XANTHINE
DEHYDROGENASE
IN CHICKEN
AND ALDEHYDE
LIVER: MOLECULAR
OXIDASE
IDENTITY?
ROGER ANDRES, HERBERT G. LEBHERZ~ AND HEINRICH URSPRUNG
Laboratory for Developmental Biology, Swiss Federal Institute of Technology, Ziirich, Switzerland
(Received 1 May, 1973) ABSTRACT. Evidence is presented to suggest that in chick liver, xanthine dehydrogenase and aldehyde oxidase activities are associated with only one protein species. The results of SDS electrophoresis of the purified material indicate a subunit MW of 120000. I. INTRODUCTION The mammalian enzymes xanthine oxidase (EC 1.2.3.2) and aldehyde oxidase (EC 1.2.3.1) are closely related molecules with broadly overlapping substrate specificities, molecular weights, and content of fiavin adenine dinucleotide, molybdenum, and iron [1]. The analogous enzymes xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX) in Drosophila are particularly interesting because their activities are dependent upon the functioning of a gene locus that is not genetically linked to the known structural genes of the two enzymes [2]. One hypothesis to account for this 'regulation' of the activities of two related enzymes by a third locus is that the two enzymes may have subunits in common [3]. Assuming that a similar relationship between the two enzymes from vertebrate species may exist, we set out to test this hypothesis by analyzing the subunits of liver XDH, which had been studied extensively in previous investigations [4], and AOX from the same organism, the chick. To our surprise, the two activities in the chick appear to be associated with only one protein. We have purified the activities to apparent homogeneity; they co-purify and co-electrophorese. These and other observations suggest that the two activities in chicken, in contrast to the rabbit, may be associated with only one protein or that they may have very similar physicochemical properties. II. MATERIALS AND METHODS Commercial chicken and rabbit liver were stored frozen at - 7 0 ~ until used. Unless stated otherwise, crude extracts were prepared by homogenizing 250 g liver in 750 ml H20 in a Waring blendor. The general purification protocol for XDH is that of [4]. XDH was assayed at 25 ~ in an incubation mixture containing one ml of 0.05 M potassium phosphate buffer, pH 7.9, 0.15 mM xanthine, 0.5 mM N A D + , 0.1 mM EDTA, and 5 to 50 lal enzyme solution. The generation of N A D H (340 m~t) or uric acid (290 mix) was recorded. AOX was assayed at 25 ~ in the same buffer, which contained phenazine methosulfate (PMS, 20 Ixgml- 1), 2,6-dichlorophenol indophenol (DCPIP, 20 txg ml-1), and acetaldehyde (50 mM); 5-200 ~tl of 81 Molecular Biology Reports 1 (1973) 81-86. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
enzyme solution were added. X D H activities are expressed as ~tmoles substrate converted permin perml enzyme solution, AOX activities as AA6o o of 1.0 pertain perml. Protein was determined by A28o/A26 o ratios according to [5]. Acrylamide disc electrophoresis was performed in a Buchler Polyanalyst apparatus; the conditions are given in the figure legends. The gels were stained for protein with Amidoschwarz and the intensity of the bands recorded with the Gilford gel-scanning attachment. X D H activity in gels was revealed in a staining mixture similar to the above reaction mixture except that it also contained PMS (0.01 mg ml-1) and nitro blue tetrazolium (NBT, 0.1 mgml-1). Unless stated otherwise, the staining mixture used to visualize AOX in gels was similar to the reaction mixture but contained, instead of DCPIP, NBT (0.1 mg ml- 1). III. RESULTS A N D DISCUSSION Fig. 1 shows electropherograms of crude extracts of chick liver and rabbit liver for comparison. Notice that both AOX (A) and X D H (B) activities are located in one and the same zone on the gels in chick liver extracts, but in two different zones in rabbit liver extracts (C, D). The AOX
Fig. 1. Acrylamide-disc electrophoresis (7.5~ gels) of particle-free, crude extracts of adult chicken (A, B) and rabbit (C, D) liver, stained for AOX (A, D) and XDH (B, C) activities, respectively.
patterns shown in (A) and (D) were obtained not only with acetaldehyde, but also with benzaldehyde, glutaraldehyde, and formaldehyde as the substrate. The single zone of both X D H and AOX activities is observed not only in extracts of adult liver, but also of embryonic liver or liver of young chicks. The specific activities of the two reactions follow a similar time-course of increase during chick liver development. Fig. 2 shows the results of an experiment in which a crude extract was maintained at 60 ~ or 65 ~ for various time periods. Clearly, under these conditions, the two activities are inactivated with identical kinetics. In this particular experiment, a particle-free, desalted (Sephadex G-25) extract 82
was used. In a series of related experiments, the heat inactivation kinetics was followed in extracts that had been desalted and equilibrated to different buffer systems (Fig. 3). Notice that in both Tris-HC1, pH 7.7, and Na+-barbital buffer, pH 7.9, the activities of XDH and AOX decrease with different slopes at 68 ~ ACTIVITY %
9 o XDH
100
9 = AOX
90
o
." ;
.
60~
80 D 0
8
70
0
o
65oc 0 13
60 i
0
20
+
i
40
60
i
80
i
100min TIME
Fig. 2.
Heat-inactivation of X D H and AOX in particle-free, desalted, crude extracts of adult chicken liver.
ACTIVITY % 100 -~
9 o XD~
60, 40
20 9 9 O.05M
TRIS-HCI
9 " O.05M
BARBITAL
, pH
7.7
, pH
7.9
!
13
2 6 min TIME
Fig. 3.
Heat-inactivation of X D H and AOX of particle-free, desalted, crude liver extracts that had been equilibrated to different buffer systems. Temperature: 68~
83
Table I shows that the two activities copurify in a m m o n i u m sulfate fractionation, D E A E cellulose chromatography, Sephadex G-200 gel filtration, and CM-cellulose chromatography. The material thus purified migrates as a major protein band and two minor contaminants (Fig. 4) that, as judged by densitometry measurements, account for less than 5700 of the total protein. When similar gels are stained for the presence of X D H or A O X activity, all three bands are
TABLE I Purification of X D H and A O X from chicken liver Enzyme
XDH
Step
Specific activity IUmg-1
Purification x
7.3
4.3
Heat 60 700 a m m o n i u m sulfate DEAE-cellulose Sephadex G-200 CM-cellulose DEAE-cellulose 60 ~ a m m o n i u m sulfate
AOX
14 69 83 230 840
8.2 41 49 130 495
840
495
Yield IU
~
103
100
75.5 41.3 11.8 3.6 1.77
73 40 23 7 3.5
1.77
3.5
Specific activity EUmg- 1
Purification x
Yield EU
0.045 0.21 0.27 0.68 2.8
8.2 a 38 49 124 510
243 127 38 10.7 5.9
71 a 37 224 6.3 3.4
2.8
510
5.9
3.4
a Arbitrarily set equal to the corresponding values for XDH; the preceding steps in AOX purification cannot be assayed meaningfully because of high blanc reactions.
Fig. 4. Acrylamide-disc electrophoresis (7.5 70 gels) of XDH purified from chicken liver according to Table I, stained for protein (A) and XDH or AOX (B), respectively. 84
developed, the great majority of activity being associated with the major protein fraction. The two minor forms are probably modifications of the major protein. The purified material has an absorption spectrum identical to the one reported for X D H in the literature [4]. Chicken liver X D H has a molecular weight of about 250000-300000 [1]. When our purified material was electrophoresed in SDS gels calibrated with E. coli Q13 R N A polymerase (which in SDS is cleaved into five subunits of MW 12000, 41000, 86000, 145000, and 150000 respectively [6]), a single dissociated protein species with an apparent MW of 120000 was detected (Fig. 5). One of the hypotheses used to explain the regulation of X D H and AOX activities in Drosophila by a genetic locus unlinked to known structural genes of the two enzymes postulates that this third locus may control the synthesis of a protein subunit which is common to both X D H and AOX. Until recently, this hypothesis was difficult to test in Drosophila because X D H was found to be very unstable and has only recently been purified to homogeneity [8]. We decided to test the 'subunit sharing' hypothesis by isolation and characterization of the two enzymes and their subunits from a source more readily suited for biochemical analysis, namely, chicken liver. Several observations suggest that in this species, the two enzyme activities are associated with
Fig. 5. SDS-electrophoresis [7] of XDH purified according to Table I, and of E. coli RNA polymerase. The enzyme solutions were placed in a boiling waterbath during 10 min, in the presence of SDS, prior to electrophoresis. The gels were stained for protein. 85
a single protein. In both crude extracts and purified material, the two activities are associated with one and the same electrophoretic species; in contrast, the two activities of rabbit liver are readily separated. The two activities copurify through fractionations involving ion-exchange columns and gel-filtration. Furthermore, the isolated protein generates only a single electrophoretic component upon dissociation in SDS. The results from our heat-denaturation studies are less clear. Although the two activities follow identical kinetics of heat inactivation at two different temperatures in water, inactivation of XDH and AOX activities is readily resolved when the experiments are conducted in several salt solutions. Although these results may point to the presence of two different proteins, they are not necessarily inconsistent with the idea that the two activities reside in one and the same protein. Ionic interactions may lead to conformational changes in the active site(s) for the two substrates leading to uncoupling of heat-inactivation of the two activities. The present observations taken together, suggest that XDH and AOX of chicken liver, in contrast to the readily separable enzymes of rabbit liver, are very similar or perhaps even identical proteins. Definitive answers to the question of molecular identity of the two activities must await further studies with more highly resolving fractionation methodology. ACKNOWLEDGEMENTS We are grateful to Mr. W. Schaffner for his gift of RNA polymerase and advice on its use for calibrating the acrylamide gel. Research supported by Swiss National Science Foundation, Project 3.247.69. REFERENCES 1. 2. 3. 4. 5. 6.
Nelson, C. A. and Handler, P., J. Biol. Chem. 243, 5368 (1968). Dickinson, W. J., Genetics 66, 487 (1970). Glassman, E., Federation Proc. 24, 1243 (1965). Rajagopalan, K. V. and Handler, P., J. Biol. Chem. 242, 4097 (1967). Warburg, O. and Christian, W., Biochem. Z. 310, 384 (1972). Berg, D., in: Methods in Enzymology (Ed. S. P. Colowick and N. O. Kaplan), Academic Press, New York, 1971, Vol. XXI, p. 506. 7. Weber, K. and Osborn, M., J. Biol. Chem. 244, 4406 (1969). 8. Seybold, W. D., Experientia, in press (1973).
86
DEHYDROGENASE
IN CHICKEN
AND ALDEHYDE
LIVER: MOLECULAR
OXIDASE
IDENTITY?
ROGER ANDRES, HERBERT G. LEBHERZ~ AND HEINRICH URSPRUNG
Laboratory for Developmental Biology, Swiss Federal Institute of Technology, Ziirich, Switzerland
(Received 1 May, 1973) ABSTRACT. Evidence is presented to suggest that in chick liver, xanthine dehydrogenase and aldehyde oxidase activities are associated with only one protein species. The results of SDS electrophoresis of the purified material indicate a subunit MW of 120000. I. INTRODUCTION The mammalian enzymes xanthine oxidase (EC 1.2.3.2) and aldehyde oxidase (EC 1.2.3.1) are closely related molecules with broadly overlapping substrate specificities, molecular weights, and content of fiavin adenine dinucleotide, molybdenum, and iron [1]. The analogous enzymes xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX) in Drosophila are particularly interesting because their activities are dependent upon the functioning of a gene locus that is not genetically linked to the known structural genes of the two enzymes [2]. One hypothesis to account for this 'regulation' of the activities of two related enzymes by a third locus is that the two enzymes may have subunits in common [3]. Assuming that a similar relationship between the two enzymes from vertebrate species may exist, we set out to test this hypothesis by analyzing the subunits of liver XDH, which had been studied extensively in previous investigations [4], and AOX from the same organism, the chick. To our surprise, the two activities in the chick appear to be associated with only one protein. We have purified the activities to apparent homogeneity; they co-purify and co-electrophorese. These and other observations suggest that the two activities in chicken, in contrast to the rabbit, may be associated with only one protein or that they may have very similar physicochemical properties. II. MATERIALS AND METHODS Commercial chicken and rabbit liver were stored frozen at - 7 0 ~ until used. Unless stated otherwise, crude extracts were prepared by homogenizing 250 g liver in 750 ml H20 in a Waring blendor. The general purification protocol for XDH is that of [4]. XDH was assayed at 25 ~ in an incubation mixture containing one ml of 0.05 M potassium phosphate buffer, pH 7.9, 0.15 mM xanthine, 0.5 mM N A D + , 0.1 mM EDTA, and 5 to 50 lal enzyme solution. The generation of N A D H (340 m~t) or uric acid (290 mix) was recorded. AOX was assayed at 25 ~ in the same buffer, which contained phenazine methosulfate (PMS, 20 Ixgml- 1), 2,6-dichlorophenol indophenol (DCPIP, 20 txg ml-1), and acetaldehyde (50 mM); 5-200 ~tl of 81 Molecular Biology Reports 1 (1973) 81-86. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland
enzyme solution were added. X D H activities are expressed as ~tmoles substrate converted permin perml enzyme solution, AOX activities as AA6o o of 1.0 pertain perml. Protein was determined by A28o/A26 o ratios according to [5]. Acrylamide disc electrophoresis was performed in a Buchler Polyanalyst apparatus; the conditions are given in the figure legends. The gels were stained for protein with Amidoschwarz and the intensity of the bands recorded with the Gilford gel-scanning attachment. X D H activity in gels was revealed in a staining mixture similar to the above reaction mixture except that it also contained PMS (0.01 mg ml-1) and nitro blue tetrazolium (NBT, 0.1 mgml-1). Unless stated otherwise, the staining mixture used to visualize AOX in gels was similar to the reaction mixture but contained, instead of DCPIP, NBT (0.1 mg ml- 1). III. RESULTS A N D DISCUSSION Fig. 1 shows electropherograms of crude extracts of chick liver and rabbit liver for comparison. Notice that both AOX (A) and X D H (B) activities are located in one and the same zone on the gels in chick liver extracts, but in two different zones in rabbit liver extracts (C, D). The AOX
Fig. 1. Acrylamide-disc electrophoresis (7.5~ gels) of particle-free, crude extracts of adult chicken (A, B) and rabbit (C, D) liver, stained for AOX (A, D) and XDH (B, C) activities, respectively.
patterns shown in (A) and (D) were obtained not only with acetaldehyde, but also with benzaldehyde, glutaraldehyde, and formaldehyde as the substrate. The single zone of both X D H and AOX activities is observed not only in extracts of adult liver, but also of embryonic liver or liver of young chicks. The specific activities of the two reactions follow a similar time-course of increase during chick liver development. Fig. 2 shows the results of an experiment in which a crude extract was maintained at 60 ~ or 65 ~ for various time periods. Clearly, under these conditions, the two activities are inactivated with identical kinetics. In this particular experiment, a particle-free, desalted (Sephadex G-25) extract 82
was used. In a series of related experiments, the heat inactivation kinetics was followed in extracts that had been desalted and equilibrated to different buffer systems (Fig. 3). Notice that in both Tris-HC1, pH 7.7, and Na+-barbital buffer, pH 7.9, the activities of XDH and AOX decrease with different slopes at 68 ~ ACTIVITY %
9 o XDH
100
9 = AOX
90
o
." ;
.
60~
80 D 0
8
70
0
o
65oc 0 13
60 i
0
20
+
i
40
60
i
80
i
100min TIME
Fig. 2.
Heat-inactivation of X D H and AOX in particle-free, desalted, crude extracts of adult chicken liver.
ACTIVITY % 100 -~
9 o XD~
60, 40
20 9 9 O.05M
TRIS-HCI
9 " O.05M
BARBITAL
, pH
7.7
, pH
7.9
!
13
2 6 min TIME
Fig. 3.
Heat-inactivation of X D H and AOX of particle-free, desalted, crude liver extracts that had been equilibrated to different buffer systems. Temperature: 68~
83
Table I shows that the two activities copurify in a m m o n i u m sulfate fractionation, D E A E cellulose chromatography, Sephadex G-200 gel filtration, and CM-cellulose chromatography. The material thus purified migrates as a major protein band and two minor contaminants (Fig. 4) that, as judged by densitometry measurements, account for less than 5700 of the total protein. When similar gels are stained for the presence of X D H or A O X activity, all three bands are
TABLE I Purification of X D H and A O X from chicken liver Enzyme
XDH
Step
Specific activity IUmg-1
Purification x
7.3
4.3
Heat 60 700 a m m o n i u m sulfate DEAE-cellulose Sephadex G-200 CM-cellulose DEAE-cellulose 60 ~ a m m o n i u m sulfate
AOX
14 69 83 230 840
8.2 41 49 130 495
840
495
Yield IU
~
103
100
75.5 41.3 11.8 3.6 1.77
73 40 23 7 3.5
1.77
3.5
Specific activity EUmg- 1
Purification x
Yield EU
0.045 0.21 0.27 0.68 2.8
8.2 a 38 49 124 510
243 127 38 10.7 5.9
71 a 37 224 6.3 3.4
2.8
510
5.9
3.4
a Arbitrarily set equal to the corresponding values for XDH; the preceding steps in AOX purification cannot be assayed meaningfully because of high blanc reactions.
Fig. 4. Acrylamide-disc electrophoresis (7.5 70 gels) of XDH purified from chicken liver according to Table I, stained for protein (A) and XDH or AOX (B), respectively. 84
developed, the great majority of activity being associated with the major protein fraction. The two minor forms are probably modifications of the major protein. The purified material has an absorption spectrum identical to the one reported for X D H in the literature [4]. Chicken liver X D H has a molecular weight of about 250000-300000 [1]. When our purified material was electrophoresed in SDS gels calibrated with E. coli Q13 R N A polymerase (which in SDS is cleaved into five subunits of MW 12000, 41000, 86000, 145000, and 150000 respectively [6]), a single dissociated protein species with an apparent MW of 120000 was detected (Fig. 5). One of the hypotheses used to explain the regulation of X D H and AOX activities in Drosophila by a genetic locus unlinked to known structural genes of the two enzymes postulates that this third locus may control the synthesis of a protein subunit which is common to both X D H and AOX. Until recently, this hypothesis was difficult to test in Drosophila because X D H was found to be very unstable and has only recently been purified to homogeneity [8]. We decided to test the 'subunit sharing' hypothesis by isolation and characterization of the two enzymes and their subunits from a source more readily suited for biochemical analysis, namely, chicken liver. Several observations suggest that in this species, the two enzyme activities are associated with
Fig. 5. SDS-electrophoresis [7] of XDH purified according to Table I, and of E. coli RNA polymerase. The enzyme solutions were placed in a boiling waterbath during 10 min, in the presence of SDS, prior to electrophoresis. The gels were stained for protein. 85
a single protein. In both crude extracts and purified material, the two activities are associated with one and the same electrophoretic species; in contrast, the two activities of rabbit liver are readily separated. The two activities copurify through fractionations involving ion-exchange columns and gel-filtration. Furthermore, the isolated protein generates only a single electrophoretic component upon dissociation in SDS. The results from our heat-denaturation studies are less clear. Although the two activities follow identical kinetics of heat inactivation at two different temperatures in water, inactivation of XDH and AOX activities is readily resolved when the experiments are conducted in several salt solutions. Although these results may point to the presence of two different proteins, they are not necessarily inconsistent with the idea that the two activities reside in one and the same protein. Ionic interactions may lead to conformational changes in the active site(s) for the two substrates leading to uncoupling of heat-inactivation of the two activities. The present observations taken together, suggest that XDH and AOX of chicken liver, in contrast to the readily separable enzymes of rabbit liver, are very similar or perhaps even identical proteins. Definitive answers to the question of molecular identity of the two activities must await further studies with more highly resolving fractionation methodology. ACKNOWLEDGEMENTS We are grateful to Mr. W. Schaffner for his gift of RNA polymerase and advice on its use for calibrating the acrylamide gel. Research supported by Swiss National Science Foundation, Project 3.247.69. REFERENCES 1. 2. 3. 4. 5. 6.
Nelson, C. A. and Handler, P., J. Biol. Chem. 243, 5368 (1968). Dickinson, W. J., Genetics 66, 487 (1970). Glassman, E., Federation Proc. 24, 1243 (1965). Rajagopalan, K. V. and Handler, P., J. Biol. Chem. 242, 4097 (1967). Warburg, O. and Christian, W., Biochem. Z. 310, 384 (1972). Berg, D., in: Methods in Enzymology (Ed. S. P. Colowick and N. O. Kaplan), Academic Press, New York, 1971, Vol. XXI, p. 506. 7. Weber, K. and Osborn, M., J. Biol. Chem. 244, 4406 (1969). 8. Seybold, W. D., Experientia, in press (1973).
86
