- Email: [email protected]
Comparison of xanthine: NAD+ oxidoreductase from liver of toad Bufo viridis and other vertebrates
362
BARBARA ZAKRZEWSKA and MARIA M. JEZEWSKA
K15/30 (bed height 20cm), previously equilibrated with 20 mM Hepps-KOH buffer pH 8.0. Desalted protein fraction II was chromatographed on DEAE-Sephacel packed into a Pharmacia Laboratory column K9/15 (bed height 7.5cm) equilibrated with 20mM Hepps-KOH buffer pH 8.0. The column was washed with the same buffer until the initially coloured effluent became colourless, and then elution was performed with 20mM Hepps-KOH buffer, pH 8.0~ containing 150 mM ammonium sulphate. The active yellow fractions were pooled and used in further experiments (fraction IIA). Spectrophotometric measurements qf various enzymic activities
(a) Dehydrogenase activity of xanthine oxidoreductase was measured with NAD + as electron acceptor and with xanthine or hypoxanthine as substrates undergoing hydroxylation. Xanthine hydroxylation was followed as the absorbance increase at 302 nm, and the two-step hypoxanthine hydroxylation to xanthine and next to uric acid, as the absorbance increases at 279 nm (sum of xanthine and uric acid) and at 302 nm (uric acid) under conditions of NADH reoxidation or of NADH accumulation (Kamifiski and Je2ewska, 1979, 1981, 1984); in the latter case, NADH formation was followed as the absorbance increase at 340 nm. (b) Oxidase activity of xanthine oxidoreductase was measured with oxygen as electron acceptor and with xanthine as the hydroxylated substrate: the reaction was followed as the absorbance increase at 302 nm. Reaction mixture for measurements (a) and (b) contained 50 mM TAPS KOH buffer, pH 8.0, xanthine or hypoxanthine in micromolar concentrations, 350/~M NAD + and the enzyme preparation (usually 11 80 pkat/ml) in a final volume of 3 ml. Under conditions of NADH reoxidation, 40 nkat/ml of lactate dehydrogenase and sodium pyruvate to final concentration of 0.5 mM were added to the incubation mixture. (c) NADH oxidase activity: NADH oxidation with oxygen as electron acceptor was measured as the absorbance decrease at 340 nm. The reaction mixture contained 50 mM TAPS KOH buffer pH8.0, 150raM NADH and the enzyme preparation 80 pkat/ml in a final volume of 3 ml. Changes in purine and NADH concentrations, representing various enzyme activities and illustrating the reaction course, were expressed in nmol/ml of the incubation mixture. Michaelis constants for xanthine and NAD + were calculated from the initial rates of uric acid formation under conditions of NADH reoxidation to avoid possible inhibition of the enzyme by accumulating NADH. The method of direct linear plotting by Cornish-Bowden and Eisenthal (1978), with statistics by Potter and Trager (1977), was used. Protein was determined by the method of Bradford (1976).
RESULTS AND DISCUSSION
At the occurrence of cold weather in Poland (October), when the toad undergoes hibernation and starvation, the xanthine-hydroxylating activity in the liver was about 0.2 nkat per g of fresh tissue; on the other hand, during the active period of life (May-September), this activity increased to 0.7-1.25 nkat/g. The enzyme fraction II was free from the urate oxidase, aldehyde oxidase and NAD+-reductase activities (measured as described previously, Kamifiski and J&ewska, 1979); it exhibited, however, the N A D H oxidase activity (with oxygen as electron
acceptor) which accounted for 50% of the xanthinehydroxylating activity. The K,o for N A D H was 3-4/~M at a N A D H concentration range of 2.2-66.3/~M, the value being close to K~ for N A D H ofxanthine oxidoreductase from rat and chicken liver (Della Corte and Stirpe, 1970). N A D H oxidation was not, however, catalyzed by functional toad liver xanthine oxidoreductase: the N A D H - o x i d a s e and xanthine-hydroxylating activities were altogether separated by chromatography of the enzyme fraction II on DEAE-Sephacel. The former activity was eluted first with H e p p s - K O H buffer, and the latter alter addition of a m m o n i u m sulphate to the eluting buffer. Xanthine oxidoreductase in protein fraction IIA was purified about 30 times as compared with the 120,000g supernatant, and was found to be completely free from the N A D H oxidase activity. The protein fraction IIa was used in further experiments. T o a d xanthine oxidoreductase appeared to occur as stable dehydrogenase (EC 1.1.1.204), similarly as this enzyme from frog of Rana species (Francois, 1973). The protein fraction II exhibited exclusively the N A D ~ - d e p e n d e n t xanthine-hydroxylating activity (105pkat/mg protein) only when D T T was present in the homogenization medium: in the absence of DTT, a low 02-dependent xanthinehydroxylating activity (1 1.5% of that of the N A D ~dependent one) appeared. This activity of the toad enzyme, in contrast to the mammalian enzyme (Della Corte and Stirpe, 1970) did not increase during storage at - 2 0 : C in a medium without DTT, albeit the N A D ~-dependent activity decreased linearly by 75% during 5 months storage. The toad enzyme was also more resistant toward Cu +: ions (oxidizing the thiol groups), than the rat and snake xanthine oxidoreductase (Kamifiski and Je2ewska, 1979, 1984, respectively). A 5 min incubation with CuSO4, even at a 80/~ M concentration, had no effect on the N A D ~dependent activity. This activity was, however destroyed by 200/xM CuSO 4 in 84%, but without transformation into the O2-dependent activity. The above data suggest that the thiol groups are not involved in the catalytic activity of the toad enzyme. Preincubation of the toad enzyme with 3/~ M allopurinol for 15rain led to its inhibition in 95%, similarly as in case of human liver xanthine oxidoreductase (Elion, 1966): in contrast, however, the toad enzyme did not convert allopurinol into oxypurinol (there was no absorbance increase at 285 nm with 35/~M allopurinol as substrate: Krenitsky et al., 1972). The differences in the effects of Cu ~2 ions and allopurinol on the enzyme from various vertebrate species may testify to dissimilarities in the molecular structure of xanthine oxidoreductase between the investigated species. The xanthine and N A D + concentrations which were saturating for the enzyme amounted to 4(~50/~M and over 60/~M, respectively; the excess of both cosubstrates neither activated nor inhibited the enzyme. The apparent K m for N A D ~ was 25.1 _+ 1.2 # M, when determined at the N A D ~ concentration range of 8.8-88.5/~M, and the apparent Km for xanthine was 7.15_+ 1.23~M, when determined at the xanthine concentration range of 4-100/zM, if saturating concentrations of the respective cosubstrates were used. Both these Km values of
Xanthine: NAD + oxidoreductas¢ from liver of toad Bufo viridis xanthine oxidoreductase from ureotelic toad remain at the level of the K='s values found for the enzyme from ureotelic rat (Della Corte and Stirpe, 1970) and ammonotelic fish (Kamifiski and Je~ewska, 1985); they substantially differ from those found for uricotelic chicken (Della Corte and Stirpe, 1970) and snake (Kamifiski and Jetewska, 1984). This seemed to support the assumption (Della Corte and Stirpe, 1970) that the kinetic properties of xanthine oxidoreductase may be related to the type of nitrogen excretion specific for the given species. The differences between animal species in other kinetic properties of this enzyme are not, however, so clear-cut; toad xanthine oxidoreductase has been found to resemble this enzyme from either uricotelic snake or ammonotelic fish and ureotelic rat (Kamifiski and Je~'ewska, 1984, 1985 and 1981, respectively). Thus, in contrast to the rat enzyme, toad xanthine oxidoreductase was saturated by a higher concentration (over 6 0 # M ) of hypoxanthine than of xanthine (40-50 #M), and hypoxanthine was hydroxylated faster than xanthine by freshly obtained enzyme preparations (Fig. 1); with their ageing, the rates of hydroxylation of both substrates became equal. In these properties the toad enzyme resembled the snake enzyme. Consequently, in the presentation of the time-course of hypoxanthine ~ xanthine -~ uric acid hydroxylation (Fig. 2(a) and (b)), the curve corresponding to xanthine accumulation in the reaction medium has an asymmetric shape, as in case of the enzyme from shake and fish. The rate of uric acid production (Figs l and 2) was at first independent from xanthine accumulation and from the initial hypoxanthine concentration, as incase of the snake enzyme. In contrast, however, this independence was limited only to a certain range of hypoxanthine
'[ I1 ; '"ITt 0 0
i 20
i i 40 610 80 Subsfratt. conc. (IJMI
DI 100
120
Fig. 1. Dependence of the rate of hypoxanthine and xanthine hydroxylation catalysed by toad liver xanthine oxidoreductase on the initial substrate concentration. The incubation mixture consisted of 3 ml of TAPS--KOH buffer, pH 8.0, containing either hypoxanthine or xanthine at concentrations given on the abeissae, 350/aM NAD +, 33 pkat of enzymic activity, and 40 nkat of lactate dehydrogenase activity plus 0.5 mM pyruvate. The reaction time was 5 rain. Spectrophotometric measurements were performed as described in Materials and Methods. ~7, xanthine hydroxylation to uric acid; [], hypoxanthine hydroxylation to xanthine + uric acid; O, uric acid formation from hypoxanthine. Arrows indicate the range of hypoxanthine concentrations at which the uric acid formation rate remained approximately constant.
363
k
lal
15
...o..~..~°
0
5
10
15
20 25
30
35
40 45
50 55 60
Tim( Imin )
20L
K
-I
0
[bl
x "
.Y
1
2
3
4
5
6
'o
7
8
9
Time. {rain 3
Fig. 2. Time-course of hypoxanthine ~ xanthine--* uric acid hydroxylation catalysed by toad liver xanthine oxidoreductase. The incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 40 nkat of lactate dehydrogenas¢ activity plus 0.5/aM pyruvate, 350#M NAD + and: (a) 9 or 18 #M hypoxanthine and 36 pkat of enzymic activity; (b) 18 or 36/aM hypoxanthine and 240 pkat of enzymic activity. Spectrophotometric measurements were carded out as described in Materials and Methods. I-q, hypoxanthine utilization; ~7, xanthine accumulation; ©, uric acid formation, at three initial 9( . . . . ), 18 (--) and 36 ( - - - ) / a M hypoxanthine concentrations.
concentrations: when the enzyme became saturated by hypoxanthine, uric acid production decrease (Fig. 1); such a decrease, found previously for the rat and fish (but not snake) xanthine oxidoreductase, suggests that the enzyme active centers are common for both substrates. In turn, the relationship between the enzymic activity and both xanthine and uric acid accumulation during the two-step hypoxanthine hydroxylation was similar to that obtained for rat xanthine oxidoreductase (Kamifiski and Je~ewska, 1981): the time-courses (Fig. 3) showed that at a given initial hypoxanthine concentration, the lower the enzymic activity the more xanthine and less uric acid accumulated in the reaction mixture. The most important difference between xanthine oxidoreductases from both the ureotelic vertebrates involved the effect of N A D H accumulating on the enzymic activity. Namely, in contrast to instantaneous inhibition of rat xanthine oxidoreductase by N A D H at nanomolar concentrations (Kamifiski and Je~ewska, 1979), the inhibition of the toad enzyme
364
BARBARA ZAKRZEWSKA a n d MARIA M . JE~EWSKA
15
,
~xx
./
-
35
_-
N/
.0- I
lo
~
&
5
.-w- ' v -
,,,, 00
,,.... 10
20
3~
•
---
-. 30 40 Time (rain I
~ 50
60
,
~/
7
/
../
o/.'__
70
Fi . Ef ec, of,he enzymic activity on t e,ime-course of hypoxanthine --*xanthine --*uric acid hydroxylation catalysed by toad liver xanthine oxidoreductases. Incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 42 nkat of lactate dehydrogenase activity, 0.5mM pyruvate, 350/~M NAD +, 18/~M hypoxanthine, and 36 (- ), 120 (...) or 240(--) pkat of enzymic activity. N, hypoxanthine utilization; W, xanthine accumulation; O, uric acid formation. Spectrophotometric measurements were carried out as described in Materials and Methods.
rose progressively (Fig. 4) with an increase in the NADH (micromolar) concentration; when the initial xanthine concentration approached Km for xanthine, there was no inhibition of the enzyme. Similar progressive inhibition was found in case of snake and fish xanthine oxidoreductases (Kamiflski and Jeiewska, 1984, 1985, respectively). The fact that enzyme inhibition was found only at high non-physiological concentrations of NADH rules out the possibility of modulation of the toad liver enzyme activity by changes in the N A D H / N A D ÷ ratio, which may occur in vivo. Therefore, toad xanthine oxidoreductase could not be a factor regulating the purine nucleotide metabolism, as it has been postulated (Jeiewska and Kamifiski, 1980) for rat xanthine oxidoreductase. The role of this enzyme seems to be different in both these ureotelic vertebrate species. The above comparison of xanthine oxidoreductases from vertebrates at various levels of evolutionary development suggests that differentiation of the kinetic properties of this enzyme is caused by many more factors than, solely, the evolving type of nitrogen excretion. In Amphibia a large variety of pteridines have been found (cf. Balinsky, 1970), including 2-amino4-hydroxypteridine and 2-amino-4,6-dihydroxypteridine (isoxanthopterine), both of which occur in the liver of Bufo viridis (Kokolis and Zafiratos, 1967). The former pteridine is known to be hydroxylated to the latter by milk xanthine oxidase (Valerino and McCormack, 1969), and probably the same reaction may be catalysed by the xanthine-hydroxylating enzyme in the liver of Bufo viridis. According to the present results, 2-amino-4-hydroxypteridine inhibited, in fact, xanthine hydroxylation catalysed by the toad enzyme; in the presence of 50/~M xanthine, inhibition rose linearly with an increase in the pteridine concentration (20, 40, 50 or 75% of inhibition for 3.3, 7.0, 10.0 or 16.6/xM pteridine,
t
i
/ ' /
/~y~:~,,l~/'// _.~.fo"f 0
, 6-
3
j
9
Time. (rain)
Fig. 4. Inhibition of toad liver xanthine oxidoreductase by NADH produced from cosubstrate NAD ÷ during oxypurine hydroxylation. Incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 350/xM NAD ÷, 240 pkat of enzymic activity, and either 142/zM xanthine or 36-/tM hypoxanthine, without the NADH-oxidizing system (open symbols) or with 40 nkat of lactate dehydrogenase activity plus 0.5mM pyruvate (black symbols); in the former case NADH accumulated in amounts either equal to those of xanthine utilized or corresponding to those of hypoxanthine utilized (xanthine + uric acid formed) plus uric acid formed, according to the substrate used. Spectrophotometric measurements were performed as described in Materials and Methods. W, V, utilization of xanthine; IS], II, utilization of hypoxanthine; O, O, formation of uric acid from hypoxanthine. Inhibition of the above reactions (%) is denoted on the curves representing experimental data obtained without the NADH oxidizing system. respectively). Assuming a competitive character of inhibition, we calculated K~ which was 0.98 ___0.35 # M for 2-amino-4-hydroxypteridine. A comparison of this value with Km = 7.15/~M for xanthine indicates that pteridine may effectively compete with xanthine for the toad enzyme. The abundance of pteridine compounds in Bufo viridis suggests the important role of xanthine oxidoreductase in the pteridine metabolism of this species.
Acknowledgement--This work was supported by the Polish Academy of Sciences within the project CPBR 3.13.1.3.3. REFERENCES
Balinsky J. B. (1970) Nitrogen metabolism in Amphibians. In Comparative Biochemistry of Nitrogen Metabolism (Edited by Campbell J. W.), Vol. 2, pp. 519-637. Academic Press, London, New York. Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248554. Cornish-Bowden A. and Eisenthal R. (1978) Estimation of Michaelis constant and maximum velocity from the direct linear plots. Biochim. biophys. Acta 523, 268-272. Della Corte E. and Stirpe F. (1970) The regulation of xanthine oxidase. Inhibition by reduced nicotinamideadenine dinucleotide of rat liver xanthine oxidase type D
Xanthine: NAD + oxidoreductase from liver of toad Bufo viridis and of chick liver xanthine dehydrogenase. Biochem. J. 117, 97-100. Elion G. B. (1966) Enzymatic and metabolic studies with allopurinol. Ann. Rheum. Dis. 25, 608-614. Franqois C. J. (1973) Activit6 oxydasique de l'oxydor6ductase de la xanthine, sp~eifique de la class des Mammif6res. Biochem. Syst. 1, 231-236. JeSewska M. M. and Kimifiski Z. W. (1980) Xanthine oxidoreductase inhibition by NADH as a regulatory factor of purine metabolism. In Purine Metabolism in Man--Ill (Edited by Rapado A., Watts R. W. E. and Debruyn Ch. H. M. M.), Adv, exp. Biol. Med., Vol. 122B, pp. 35-40. Plenum Press, New York. Kamifiski Z. W. and Je~ewska M. M. (1979) Intermediate dehydrogenase-oxidase form of xanthine oxidoreduetase in rat liver. Biochem. J. 181, 177-182. Kamifiski Z. W. and Jezewska M. M. (1981) Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect. Biochem. J. 200, 597-603.
365
Kamiflski Z. W. and Je2ewska M. M. (1984) XanthineNAD + oxidoreductase in the liver of grass snake Natrix natrix. Comp. Biochem. Physiol. 7811, 447-451. Kamiflski Z. W. and Je~ewska M. M. (1985) Xanthine: NAD + oxidoreductase in the liver of the teleostean fish Cyprinus carpio. Comp. Biochem. Physiol. 80B, 371-375. Kokolis N. and Zafiratos C. (1967) Organ-specific patterns of pteridines in Bufo viridis. Comp. Biochem. Physiol. 21, 373-382. Krenitsky T. A., Neil S. M., Elion G. B. and Hitchings G. B. (1972) A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Archs. Biochem. Biophys. 150, 585-599. Potter W. R. and Trager W. F. (1977) Improved nonparametric statistical method for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot. Biochem. J. 161, 293-302. Valerino D. M. and McCormack J. J. (1969) Studies of the oxidation of some aminopteridines by xanthine oxidase. Biochim. biophys. Acta 184, 154-163.
BARBARA ZAKRZEWSKA and MARIA M. JEZEWSKA
K15/30 (bed height 20cm), previously equilibrated with 20 mM Hepps-KOH buffer pH 8.0. Desalted protein fraction II was chromatographed on DEAE-Sephacel packed into a Pharmacia Laboratory column K9/15 (bed height 7.5cm) equilibrated with 20mM Hepps-KOH buffer pH 8.0. The column was washed with the same buffer until the initially coloured effluent became colourless, and then elution was performed with 20mM Hepps-KOH buffer, pH 8.0~ containing 150 mM ammonium sulphate. The active yellow fractions were pooled and used in further experiments (fraction IIA). Spectrophotometric measurements qf various enzymic activities
(a) Dehydrogenase activity of xanthine oxidoreductase was measured with NAD + as electron acceptor and with xanthine or hypoxanthine as substrates undergoing hydroxylation. Xanthine hydroxylation was followed as the absorbance increase at 302 nm, and the two-step hypoxanthine hydroxylation to xanthine and next to uric acid, as the absorbance increases at 279 nm (sum of xanthine and uric acid) and at 302 nm (uric acid) under conditions of NADH reoxidation or of NADH accumulation (Kamifiski and Je2ewska, 1979, 1981, 1984); in the latter case, NADH formation was followed as the absorbance increase at 340 nm. (b) Oxidase activity of xanthine oxidoreductase was measured with oxygen as electron acceptor and with xanthine as the hydroxylated substrate: the reaction was followed as the absorbance increase at 302 nm. Reaction mixture for measurements (a) and (b) contained 50 mM TAPS KOH buffer, pH 8.0, xanthine or hypoxanthine in micromolar concentrations, 350/~M NAD + and the enzyme preparation (usually 11 80 pkat/ml) in a final volume of 3 ml. Under conditions of NADH reoxidation, 40 nkat/ml of lactate dehydrogenase and sodium pyruvate to final concentration of 0.5 mM were added to the incubation mixture. (c) NADH oxidase activity: NADH oxidation with oxygen as electron acceptor was measured as the absorbance decrease at 340 nm. The reaction mixture contained 50 mM TAPS KOH buffer pH8.0, 150raM NADH and the enzyme preparation 80 pkat/ml in a final volume of 3 ml. Changes in purine and NADH concentrations, representing various enzyme activities and illustrating the reaction course, were expressed in nmol/ml of the incubation mixture. Michaelis constants for xanthine and NAD + were calculated from the initial rates of uric acid formation under conditions of NADH reoxidation to avoid possible inhibition of the enzyme by accumulating NADH. The method of direct linear plotting by Cornish-Bowden and Eisenthal (1978), with statistics by Potter and Trager (1977), was used. Protein was determined by the method of Bradford (1976).
RESULTS AND DISCUSSION
At the occurrence of cold weather in Poland (October), when the toad undergoes hibernation and starvation, the xanthine-hydroxylating activity in the liver was about 0.2 nkat per g of fresh tissue; on the other hand, during the active period of life (May-September), this activity increased to 0.7-1.25 nkat/g. The enzyme fraction II was free from the urate oxidase, aldehyde oxidase and NAD+-reductase activities (measured as described previously, Kamifiski and J&ewska, 1979); it exhibited, however, the N A D H oxidase activity (with oxygen as electron
acceptor) which accounted for 50% of the xanthinehydroxylating activity. The K,o for N A D H was 3-4/~M at a N A D H concentration range of 2.2-66.3/~M, the value being close to K~ for N A D H ofxanthine oxidoreductase from rat and chicken liver (Della Corte and Stirpe, 1970). N A D H oxidation was not, however, catalyzed by functional toad liver xanthine oxidoreductase: the N A D H - o x i d a s e and xanthine-hydroxylating activities were altogether separated by chromatography of the enzyme fraction II on DEAE-Sephacel. The former activity was eluted first with H e p p s - K O H buffer, and the latter alter addition of a m m o n i u m sulphate to the eluting buffer. Xanthine oxidoreductase in protein fraction IIA was purified about 30 times as compared with the 120,000g supernatant, and was found to be completely free from the N A D H oxidase activity. The protein fraction IIa was used in further experiments. T o a d xanthine oxidoreductase appeared to occur as stable dehydrogenase (EC 1.1.1.204), similarly as this enzyme from frog of Rana species (Francois, 1973). The protein fraction II exhibited exclusively the N A D ~ - d e p e n d e n t xanthine-hydroxylating activity (105pkat/mg protein) only when D T T was present in the homogenization medium: in the absence of DTT, a low 02-dependent xanthinehydroxylating activity (1 1.5% of that of the N A D ~dependent one) appeared. This activity of the toad enzyme, in contrast to the mammalian enzyme (Della Corte and Stirpe, 1970) did not increase during storage at - 2 0 : C in a medium without DTT, albeit the N A D ~-dependent activity decreased linearly by 75% during 5 months storage. The toad enzyme was also more resistant toward Cu +: ions (oxidizing the thiol groups), than the rat and snake xanthine oxidoreductase (Kamifiski and Je2ewska, 1979, 1984, respectively). A 5 min incubation with CuSO4, even at a 80/~ M concentration, had no effect on the N A D ~dependent activity. This activity was, however destroyed by 200/xM CuSO 4 in 84%, but without transformation into the O2-dependent activity. The above data suggest that the thiol groups are not involved in the catalytic activity of the toad enzyme. Preincubation of the toad enzyme with 3/~ M allopurinol for 15rain led to its inhibition in 95%, similarly as in case of human liver xanthine oxidoreductase (Elion, 1966): in contrast, however, the toad enzyme did not convert allopurinol into oxypurinol (there was no absorbance increase at 285 nm with 35/~M allopurinol as substrate: Krenitsky et al., 1972). The differences in the effects of Cu ~2 ions and allopurinol on the enzyme from various vertebrate species may testify to dissimilarities in the molecular structure of xanthine oxidoreductase between the investigated species. The xanthine and N A D + concentrations which were saturating for the enzyme amounted to 4(~50/~M and over 60/~M, respectively; the excess of both cosubstrates neither activated nor inhibited the enzyme. The apparent K m for N A D ~ was 25.1 _+ 1.2 # M, when determined at the N A D ~ concentration range of 8.8-88.5/~M, and the apparent Km for xanthine was 7.15_+ 1.23~M, when determined at the xanthine concentration range of 4-100/zM, if saturating concentrations of the respective cosubstrates were used. Both these Km values of
Xanthine: NAD + oxidoreductas¢ from liver of toad Bufo viridis xanthine oxidoreductase from ureotelic toad remain at the level of the K='s values found for the enzyme from ureotelic rat (Della Corte and Stirpe, 1970) and ammonotelic fish (Kamifiski and Je~ewska, 1985); they substantially differ from those found for uricotelic chicken (Della Corte and Stirpe, 1970) and snake (Kamifiski and Jetewska, 1984). This seemed to support the assumption (Della Corte and Stirpe, 1970) that the kinetic properties of xanthine oxidoreductase may be related to the type of nitrogen excretion specific for the given species. The differences between animal species in other kinetic properties of this enzyme are not, however, so clear-cut; toad xanthine oxidoreductase has been found to resemble this enzyme from either uricotelic snake or ammonotelic fish and ureotelic rat (Kamifiski and Je~'ewska, 1984, 1985 and 1981, respectively). Thus, in contrast to the rat enzyme, toad xanthine oxidoreductase was saturated by a higher concentration (over 6 0 # M ) of hypoxanthine than of xanthine (40-50 #M), and hypoxanthine was hydroxylated faster than xanthine by freshly obtained enzyme preparations (Fig. 1); with their ageing, the rates of hydroxylation of both substrates became equal. In these properties the toad enzyme resembled the snake enzyme. Consequently, in the presentation of the time-course of hypoxanthine ~ xanthine -~ uric acid hydroxylation (Fig. 2(a) and (b)), the curve corresponding to xanthine accumulation in the reaction medium has an asymmetric shape, as in case of the enzyme from shake and fish. The rate of uric acid production (Figs l and 2) was at first independent from xanthine accumulation and from the initial hypoxanthine concentration, as incase of the snake enzyme. In contrast, however, this independence was limited only to a certain range of hypoxanthine
'[ I1 ; '"ITt 0 0
i 20
i i 40 610 80 Subsfratt. conc. (IJMI
DI 100
120
Fig. 1. Dependence of the rate of hypoxanthine and xanthine hydroxylation catalysed by toad liver xanthine oxidoreductase on the initial substrate concentration. The incubation mixture consisted of 3 ml of TAPS--KOH buffer, pH 8.0, containing either hypoxanthine or xanthine at concentrations given on the abeissae, 350/aM NAD +, 33 pkat of enzymic activity, and 40 nkat of lactate dehydrogenase activity plus 0.5 mM pyruvate. The reaction time was 5 rain. Spectrophotometric measurements were performed as described in Materials and Methods. ~7, xanthine hydroxylation to uric acid; [], hypoxanthine hydroxylation to xanthine + uric acid; O, uric acid formation from hypoxanthine. Arrows indicate the range of hypoxanthine concentrations at which the uric acid formation rate remained approximately constant.
363
k
lal
15
...o..~..~°
0
5
10
15
20 25
30
35
40 45
50 55 60
Tim( Imin )
20L
K
-I
0
[bl
x "
.Y
1
2
3
4
5
6
'o
7
8
9
Time. {rain 3
Fig. 2. Time-course of hypoxanthine ~ xanthine--* uric acid hydroxylation catalysed by toad liver xanthine oxidoreductase. The incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 40 nkat of lactate dehydrogenas¢ activity plus 0.5/aM pyruvate, 350#M NAD + and: (a) 9 or 18 #M hypoxanthine and 36 pkat of enzymic activity; (b) 18 or 36/aM hypoxanthine and 240 pkat of enzymic activity. Spectrophotometric measurements were carded out as described in Materials and Methods. I-q, hypoxanthine utilization; ~7, xanthine accumulation; ©, uric acid formation, at three initial 9( . . . . ), 18 (--) and 36 ( - - - ) / a M hypoxanthine concentrations.
concentrations: when the enzyme became saturated by hypoxanthine, uric acid production decrease (Fig. 1); such a decrease, found previously for the rat and fish (but not snake) xanthine oxidoreductase, suggests that the enzyme active centers are common for both substrates. In turn, the relationship between the enzymic activity and both xanthine and uric acid accumulation during the two-step hypoxanthine hydroxylation was similar to that obtained for rat xanthine oxidoreductase (Kamifiski and Je~ewska, 1981): the time-courses (Fig. 3) showed that at a given initial hypoxanthine concentration, the lower the enzymic activity the more xanthine and less uric acid accumulated in the reaction mixture. The most important difference between xanthine oxidoreductases from both the ureotelic vertebrates involved the effect of N A D H accumulating on the enzymic activity. Namely, in contrast to instantaneous inhibition of rat xanthine oxidoreductase by N A D H at nanomolar concentrations (Kamifiski and Je~ewska, 1979), the inhibition of the toad enzyme
364
BARBARA ZAKRZEWSKA a n d MARIA M . JE~EWSKA
15
,
~xx
./
-
35
_-
N/
.0- I
lo
~
&
5
.-w- ' v -
,,,, 00
,,.... 10
20
3~
•
---
-. 30 40 Time (rain I
~ 50
60
,
~/
7
/
../
o/.'__
70
Fi . Ef ec, of,he enzymic activity on t e,ime-course of hypoxanthine --*xanthine --*uric acid hydroxylation catalysed by toad liver xanthine oxidoreductases. Incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 42 nkat of lactate dehydrogenase activity, 0.5mM pyruvate, 350/~M NAD +, 18/~M hypoxanthine, and 36 (- ), 120 (...) or 240(--) pkat of enzymic activity. N, hypoxanthine utilization; W, xanthine accumulation; O, uric acid formation. Spectrophotometric measurements were carried out as described in Materials and Methods.
rose progressively (Fig. 4) with an increase in the NADH (micromolar) concentration; when the initial xanthine concentration approached Km for xanthine, there was no inhibition of the enzyme. Similar progressive inhibition was found in case of snake and fish xanthine oxidoreductases (Kamiflski and Jeiewska, 1984, 1985, respectively). The fact that enzyme inhibition was found only at high non-physiological concentrations of NADH rules out the possibility of modulation of the toad liver enzyme activity by changes in the N A D H / N A D ÷ ratio, which may occur in vivo. Therefore, toad xanthine oxidoreductase could not be a factor regulating the purine nucleotide metabolism, as it has been postulated (Jeiewska and Kamifiski, 1980) for rat xanthine oxidoreductase. The role of this enzyme seems to be different in both these ureotelic vertebrate species. The above comparison of xanthine oxidoreductases from vertebrates at various levels of evolutionary development suggests that differentiation of the kinetic properties of this enzyme is caused by many more factors than, solely, the evolving type of nitrogen excretion. In Amphibia a large variety of pteridines have been found (cf. Balinsky, 1970), including 2-amino4-hydroxypteridine and 2-amino-4,6-dihydroxypteridine (isoxanthopterine), both of which occur in the liver of Bufo viridis (Kokolis and Zafiratos, 1967). The former pteridine is known to be hydroxylated to the latter by milk xanthine oxidase (Valerino and McCormack, 1969), and probably the same reaction may be catalysed by the xanthine-hydroxylating enzyme in the liver of Bufo viridis. According to the present results, 2-amino-4-hydroxypteridine inhibited, in fact, xanthine hydroxylation catalysed by the toad enzyme; in the presence of 50/~M xanthine, inhibition rose linearly with an increase in the pteridine concentration (20, 40, 50 or 75% of inhibition for 3.3, 7.0, 10.0 or 16.6/xM pteridine,
t
i
/ ' /
/~y~:~,,l~/'// _.~.fo"f 0
, 6-
3
j
9
Time. (rain)
Fig. 4. Inhibition of toad liver xanthine oxidoreductase by NADH produced from cosubstrate NAD ÷ during oxypurine hydroxylation. Incubation mixture consisted of 3 ml of TAPS-KOH buffer, pH 8.0, containing 350/xM NAD ÷, 240 pkat of enzymic activity, and either 142/zM xanthine or 36-/tM hypoxanthine, without the NADH-oxidizing system (open symbols) or with 40 nkat of lactate dehydrogenase activity plus 0.5mM pyruvate (black symbols); in the former case NADH accumulated in amounts either equal to those of xanthine utilized or corresponding to those of hypoxanthine utilized (xanthine + uric acid formed) plus uric acid formed, according to the substrate used. Spectrophotometric measurements were performed as described in Materials and Methods. W, V, utilization of xanthine; IS], II, utilization of hypoxanthine; O, O, formation of uric acid from hypoxanthine. Inhibition of the above reactions (%) is denoted on the curves representing experimental data obtained without the NADH oxidizing system. respectively). Assuming a competitive character of inhibition, we calculated K~ which was 0.98 ___0.35 # M for 2-amino-4-hydroxypteridine. A comparison of this value with Km = 7.15/~M for xanthine indicates that pteridine may effectively compete with xanthine for the toad enzyme. The abundance of pteridine compounds in Bufo viridis suggests the important role of xanthine oxidoreductase in the pteridine metabolism of this species.
Acknowledgement--This work was supported by the Polish Academy of Sciences within the project CPBR 3.13.1.3.3. REFERENCES
Balinsky J. B. (1970) Nitrogen metabolism in Amphibians. In Comparative Biochemistry of Nitrogen Metabolism (Edited by Campbell J. W.), Vol. 2, pp. 519-637. Academic Press, London, New York. Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248554. Cornish-Bowden A. and Eisenthal R. (1978) Estimation of Michaelis constant and maximum velocity from the direct linear plots. Biochim. biophys. Acta 523, 268-272. Della Corte E. and Stirpe F. (1970) The regulation of xanthine oxidase. Inhibition by reduced nicotinamideadenine dinucleotide of rat liver xanthine oxidase type D
Xanthine: NAD + oxidoreductase from liver of toad Bufo viridis and of chick liver xanthine dehydrogenase. Biochem. J. 117, 97-100. Elion G. B. (1966) Enzymatic and metabolic studies with allopurinol. Ann. Rheum. Dis. 25, 608-614. Franqois C. J. (1973) Activit6 oxydasique de l'oxydor6ductase de la xanthine, sp~eifique de la class des Mammif6res. Biochem. Syst. 1, 231-236. JeSewska M. M. and Kimifiski Z. W. (1980) Xanthine oxidoreductase inhibition by NADH as a regulatory factor of purine metabolism. In Purine Metabolism in Man--Ill (Edited by Rapado A., Watts R. W. E. and Debruyn Ch. H. M. M.), Adv, exp. Biol. Med., Vol. 122B, pp. 35-40. Plenum Press, New York. Kamifiski Z. W. and Je~ewska M. M. (1979) Intermediate dehydrogenase-oxidase form of xanthine oxidoreduetase in rat liver. Biochem. J. 181, 177-182. Kamifiski Z. W. and Jezewska M. M. (1981) Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect. Biochem. J. 200, 597-603.
365
Kamiflski Z. W. and Je2ewska M. M. (1984) XanthineNAD + oxidoreductase in the liver of grass snake Natrix natrix. Comp. Biochem. Physiol. 7811, 447-451. Kamiflski Z. W. and Je~ewska M. M. (1985) Xanthine: NAD + oxidoreductase in the liver of the teleostean fish Cyprinus carpio. Comp. Biochem. Physiol. 80B, 371-375. Kokolis N. and Zafiratos C. (1967) Organ-specific patterns of pteridines in Bufo viridis. Comp. Biochem. Physiol. 21, 373-382. Krenitsky T. A., Neil S. M., Elion G. B. and Hitchings G. B. (1972) A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Archs. Biochem. Biophys. 150, 585-599. Potter W. R. and Trager W. F. (1977) Improved nonparametric statistical method for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot. Biochem. J. 161, 293-302. Valerino D. M. and McCormack J. J. (1969) Studies of the oxidation of some aminopteridines by xanthine oxidase. Biochim. biophys. Acta 184, 154-163.