A inteligibilidade da palavra em igrejas católicas, através de análises de carácter objectivo e subjectivo
Lencastre, Margarida Maria Mendes de Freitas de Queiroz e
1988-01-01
Separation and differential sensitivity toward avidin of carbamyl phosphate synthetase and urea amidolyase 
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Date
1969-06
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FEBS Letters
11947016
Rognes, Sven E., Roon, Robert J., Levenberg, Bruce (1969/06)."Separation and differential sensitivity toward avidin of carbamyl phosphate synthetase and urea amidolyase." FEBS Letters 3(4): 233-236.
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Volume 3, number 4 FEBS LETTERS June 1969
SEPARATION AND DIFFERENTIAL SENSITIVITY TOWARD AVIDIN OF
CARBAMYL PHOSPHATE SYNTHETASE AND UREA AMIDOLYASE
Sven E.ROGNES, Robert J.ROON and Bruce LEVENBERG * Department of Botany, University of Oslo, Oslo, Norway
and the Department of Biological G%emistry, The University of Michigan, Ann Arbor, Michigan, USA
Received 10 May 1969
1. Introduction
In a recent communication from this laboratory [I] , we have described a new, inducible enzyme, ATP:urea amidolyase (UALase), from urease-negative yeast and unicellular green algae, which catalyzes the reaction
urea + ATP Mg*’ K+ -> CO2 t 2NH3 + ADP + Pi
This enzyme is remarkably sensitive to inhibition by highly purified egg white avidin, a phenomenon which can be completely prevented by inclusion of excess biotin in the assay system. Inasmuch as avidin has been found to selectively inhibit all known biotin- enzymes and, indeed, is now accepted as a diagnostic tool for the detection of such reactions [2], the pro- posal was made that UALase belongs to that class of enzymes which contain biotin in the form of a bound, functionally-active prosthetic group.
A recent publication by Wellner, Santos and Meis- ter [3], asserting that the glutamine-dependent car- bamyl phosphate synthetase (CPSase, EC 2.7.2.5) of Escherichia coli may also be a biotinznzyme, has focused our attention on the relevant question of whether these two enzymic processes might possibly represent analogous activities of the same protein molecule. At this time we wish to report results which indicate that not only is each reaction catalyzed by a
* U.S.Public Health Service Career Development Awardee, GM-3 115-K-3.
North-Holland Publishing Company - Amsterdam
different, separable protein entity, but also that the glutamine-dependent CPSases present in extracts of three widely-different organisms - a yeast (Candida utilis), a bacterium (Aerobacter aerogenes), and a fungus (Agaricus bisponts) - do not appear to be biotinenzymes.
2. Materials and methods
2.1. Source of organisms and preparation of enzymes Candida utilis (ATCC No. 8205) was harvested af-
ter growth on urea as sole nitrogen source and dis- rupted by sonic oscillation as described [4]. The crude extract was carried through step 3 (ammonium sulfate fractionation) of the scheme for purification of UALase [4] , and the resulting preparation was em- ployed directly for gel-filtration as described in fig. 1.
Agaricus bisponts (the mushroom of commerce in the U.S.) was obtained from a local food store. In the manner described previously [5] , an extract was pre- pared from 100 g of sporophores and concentrated in CPSase activity by precipitation of proteins with am- monium sulfate. The precipitate was washedwith cold, 75% saturated ammonium sulfate and dissolved in 6 ml of 0.02 M potassium phosphate buffer, pH 7.5. After clarifying the material by centrifugation, the supernatant was used directly in the experiment described in fig. 2.
A strain of Aerobacter aerogenes (obtained from the culture collection of the Department of Micro- biology, The University of Michigan) was grown aero- bically on Enterobacter medium No. 48 [6] . Cells were
233
Volume 3, number 4 FEBS LETTERS June 1969
32 r *rl”1”!“l
24m3236404440525660
Tube number
Fig. 1. Separation of C.urilis UALase and CPSase by gel-filtra- tion on Sephadex. The yeast protein fraction (see section 2.1) was dissolved in 12 ml of a chilled solution of 0.02 M potas- sium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol (Buffer A), and placed on a column (3 X 50 cm) of Sephadex G-200 previously equilibrated against this same solution. Elu- tion with Buffer A was carried out at 3’, 5 ml fractions being collected at 20 min intervals. Enzyme activities (units ex- pressed as micromoles of product formed per min per fraction) were determined as given in section 2.2, using aliquots of
suitable size taken from alternate tubes.
centrifuged in late log phase, suspended in 0.05 M potassium phosphate buffer, pH 7.5, and disrupted by sonic oscillation. The extract was centrifuged at 20000 X g and the supernatant was treated with solid ammonium sulfate to 67% saturation. The re- sulting precipitate was dissolved in 0.1 M tris-HCl buffer, pH 7.5, containing 10 mM KCl, 10 mM MgSO,, and 5 mM pmercaptoethanol, dialyzed over- night versus two changes of this same solution, and assayed directly for glutamine-dependent CPSase activity.
Ornithine transcarbamylase (OTCase, EC 2.1.3.3) was prepared as described in ref. [5] .
2.2. Enzymatic assays UALase activity was determined by the radio-
active technique previously described [ 11, which is based on the rate of release of 14CO2 from 14C-urea under a standard set of conditions. In the avidin in- hibition studies, glutamine-dependent CPSase activity ,was measured by the isotopic procedure [5] . For de-
Fig. 2. Differential sensitivity toward avidin of UALase and glutamine-dependent CPSases from yeast, fungal and bacte- rial sources. The standard incubation systems referred to in section 2.2 were employed, with supplements of 200 pg of avidin and/or lOOir,rg of biotin included as indicated. A mix- ture of the contents of tubes 28 and 38 from the Sephadex column eluate (see fig. 1) was used as the source of the C. utilis enzymes; the remaining preparations were obtained as given in section 2.1. Control values (i.e. enzyme activities in the absence of avidin and biotin) were as follows (in mi- cromoles of carbamyl phosphate or CO2 formed per hr per mg protein): 27.6 for UALase from C. utilis; 0.35, 0.12, and 0.08 for CPSase from C. utilis, A. aerogenes, and A. bisporus, respectively. Extracts of the latter two organisms contained
no detectable UALase activity.
tecting this enzyme in effluents from the Sephadex column, the calorimetric method for citrulline de- termination was employed [ 71. In either case incu- bations were carried out in the presence of an excess of ornithine and OTCase.
2.3. Chemicals 14C-urea was purchased from Schwarz BioRe-
search, I4C-sodium bicarbonate from New England Nuclear Corp., d-biotin from Hoffman-LaRoche, highly purified avidin (12 units per mg) from Worth- ington Biochemical Corp., and Sephadex from Phar- macia Fine Chemicals, Inc.
3. Results
In fig. 1 is shown the separation of UALase from CPSase which can be achieved by passage of the C. utilis enzyme preparation over a column of Sephadex
234
Volume 3, number 4 FEBS LETTERS June 1969
G-200. Although previous studies [ 1, 81 had indi- cated that highly purified samples of yeast UALase and E. coli CPSase were not contaminated by the catalytic activities of one another, the results shown here clearly establish that, within the same cell, one is dealing with distinctly separable proteins.
The differential effect of avidin on the two en- zyme activities in C. utilis is illustrated in fig. 2, along with data from similar studies on the glutamine- dependent CPSase activities of Aerobacter aerogenes * and Agaricus bispoms. The comparatively high level of avidin employed resulted in total inhibition of UALase activity; prior inclusion of excess biotin, although without effect on the uninhibited reaction, could completely nullify the inhibitory property of avidin. In contrast, CPSase remained either un- affected by this same high level of avidin (A. bi- spotus) or suffered only a 16% loss in activity (A. aerogenes, C. utilis). This small inhibition could not be prevented by the presence of biotin.
4. Discussion and conclusions
From a structural standpoint, the urea molecule can be looked upon as a nitrogen-system counter- part of carbonic acid, wherein -OH groups are re- placed by -NH2. Hence it may not be unreasonable to speculate that an analogy might exist between the mechanisms of urea cleavage and of carbon di- oxide activation catalyzed, respectively, by UALase and all of the five known biotin-contining carboxy- lases [2] . Providing that biotin were involved in the C02-fixing activity of CPSase, such an analogy could be extended to include this enzyme as well. Indeed, one might envision even a closer connection between CPSase and UALase inasmuch as each of these enzymes, in contrast to the carboxylases, con- cerns a reaction dealing primarily with the meta- bolism of the carbon-nitrogen bond and, further- more, one in which the presence only of cosubstrate (i.e. bicarbonate or urea) is required to effect the cleavage of ATP [ 1, lo] . The sequence of interme- diate steps proposed for the action of CPSase in- cludes the formation of an enzyme-bound carbamyl
* Urease (EC 3.5.1.5) is present in extracts of this organism when cells are grown on urea as sole nitrogen source [9].
(or carbamate) moiety [ 1 l] . If this were linked to the functional N-( 1) position of the biotin residue, as is CO2 in the carboxylases [lo] , the resulting bound intermediate would essentially be identical to that favored by us, purely on theoretical grounds, for the UALase-catalyzed reaction. For these rea- sons it was considered crucial to ascertain (a) whether UALase and CPSase might conceivably be one and the same protein, and (b) whether biotin is indeed a functionally-active component of C utilis CPSase.
Our results indicate that yeast UALase and CPSase are activities associated with distinctly dif- ferent proteins, as shown by the facile separation of these enzymes on a column of Sephadex G-200. Examination of the effects of avidin on each acti- vity under essentially identical conditions has re- vealed further fundamental differences. No evidence could be obtained from avidin inhibition studies to implicate biotin in the catalytic activity of glutamine- dependent CPSase originating from either a yeast, bacterial, or fungal source. In contrast, yeast UALase, as reported previously [ 1 ] , could be inhibited com- pletely by avidin under these same conditions.
A number of other reports have recently ap- peared which likewise are at variance with the con- clusions drawn by Wellner et al. [3]. Peng and Jones [ 121 could find no significant effect of avidin on either frog liver (ammonia-dependent) or pigeon liver (glutamine-dependent) CPSase systems. Huston [13] has reported similar results with beef liver (ammonia-dependent) CPSase and, furthermore, could obtain no evidence that biotin was present in the purified E. coli enzyme or that avidin inhibited its activity. Guthijhrlein and Knappe [ 141 have noted a similar result with purified rat liver (ammo nia-dependent) CPSase. The present observations are in line with these reports. They have forced us to reconsider certain thoughts we may have entertained as to the possible similarity in mechanism between the reactions catalyzed by UALase and CPSase. Stud- ies currently in progress, using larger quantities of more highly purified preparations of UALase, hope- fully will aid in clarifying the functional role of bio- tin in this enzyme.
Acknowledgements
We are grateful to Miss Mary Kathleen Costello
235
Volume 3, number 4 FEBS LETTERS
for excellent technical assistance. This work was sup- [51 ported, in part, by a grant (GM-13325) from the U.S. 161 Public Health Service. One of us (S.E.R.) is indebted to the Norwegian Research Council for Science and 171
the Humanities (NAVF) for funds enabling him to PI participate in this investigation. [91
[lOI
References
[I] R.J.Roon and B.Levenberg, J. Biol. Chem. 243 (1968) 1111
I21
131
[41
5213. H.G.Wood and M.F.Utter, in: Essays in Biochemistry, Vol. 1, eds. P.N.CampbeB and G.D.Greville (Academic Press, London, 1965) pp. l-27. V.P.Wellner, J.LSantos and A.Meister, Biochem. 7 (1968) 2848. R.J.Roon and B.Levenberg, in: Methods in Enzymology, Volume on Metabolism of Amino Acids and Amines, eds. H.Tabor and C.W.Tabor (Academic Press, New York, (1969) in press.
j12j
[I31 !I41
June 1969
B.Levenberg, J. Biol. Chem. 237 (1962) 2590. American Type Culture Collection, catalog of strains, 8th Edition (1968) 133. J.C.Gerhart and A.B.Pardee, J. Biol. Chem. 237 (1962) 891. R.J.Roon, unpublished data. S.E.Rognes, unpublished experiments. F.Lynen, J.Knappe and E.Lorch, in: Mechanism of Action of Water-soluble Vitamins (Ciba Foundation Study Group No. 11) (Little, Brown and Co., Boston, 1961) pp. 80-105. P.M.Anderson and A.Meister, Biochem. 4 (1965) 2803. L.Peng and M.E.Jones, Biochem. Biophys. Res. Commun. 34( 1969) 335. R.B.Huston, Federation Proc. 28 (1969) 862. G.Guthohrlein and J.Knappe, European J. Biochem. 7 (1968) 119.
236
SEPARATION AND DIFFERENTIAL SENSITIVITY TOWARD AVIDIN OF
CARBAMYL PHOSPHATE SYNTHETASE AND UREA AMIDOLYASE
Sven E.ROGNES, Robert J.ROON and Bruce LEVENBERG * Department of Botany, University of Oslo, Oslo, Norway
and the Department of Biological G%emistry, The University of Michigan, Ann Arbor, Michigan, USA
Received 10 May 1969
1. Introduction
In a recent communication from this laboratory [I] , we have described a new, inducible enzyme, ATP:urea amidolyase (UALase), from urease-negative yeast and unicellular green algae, which catalyzes the reaction
urea + ATP Mg*’ K+ -> CO2 t 2NH3 + ADP + Pi
This enzyme is remarkably sensitive to inhibition by highly purified egg white avidin, a phenomenon which can be completely prevented by inclusion of excess biotin in the assay system. Inasmuch as avidin has been found to selectively inhibit all known biotin- enzymes and, indeed, is now accepted as a diagnostic tool for the detection of such reactions [2], the pro- posal was made that UALase belongs to that class of enzymes which contain biotin in the form of a bound, functionally-active prosthetic group.
A recent publication by Wellner, Santos and Meis- ter [3], asserting that the glutamine-dependent car- bamyl phosphate synthetase (CPSase, EC 2.7.2.5) of Escherichia coli may also be a biotinznzyme, has focused our attention on the relevant question of whether these two enzymic processes might possibly represent analogous activities of the same protein molecule. At this time we wish to report results which indicate that not only is each reaction catalyzed by a
* U.S.Public Health Service Career Development Awardee, GM-3 115-K-3.
North-Holland Publishing Company - Amsterdam
different, separable protein entity, but also that the glutamine-dependent CPSases present in extracts of three widely-different organisms - a yeast (Candida utilis), a bacterium (Aerobacter aerogenes), and a fungus (Agaricus bisponts) - do not appear to be biotinenzymes.
2. Materials and methods
2.1. Source of organisms and preparation of enzymes Candida utilis (ATCC No. 8205) was harvested af-
ter growth on urea as sole nitrogen source and dis- rupted by sonic oscillation as described [4]. The crude extract was carried through step 3 (ammonium sulfate fractionation) of the scheme for purification of UALase [4] , and the resulting preparation was em- ployed directly for gel-filtration as described in fig. 1.
Agaricus bisponts (the mushroom of commerce in the U.S.) was obtained from a local food store. In the manner described previously [5] , an extract was pre- pared from 100 g of sporophores and concentrated in CPSase activity by precipitation of proteins with am- monium sulfate. The precipitate was washedwith cold, 75% saturated ammonium sulfate and dissolved in 6 ml of 0.02 M potassium phosphate buffer, pH 7.5. After clarifying the material by centrifugation, the supernatant was used directly in the experiment described in fig. 2.
A strain of Aerobacter aerogenes (obtained from the culture collection of the Department of Micro- biology, The University of Michigan) was grown aero- bically on Enterobacter medium No. 48 [6] . Cells were
233
Volume 3, number 4 FEBS LETTERS June 1969
32 r *rl”1”!“l
24m3236404440525660
Tube number
Fig. 1. Separation of C.urilis UALase and CPSase by gel-filtra- tion on Sephadex. The yeast protein fraction (see section 2.1) was dissolved in 12 ml of a chilled solution of 0.02 M potas- sium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol (Buffer A), and placed on a column (3 X 50 cm) of Sephadex G-200 previously equilibrated against this same solution. Elu- tion with Buffer A was carried out at 3’, 5 ml fractions being collected at 20 min intervals. Enzyme activities (units ex- pressed as micromoles of product formed per min per fraction) were determined as given in section 2.2, using aliquots of
suitable size taken from alternate tubes.
centrifuged in late log phase, suspended in 0.05 M potassium phosphate buffer, pH 7.5, and disrupted by sonic oscillation. The extract was centrifuged at 20000 X g and the supernatant was treated with solid ammonium sulfate to 67% saturation. The re- sulting precipitate was dissolved in 0.1 M tris-HCl buffer, pH 7.5, containing 10 mM KCl, 10 mM MgSO,, and 5 mM pmercaptoethanol, dialyzed over- night versus two changes of this same solution, and assayed directly for glutamine-dependent CPSase activity.
Ornithine transcarbamylase (OTCase, EC 2.1.3.3) was prepared as described in ref. [5] .
2.2. Enzymatic assays UALase activity was determined by the radio-
active technique previously described [ 11, which is based on the rate of release of 14CO2 from 14C-urea under a standard set of conditions. In the avidin in- hibition studies, glutamine-dependent CPSase activity ,was measured by the isotopic procedure [5] . For de-
Fig. 2. Differential sensitivity toward avidin of UALase and glutamine-dependent CPSases from yeast, fungal and bacte- rial sources. The standard incubation systems referred to in section 2.2 were employed, with supplements of 200 pg of avidin and/or lOOir,rg of biotin included as indicated. A mix- ture of the contents of tubes 28 and 38 from the Sephadex column eluate (see fig. 1) was used as the source of the C. utilis enzymes; the remaining preparations were obtained as given in section 2.1. Control values (i.e. enzyme activities in the absence of avidin and biotin) were as follows (in mi- cromoles of carbamyl phosphate or CO2 formed per hr per mg protein): 27.6 for UALase from C. utilis; 0.35, 0.12, and 0.08 for CPSase from C. utilis, A. aerogenes, and A. bisporus, respectively. Extracts of the latter two organisms contained
no detectable UALase activity.
tecting this enzyme in effluents from the Sephadex column, the calorimetric method for citrulline de- termination was employed [ 71. In either case incu- bations were carried out in the presence of an excess of ornithine and OTCase.
2.3. Chemicals 14C-urea was purchased from Schwarz BioRe-
search, I4C-sodium bicarbonate from New England Nuclear Corp., d-biotin from Hoffman-LaRoche, highly purified avidin (12 units per mg) from Worth- ington Biochemical Corp., and Sephadex from Phar- macia Fine Chemicals, Inc.
3. Results
In fig. 1 is shown the separation of UALase from CPSase which can be achieved by passage of the C. utilis enzyme preparation over a column of Sephadex
234
Volume 3, number 4 FEBS LETTERS June 1969
G-200. Although previous studies [ 1, 81 had indi- cated that highly purified samples of yeast UALase and E. coli CPSase were not contaminated by the catalytic activities of one another, the results shown here clearly establish that, within the same cell, one is dealing with distinctly separable proteins.
The differential effect of avidin on the two en- zyme activities in C. utilis is illustrated in fig. 2, along with data from similar studies on the glutamine- dependent CPSase activities of Aerobacter aerogenes * and Agaricus bispoms. The comparatively high level of avidin employed resulted in total inhibition of UALase activity; prior inclusion of excess biotin, although without effect on the uninhibited reaction, could completely nullify the inhibitory property of avidin. In contrast, CPSase remained either un- affected by this same high level of avidin (A. bi- spotus) or suffered only a 16% loss in activity (A. aerogenes, C. utilis). This small inhibition could not be prevented by the presence of biotin.
4. Discussion and conclusions
From a structural standpoint, the urea molecule can be looked upon as a nitrogen-system counter- part of carbonic acid, wherein -OH groups are re- placed by -NH2. Hence it may not be unreasonable to speculate that an analogy might exist between the mechanisms of urea cleavage and of carbon di- oxide activation catalyzed, respectively, by UALase and all of the five known biotin-contining carboxy- lases [2] . Providing that biotin were involved in the C02-fixing activity of CPSase, such an analogy could be extended to include this enzyme as well. Indeed, one might envision even a closer connection between CPSase and UALase inasmuch as each of these enzymes, in contrast to the carboxylases, con- cerns a reaction dealing primarily with the meta- bolism of the carbon-nitrogen bond and, further- more, one in which the presence only of cosubstrate (i.e. bicarbonate or urea) is required to effect the cleavage of ATP [ 1, lo] . The sequence of interme- diate steps proposed for the action of CPSase in- cludes the formation of an enzyme-bound carbamyl
* Urease (EC 3.5.1.5) is present in extracts of this organism when cells are grown on urea as sole nitrogen source [9].
(or carbamate) moiety [ 1 l] . If this were linked to the functional N-( 1) position of the biotin residue, as is CO2 in the carboxylases [lo] , the resulting bound intermediate would essentially be identical to that favored by us, purely on theoretical grounds, for the UALase-catalyzed reaction. For these rea- sons it was considered crucial to ascertain (a) whether UALase and CPSase might conceivably be one and the same protein, and (b) whether biotin is indeed a functionally-active component of C utilis CPSase.
Our results indicate that yeast UALase and CPSase are activities associated with distinctly dif- ferent proteins, as shown by the facile separation of these enzymes on a column of Sephadex G-200. Examination of the effects of avidin on each acti- vity under essentially identical conditions has re- vealed further fundamental differences. No evidence could be obtained from avidin inhibition studies to implicate biotin in the catalytic activity of glutamine- dependent CPSase originating from either a yeast, bacterial, or fungal source. In contrast, yeast UALase, as reported previously [ 1 ] , could be inhibited com- pletely by avidin under these same conditions.
A number of other reports have recently ap- peared which likewise are at variance with the con- clusions drawn by Wellner et al. [3]. Peng and Jones [ 121 could find no significant effect of avidin on either frog liver (ammonia-dependent) or pigeon liver (glutamine-dependent) CPSase systems. Huston [13] has reported similar results with beef liver (ammonia-dependent) CPSase and, furthermore, could obtain no evidence that biotin was present in the purified E. coli enzyme or that avidin inhibited its activity. Guthijhrlein and Knappe [ 141 have noted a similar result with purified rat liver (ammo nia-dependent) CPSase. The present observations are in line with these reports. They have forced us to reconsider certain thoughts we may have entertained as to the possible similarity in mechanism between the reactions catalyzed by UALase and CPSase. Stud- ies currently in progress, using larger quantities of more highly purified preparations of UALase, hope- fully will aid in clarifying the functional role of bio- tin in this enzyme.
Acknowledgements
We are grateful to Miss Mary Kathleen Costello
235
Volume 3, number 4 FEBS LETTERS
for excellent technical assistance. This work was sup- [51 ported, in part, by a grant (GM-13325) from the U.S. 161 Public Health Service. One of us (S.E.R.) is indebted to the Norwegian Research Council for Science and 171
the Humanities (NAVF) for funds enabling him to PI participate in this investigation. [91
[lOI
References
[I] R.J.Roon and B.Levenberg, J. Biol. Chem. 243 (1968) 1111
I21
131
[41
5213. H.G.Wood and M.F.Utter, in: Essays in Biochemistry, Vol. 1, eds. P.N.CampbeB and G.D.Greville (Academic Press, London, 1965) pp. l-27. V.P.Wellner, J.LSantos and A.Meister, Biochem. 7 (1968) 2848. R.J.Roon and B.Levenberg, in: Methods in Enzymology, Volume on Metabolism of Amino Acids and Amines, eds. H.Tabor and C.W.Tabor (Academic Press, New York, (1969) in press.
j12j
[I31 !I41
June 1969
B.Levenberg, J. Biol. Chem. 237 (1962) 2590. American Type Culture Collection, catalog of strains, 8th Edition (1968) 133. J.C.Gerhart and A.B.Pardee, J. Biol. Chem. 237 (1962) 891. R.J.Roon, unpublished data. S.E.Rognes, unpublished experiments. F.Lynen, J.Knappe and E.Lorch, in: Mechanism of Action of Water-soluble Vitamins (Ciba Foundation Study Group No. 11) (Little, Brown and Co., Boston, 1961) pp. 80-105. P.M.Anderson and A.Meister, Biochem. 4 (1965) 2803. L.Peng and M.E.Jones, Biochem. Biophys. Res. Commun. 34( 1969) 335. R.B.Huston, Federation Proc. 28 (1969) 862. G.Guthohrlein and J.Knappe, European J. Biochem. 7 (1968) 119.
236
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