14. 15. NO disrupts mitochondrial function by reversibly inactivating cytochrome c oxidase thus, stimulating superoxide anion production by the respiratory chain 16. The resulting superoxide anion through the formation of ONOO may be responsible for irreversible inhibition of complexes I and III of mitochondrial electron transport chain17

• It has also been reported that mitochondrial proteins are nitrated in vitro and in vivo, including aconitase, cytochrome c, voltage dependent anion channel, ATPase and succinyI-CoA oxoacid-CoA transferaseI8

,19. In our study, the decrease in ATP concentration is possibly due to disruption of electron transport chain of mitochondria and our results are in accordance to earlier reports. The decrease in ATP concentration in the presence of glucose may be attributed to the hydrolysis of ATP by W-ATPase of plasma membrane for uptake of glucose by Candida cells. It has been demonstrated that increase in W extrusion rate is around 9 folds in the presence of glucose5

. Nutrients like glucose, proline, glutamic acid and arginine acts as strong stimulator for W extrusion by Candida albicans'lIl and for It extrusion definitely the ATP will be utilized, therefore we found lower concentration of ATP when cells were incubated with glucose.

Several workers have reported the effect of NO on activity of Na, K-ATPase and Ca2+-ATPase isolated from various tissues. However, less attention has been

paid to H+ -ATPase from yeast plasma membrane. For the first time, we observed the effect of NO on activity of plasma membrane H+ -A TPase from Candida albicans. We found that there was a decrease in activity of W-ATPase in presence of NO, and vanadate did not have any independent or cumulative effect in SNP exposed cells. This may be correlated to earlier reports that vanadate displays non-competitive inhibition, indicating that it does not bind to the catalytic site of the enzyme21

.24

. Also, it has been reported that vanadate (0.5 mM) inhibits around 70% of ATPase activitl5

. In our results the inhibition in ATPase activity in the presence of SNP is almost equal to the vanadate present condition, reinforcing earlier reports that vanadate acts as non-competitive inhibitor. Inhibition of ATPase activity in presence of SNP may be due to nitrosylation of tyrosine residues of H+ -ATPase. Earlier it has been reported that ONOO' (an intermediate of NO) affects both free and protein bound tyrosine with subsequent alterations of protein phosphorylation or perturbation of protein tertiary structure. Mechanism by which NO modulates the activity of P-type ATPase, especially W -ATPase is not fully understood. Evidence indicates that NO generating compounds inhibit the activity of purified Na,K-ATPase from porcine cerebral cortex26 and bovine brain and dog kidnel7

, Ca2+ -A TPase28 and other membrane P-type ATPase. Survival of few cells when grown in presence of SNP may be explained in the light of the fact, that NO acts as a potent effector molecule and it has been demonstranted that NO has antimicrobial activity against a remarkably broad range of pathogenic microorganisms including viruses, bacteria, fungi and parasite. NO is a short­lived free radical that may interact with reactive oxygen intermediates to form more toxic species. The reaction of NO with superoxide anion produces ON02

., which can decompose to generate a strong oxidant such as hydroxyl radical29

. Like Na,K­ATPase and Ca2+-ATPase, W-ATPase share similar SH groups and it seems that inhibition in W-ATPase activity observed in the present study is due to oxidation of thiol groups and nitrosylation of tyrosine residues of the enzyme. Our results supported by earlier findings of Sato et aP6 and Boldyrev et az27 for Na,K-ATPase and Muriel and Sandovaes for Ca2

+_

ATPase. In our in vitro ATPase assay experiments, it was observed that ATPase activity increased in presence of glucose like in vivo condition. This result may be explained on the basis of earlier findings,

878 INDIAN J EXP BIOL, OCTOBER 2005

which reveals that ATPase actIVIty of the plasma membrane H+-ATPase in isolated membranes from yeast in presence of glucose is 3-5 times higher than in glucose absent condition29

. Glucose-activated ATPase has a pH optimum around 6, whereas the non-activated enzyme has a pH around 5.5. Lapathitis and Kotyk30 have reported that the highest rate of ATP hydrolysis in vitro is found with trkl delta trk2 delta mutants, where glucose induced acidification is lowest. We too observed that the rate of ATP hydrolysis increased in presence of glucose, which might be associated with a decline in phosphorylation level of the enzyme because phosphorylated enzymes have lower H+-ATPase activit/I . Our results are in accordance with the earlier findings on H+-ATPase of yeast.

Conclusion The present study indicated that NO had a significant

effect on ATP synthesis and activity of W- ATPase. In the presence of NO, the ATP concentration was decreased indicating that it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with W - ATPase of plasma membrane by changing its conformation resulting in decreased activity.

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