Comp. Biochem. Physiol., Vol. 63B. pp. 369 to 371
0305-0491/79/0107-0369502.00/0
© Pergamon Press Lid 1979, Printed in Great Britain
PURINE BIOSYNTHESIS IN HELIX ASPERSA: METABOLIC FATE OF LABELLED PRECURSORS SANDRA W. CLARK and FREDERICK B. RUDOLPH* Department of Biochemistry, Rice University, Houston, TX 77001, U.S.A. (Received 4 December 1978)
Abstract--l. The hepatopancreas of terrestial snails actively synthesizes purines from labelled formic acid. 2. The guanine nucleotides are initially labelled to the highest specific activity followed by adenine and hypoxanthine. 3. The labelling patterns suggest that uric acid synthesis is the primary catabolic process for nitrogen excretion while guanine excretion is due to an inability to reutilize the base.
INTRODUCTION Terrestial snails are uricotelic species (Delauney, 1931) excreeting uric acid, xanthine and guanine as major end-products of nitrogen metabolism. The multiple excretion products suggest an unusual regulation of the catabolic pathways compared to other species. Generally, uric acid or occasionally guanine are the major excretion products with very little of any other base excreted. Speeg & Campbell (1966) have evaluated the nitrogen excretion of the snail and found that, in the kidney where the waste products accumulate, purines represent 90% of the total nitrogen present with uric acid accounting for about two-thirds of the total and approximately equal amounts of xanthine and guanine representing the other third. The hepatopancreas is the major organ for both de noco synthesis and catabolism of purines (Barankiewicz, 1973; Jezewska et al., 1964). To evaluate the ' various pathways of pt~rine metabolism in the snail, the animals were injected with ~4C-labelled formic acid and the distribution of the label in synthesized purines determined in the hepatopancreas. Pool sizes of the various soluble purines were also determined. MATERIALS AND METHODS Helix aspersa were collected locally and maintained under controlled laboratory conditions on a diet of lettuce. The snails were injected in the foot pad with 10/~1 of l~C-labeiled formate (10#Ci, specific activity of 60.7 #Ci/ gmol, supplied by New England Nuclear). Duplicate snails were sacrificed at various time periods. The hepatopancreas was removed immediately and minced in 1.5 mi of cold 0.5 M perchloric acid. The tissue was then homogenized with a Tekmar Tissumizer followed by centrifugation at 32,000@ for 1 hr. The clear supei~natant was collected and total radioactivity determined on an aliquot. For determination of the incorporation of the labelled formate into purine bases in the soluble fraction of the homogenates, an aliquot of the acid extract was heated at 100°C for 1 hr. After centrifugation, the supernatant was applied to a Dowex 50 column (200-400 mesh, H + form, 0.5 x 5 cm). The initial wash was collected for uric acid determination described below and the absorbed purine bases were eluted with 6 N hydrochloric acid. The pooled
* To whom correspondence should be addressed. 369
base fractions were lyophilized and then dissolved in 0.2 ml of H 2 0 . A 0.1 ml aliquot of the eluted base fraction was then applied to a Partisil 10-CX cation exchange column (0.5 x 25cm, Whatman) using a Glenco high-pressure liquid chromatography system. The column was equilibrated with 0.01 M NH4H2PO4, pH 3.5 and elution was done with the same buffer. Xanthine, hypoxanthine, guanine and adenine eluted from the column in that order. The concentrations of the bases and their identity were determined from the ultraviolet spectra in acid and base (Speeg & Campbell, 1966). Uric acid was determined by first extraction of the initial wash from the Dowex-50 column with 0.4 M alamine in freon to remove the perchloric acid. The sample was then applied to an anion-exchange column (Partisil 10-AX, 0.5 x 25cm, Whatman) using the Glenco high-pressure chromatography system. The column was equilibrated with 0.005 M potassium phosphate, pH 4.7, and the uric acid eluted with this buffer. The identity and amount of uric acid was determined spectroscopically as described above for the other purines. Various unknown compounds were encountered in the chromatographic procedures. To ensure that no contamination of the isolated bases added to the apparent label present, the isolated fractions were evaporated and then applied to a thin-layer cellulose sheet (E. Merck). The chromatographs were de~eloped with an isopropanol(170); water(36); hydrochloric acid(44) system. The amount of 14C was determined in each base from the chromatograph. The specific activity of each base was calculated from its concentration determined from the high-pressure chromatography system and the I~C present as determined from the second chromatograph. Tests were made with known amounts of labelled compounds and greater than 95T,, of the label was recovered. RESULTS AND DISCUSSION The incorporation of injected l'tC-labelled formic acid into the hepatopancreas with time is given in Table 1. The results are similar to those reported by Jezewska et al. (1964) for the incorporation of labelled glycine into bases in the snail. A reasonable amount of the label is incorporated allowing analysis of the metabolism in the hepatopancreas. The highest amount of label is present after 4 hr and as will be discussed below the highest specific activity of the bases occurs then. The persistence of the label even
370
SANDRA W. CLARK and FREDERICK B. RUDOLPH
Table 1. Incorporation of ~'~C-labelled formate into the hepatopancreas* Time after injection (hr)
Total counts (dpm)
Per cent of dose (%)
2 4 8 24
392,000 604,000 292,000 256,000
1.8 2.7 1.3 1.2
* The per cent represents total soluble counts in the hepatopancreas. Results from at least 2 animals were averaged to obtain the number reported. after 24 hr suggests incorporation into nucleic acids and continual turnover and salvage. Dietary and other sources of purines are not likely to have much effect on purine synthesis in the hepatopancreas if it is the major site of purines for nitrogen excretion. From the chromatographic separation of the bases extracted from the hepatopancreas, the total pool sizes for the various purines were determined (Table 2). It should be emphasized that these numbers represent the total of the free bases, nucleosides and nucleotides in the intact cell. Since it is a soluble extract, the purines in DNA and RNA are not included. The concentration of the adenine compounds is highest (1.12/~molg wet tissue) as would be expected with the guanine pool about 30% of the adenine pool. The pool sizes of xanthine and uric acid are somewhat variable. They frequently occur as the free bases in vivo and their concentrations will relate to circulating and kidney levels. A determination of adenine and guanine nucleotides (AMP, ADP, ATP, GMP, GDP, GTP) was done on an 8ohr incubation sample. The majority of the label was in the phosphorylated compounds (about 80%) for both adenine and guanine with very low levels of G M P and AMP. The di- and triphosphates were about the same concentration. Very low levels
of other phosphorylated purines were present. This supports the idea that xanthine and uric acid are catabolic products synthesized for nitrogen excretion while guanine is excreted only when in excess of metabolic requirements. The snail cannot deaminate guanine so the base has to be excreted intact (Barankiewicz, 1973). The relative rates of synthesis and catabolism of the various purines can be evaluated from the specific activity data of Table 3. The guanine pool is labelled very rapidly to a higher specific activity than either adenine or hypoxanthine. The lower labelling of hypoxanthine was unexpected since I M P is a precursor of G M P but is likely due to a considerable pool of either inosine or hypoxanthine being catabolized. The levels of I M P in the cell are too low to make meaningful measurements so the first labelling into purines cannot be determined. The specific activity of the catabolic products xanthine and uric acid do not reach a maximum until 4 h r after injection. The turnover in the adenine pool is quite slow, consistent with the many roles of adenine nucleotides and likely continuous salvage of the base. The guanine pool decreases more rapidly than adenine due to the smaller pool and incorporation of a higher percentage of the guanine into RNA and DNA. The rapid labelling of guanine in the hepatopancreas is in contrast to that observed in the kidney of the snail. Speeg & Campbell (1966) have observed that the uric acid was most rapidly labelled. This is consistent with the suggestion above that guanine is not a primary nitrogen excretion product. Uric acid and xanthine derived from I M P are synthesized more rapidly than guanine is derived from GMP. This is indicated in the data of Table 3 where the label in xanthine and uric acid is low after 24 hr. In summary, the purine metabolism of the snail has to be considered both from the standpoint of nitrogen excretion and synthesis of bases for cellular function. The results of this report clearly indicate
Table 2. Pool sizes of purines bases in the hepatopancreas* Time after injection (hr)
Adenine
Guanine
Base (~mol/g wet tissue) Hypoxanthine
Xanthine
Uric acid
2 4 8 24 Average
1.20 1.02 1.27 1.130 1.12 + 0.13
0.38 0.24 0.41 0.23 0.32 _ 0.09
0.38 0.21 0.40 0.23 0.31 +__0.10
0.64 0.49 0.79 0.15 0.51 + 0.22
1.78 0.45 l. 11 0.08 0.65 _+ 0.51
* The numbers represent the average of at least two determinations as described in Materials and Methods. Table 3. Specific activities of purine bases in the hepatopancreas after ~4C-formate injection* Time after injection (hr)
Adenine
Guanine
2 4 8 24
26,300 65,500 74,000 67,200
53,900 238,400 49,500 43,700
Specific activity (cpm//~mol) Hypoxanthine Xanthine 33,000 145,600 78,000 51,800
12,700 37,200 13,000 12,000
Uric acid 6000 23,000 21,000 trace
* The amount of label and concentration of each base was determined as described in Materials and Methods.
Purine biosynthesis in Helix aspersa that the excretion of guanine by the snail is a metabolic accident resulting from the loss of the ability to salvage the guanine base. Both the synthesis data described here for the hepatopancreas and the data of Spceg & Campbell (1966) are supportive of this conclusion.
Acknowledgements--This research was supported in part by Grant No. CA 14030 from the National Cancer Institute and Grant No. C-582 from the Robert A. Welch
371
Foundation. We wish to thank Dr J. W. Campbell for interest and assistance in this study. REFERENCE~ BARANKIEWICZJ. (1973) Doctor's Dissertation. Inst. Biochem. Biophys., Polish Academy of Sciences. D~LAUN^YH. (1931) B~ol. Rev. 6, 26~-301. JEZEWSKAM. M, GORZKOWSK!B. & HELLERJ. (1964) Acta biochim, pol. II, 135-138. SPEEGK. V. JR. & CAMPBELLJ. W. (1966) Comp. Biochem. Physiol. ~6, 579-595.
0305-0491/79/0107-0369502.00/0
© Pergamon Press Lid 1979, Printed in Great Britain
PURINE BIOSYNTHESIS IN HELIX ASPERSA: METABOLIC FATE OF LABELLED PRECURSORS SANDRA W. CLARK and FREDERICK B. RUDOLPH* Department of Biochemistry, Rice University, Houston, TX 77001, U.S.A. (Received 4 December 1978)
Abstract--l. The hepatopancreas of terrestial snails actively synthesizes purines from labelled formic acid. 2. The guanine nucleotides are initially labelled to the highest specific activity followed by adenine and hypoxanthine. 3. The labelling patterns suggest that uric acid synthesis is the primary catabolic process for nitrogen excretion while guanine excretion is due to an inability to reutilize the base.
INTRODUCTION Terrestial snails are uricotelic species (Delauney, 1931) excreeting uric acid, xanthine and guanine as major end-products of nitrogen metabolism. The multiple excretion products suggest an unusual regulation of the catabolic pathways compared to other species. Generally, uric acid or occasionally guanine are the major excretion products with very little of any other base excreted. Speeg & Campbell (1966) have evaluated the nitrogen excretion of the snail and found that, in the kidney where the waste products accumulate, purines represent 90% of the total nitrogen present with uric acid accounting for about two-thirds of the total and approximately equal amounts of xanthine and guanine representing the other third. The hepatopancreas is the major organ for both de noco synthesis and catabolism of purines (Barankiewicz, 1973; Jezewska et al., 1964). To evaluate the ' various pathways of pt~rine metabolism in the snail, the animals were injected with ~4C-labelled formic acid and the distribution of the label in synthesized purines determined in the hepatopancreas. Pool sizes of the various soluble purines were also determined. MATERIALS AND METHODS Helix aspersa were collected locally and maintained under controlled laboratory conditions on a diet of lettuce. The snails were injected in the foot pad with 10/~1 of l~C-labeiled formate (10#Ci, specific activity of 60.7 #Ci/ gmol, supplied by New England Nuclear). Duplicate snails were sacrificed at various time periods. The hepatopancreas was removed immediately and minced in 1.5 mi of cold 0.5 M perchloric acid. The tissue was then homogenized with a Tekmar Tissumizer followed by centrifugation at 32,000@ for 1 hr. The clear supei~natant was collected and total radioactivity determined on an aliquot. For determination of the incorporation of the labelled formate into purine bases in the soluble fraction of the homogenates, an aliquot of the acid extract was heated at 100°C for 1 hr. After centrifugation, the supernatant was applied to a Dowex 50 column (200-400 mesh, H + form, 0.5 x 5 cm). The initial wash was collected for uric acid determination described below and the absorbed purine bases were eluted with 6 N hydrochloric acid. The pooled
* To whom correspondence should be addressed. 369
base fractions were lyophilized and then dissolved in 0.2 ml of H 2 0 . A 0.1 ml aliquot of the eluted base fraction was then applied to a Partisil 10-CX cation exchange column (0.5 x 25cm, Whatman) using a Glenco high-pressure liquid chromatography system. The column was equilibrated with 0.01 M NH4H2PO4, pH 3.5 and elution was done with the same buffer. Xanthine, hypoxanthine, guanine and adenine eluted from the column in that order. The concentrations of the bases and their identity were determined from the ultraviolet spectra in acid and base (Speeg & Campbell, 1966). Uric acid was determined by first extraction of the initial wash from the Dowex-50 column with 0.4 M alamine in freon to remove the perchloric acid. The sample was then applied to an anion-exchange column (Partisil 10-AX, 0.5 x 25cm, Whatman) using the Glenco high-pressure chromatography system. The column was equilibrated with 0.005 M potassium phosphate, pH 4.7, and the uric acid eluted with this buffer. The identity and amount of uric acid was determined spectroscopically as described above for the other purines. Various unknown compounds were encountered in the chromatographic procedures. To ensure that no contamination of the isolated bases added to the apparent label present, the isolated fractions were evaporated and then applied to a thin-layer cellulose sheet (E. Merck). The chromatographs were de~eloped with an isopropanol(170); water(36); hydrochloric acid(44) system. The amount of 14C was determined in each base from the chromatograph. The specific activity of each base was calculated from its concentration determined from the high-pressure chromatography system and the I~C present as determined from the second chromatograph. Tests were made with known amounts of labelled compounds and greater than 95T,, of the label was recovered. RESULTS AND DISCUSSION The incorporation of injected l'tC-labelled formic acid into the hepatopancreas with time is given in Table 1. The results are similar to those reported by Jezewska et al. (1964) for the incorporation of labelled glycine into bases in the snail. A reasonable amount of the label is incorporated allowing analysis of the metabolism in the hepatopancreas. The highest amount of label is present after 4 hr and as will be discussed below the highest specific activity of the bases occurs then. The persistence of the label even
370
SANDRA W. CLARK and FREDERICK B. RUDOLPH
Table 1. Incorporation of ~'~C-labelled formate into the hepatopancreas* Time after injection (hr)
Total counts (dpm)
Per cent of dose (%)
2 4 8 24
392,000 604,000 292,000 256,000
1.8 2.7 1.3 1.2
* The per cent represents total soluble counts in the hepatopancreas. Results from at least 2 animals were averaged to obtain the number reported. after 24 hr suggests incorporation into nucleic acids and continual turnover and salvage. Dietary and other sources of purines are not likely to have much effect on purine synthesis in the hepatopancreas if it is the major site of purines for nitrogen excretion. From the chromatographic separation of the bases extracted from the hepatopancreas, the total pool sizes for the various purines were determined (Table 2). It should be emphasized that these numbers represent the total of the free bases, nucleosides and nucleotides in the intact cell. Since it is a soluble extract, the purines in DNA and RNA are not included. The concentration of the adenine compounds is highest (1.12/~molg wet tissue) as would be expected with the guanine pool about 30% of the adenine pool. The pool sizes of xanthine and uric acid are somewhat variable. They frequently occur as the free bases in vivo and their concentrations will relate to circulating and kidney levels. A determination of adenine and guanine nucleotides (AMP, ADP, ATP, GMP, GDP, GTP) was done on an 8ohr incubation sample. The majority of the label was in the phosphorylated compounds (about 80%) for both adenine and guanine with very low levels of G M P and AMP. The di- and triphosphates were about the same concentration. Very low levels
of other phosphorylated purines were present. This supports the idea that xanthine and uric acid are catabolic products synthesized for nitrogen excretion while guanine is excreted only when in excess of metabolic requirements. The snail cannot deaminate guanine so the base has to be excreted intact (Barankiewicz, 1973). The relative rates of synthesis and catabolism of the various purines can be evaluated from the specific activity data of Table 3. The guanine pool is labelled very rapidly to a higher specific activity than either adenine or hypoxanthine. The lower labelling of hypoxanthine was unexpected since I M P is a precursor of G M P but is likely due to a considerable pool of either inosine or hypoxanthine being catabolized. The levels of I M P in the cell are too low to make meaningful measurements so the first labelling into purines cannot be determined. The specific activity of the catabolic products xanthine and uric acid do not reach a maximum until 4 h r after injection. The turnover in the adenine pool is quite slow, consistent with the many roles of adenine nucleotides and likely continuous salvage of the base. The guanine pool decreases more rapidly than adenine due to the smaller pool and incorporation of a higher percentage of the guanine into RNA and DNA. The rapid labelling of guanine in the hepatopancreas is in contrast to that observed in the kidney of the snail. Speeg & Campbell (1966) have observed that the uric acid was most rapidly labelled. This is consistent with the suggestion above that guanine is not a primary nitrogen excretion product. Uric acid and xanthine derived from I M P are synthesized more rapidly than guanine is derived from GMP. This is indicated in the data of Table 3 where the label in xanthine and uric acid is low after 24 hr. In summary, the purine metabolism of the snail has to be considered both from the standpoint of nitrogen excretion and synthesis of bases for cellular function. The results of this report clearly indicate
Table 2. Pool sizes of purines bases in the hepatopancreas* Time after injection (hr)
Adenine
Guanine
Base (~mol/g wet tissue) Hypoxanthine
Xanthine
Uric acid
2 4 8 24 Average
1.20 1.02 1.27 1.130 1.12 + 0.13
0.38 0.24 0.41 0.23 0.32 _ 0.09
0.38 0.21 0.40 0.23 0.31 +__0.10
0.64 0.49 0.79 0.15 0.51 + 0.22
1.78 0.45 l. 11 0.08 0.65 _+ 0.51
* The numbers represent the average of at least two determinations as described in Materials and Methods. Table 3. Specific activities of purine bases in the hepatopancreas after ~4C-formate injection* Time after injection (hr)
Adenine
Guanine
2 4 8 24
26,300 65,500 74,000 67,200
53,900 238,400 49,500 43,700
Specific activity (cpm//~mol) Hypoxanthine Xanthine 33,000 145,600 78,000 51,800
12,700 37,200 13,000 12,000
Uric acid 6000 23,000 21,000 trace
* The amount of label and concentration of each base was determined as described in Materials and Methods.
Purine biosynthesis in Helix aspersa that the excretion of guanine by the snail is a metabolic accident resulting from the loss of the ability to salvage the guanine base. Both the synthesis data described here for the hepatopancreas and the data of Spceg & Campbell (1966) are supportive of this conclusion.
Acknowledgements--This research was supported in part by Grant No. CA 14030 from the National Cancer Institute and Grant No. C-582 from the Robert A. Welch
371
Foundation. We wish to thank Dr J. W. Campbell for interest and assistance in this study. REFERENCE~ BARANKIEWICZJ. (1973) Doctor's Dissertation. Inst. Biochem. Biophys., Polish Academy of Sciences. D~LAUN^YH. (1931) B~ol. Rev. 6, 26~-301. JEZEWSKAM. M, GORZKOWSK!B. & HELLERJ. (1964) Acta biochim, pol. II, 135-138. SPEEGK. V. JR. & CAMPBELLJ. W. (1966) Comp. Biochem. Physiol. ~6, 579-595.
