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Uric acid in nucleic and amino acid synthesis in the boll weevil, Anthonomus grandis
J. Insect Physiol., 1973, Vol. 19, pp.
1569 to 1574. Pergamon Press. Printed in Great Britain
URIC ACID IN NUCLEIC AND AMINO ACID SYNTHESIS IN THE BOLL WEEVIL, ANTHONOMUS GRANDIS NORMAN Roll Weevil
Research
MITLIN
and
GLENN
WIYGUL
Laboratory, Agr. Res. Serv., U.S.D.A., Mississippi 39762, U.S.A.
(Received 19January
Mississippi
State,
1973)
fibstract-When
uric acid-2-i% was injected into the boll weevil, Anthonomus to RNA, DNA, amino acid, and i4C0, at the end of ;I hr. The free amino acids, lipoamino acids, and protein amino acids were all labelled with the free amino acids showing the highest specific activity. Incorporation in DNA was slight, but it was extensive in RNA ; cytidylic acid showed the greatest amount of incorporation.
grandis, it was metabolized
INTRODUCTION IN A preliminary study, MITLIN and MAULDIN (1966) reported that uric acid in the boll weevil, Anthonomusgrandis Boheman, was in a dynamic state and served as a nutritional source. This participation of uric acid in the metabolic processes is not unique to this insect since HILLIARD and BUTZ (1969) reported a daily fluctuation in the concentration of uric acid in the haemolymph of the American cockroach, Peripltzneta ame-ricuna, and MCENROE (1966) reported that labelled uric acid was metabolized to 14C0, in the same insect. However, the earlier study was made with less precise thin-layer chromatography. It was therein reported that after the injiections of uric acid-2-14C, 6 free amino acids were consistently found to be radioactive. That study was therefore expanded, and more accurate gas chromatographic methods were used to get quantitative data for the free amino acids and also for the amino acids of the lipoprotein and protein fractions of the weevil. Additionally, we were interested to see to what extent uric acid contributed to nucleic acid synthesis within the conditions of the experiment.
MATERIALS
AND
METHODS
The insects used were newly emerged adult ebony male boll weevils derived from t.he laboratory colony and reared by the method of GAST (1966). The uric acid-2-14C used in the test was purchased from Amersham-Searle, Arlington Heights, Ill., U.S.A. It had a specific activity of 52.0 mCi/m-mole. Radiopurity was in excess of 97% and was determined by paper chromatography in four systems: (1) n-butanol-ethoxyethanol-acetic acid-water (70 : 70 : 20 : 40), (2) ethanol-ammonia-water (80 : 4 : 16), (3) methanol-pyridine-water (160 : 8 : 80), (4) tervt-butanol-ethyl methyl ketone-formic acid-water (40 : 30 :15 : 15). 1569
1570
NORMANMITLIN ANDGLENN WIYGUL
Experimental procedure For each experiment, 80 to 100 weevils were each injected with 0.7 ~1 of solution of labelled uric acid dissolved in 0.1 N NaOH (preliminary tests showed the dose was nontoxic) with a Dutky-Fest microinjector, held for 2 hr, and then frozen. The frozen weevils were subsequently ground in a glass homogenizer with 0.25 N perchloric acid and centrifuged. The supernatants contained the free amino acids. The residue was extracted with ethanol and ethanol-ether for lipoprotein and then with 5% trichloroacetic acid to obtain the nucleic acids. The nucleic acid fraction was discarded, and the lipoprotein and the final protein residue were retained for hydrolysis. Alternatively, to obtain RNA and DNA separately as hydrolysates, we extracted the free amino acids and lipoproteins from other groups of weevils as before. Then tissue residue was treated with 1.0 N KOH and held for 3 days at 37°C (SCHMIDT and THANNHAUSER, 1945). After the RNA hydrolysate was removed, the DNA residue was hydrolysed with trifluoroacetic acid to free the constituent bases (KLEINSCHMIDT and MANTHEY, 1958). Also, the RNA hydrolysate (nucleotides) was further hydrolysed with hydrochloric acid to obtain the purine bases and pyrimidine nucleotides. The lipoprotein fractions obtained as noted were taken to dryness. Then these and the protein fractions were hydrolysed to free amino acids by adding to them an excess of 6 N HCl in ampoules which were then filled with nitrogen and sealed. After the ampoules were heated for 18 hr at llO”C, they were chilled and opened, and the humin was filtered off. The lipoprotein hydrolysates were partitioned with ethyl ether to remove the fatty acids, and the amino acid fraction thereof and the protein hydrolysates were evaporated to dryness on a rotary evaporator, washed with water, and evaporated; the amino acids were finally taken up in 0.1 N HCl. Chromatography The nucleic acid bases and nucleotides were chromatographed by WYATT’S (1951) procedure on paper. The resultant spots (located by shortwave ultraviolet light) were cut out, and each was rolled in a cylinder, placed in a counting vial containing scintillation fluid, and counted. The amino acids as IV-trifluoroacetyl n butyl ester derivatives were chromatographed by gas-liquid chromatography after preparation by the method of ZUMWALT et al. (1970). All three fractions, the free amino acids and the lipoprotein and protein hydrolysates, were cleaned by passing them through a column containing Dowex 50@ (lOO/ZOO mesh) anion exchange resin as set forth in the method. A Micro-Tek@ Model GC 2000 R dual hydrogen flame gas chromatograph was used, first with a 6 ft x $ in. coiled glass column and later, when experience showed that better resolution could be achieved, with an 8 ft x & in. glass column packed with Tabsorb @. The second column was a 6 ft x t in. coiled glass column packed with SO/l00 mesh OV 17 (1%) on chromosorb G. The chromatograph was equipped with a stream splitter, which allowed us to capture the radioactive
URIC ACID IN NUCLEIC
fractions directly then be counted.
AND AMINO ACID SYNTHESIS
into a vial containing
a toluene-based
IN THE BOLL WEEVIL
scintillation
1571
fluid that could
Measurement of 14C0, Individual weevils were injected with labelled uric acid and housed in a CO, collection apparatus. The expired 14C0, was captured in a methyl cellosolveethano!amine (7 : 3) mixture. Aliquots of the fluid were incorporated into scintillation fluid and counted. RESULTS
AND DISCUSSION
Nucleic acids Both the RNA and DNA fractions from injected weevils were radioactive though so little activity was found in the DNA fraction that we could not obtain precise figures for the percentages of each base. This result was not unexpected since we know from earlier studies that little DNA is synthesized within 2 hr (MITLIN and WIYGUL, 1969). The RNA, however, was highly labelled, the amount of incorporation representing 0.2-0.4 per cent of the injected dose. On an average, 0.02 per cent of the injected radioactivity of uric acid was found as DNA and 1.6 per cent as RNA. The average percentages of radioactivity in the constituent bases and in nucleotides of RNA were: guanine 11.5, adenine 22.8, cytidylic acid 45.8, and uridylic acid 19% The ratios of incorporation were . . cytidyhc acid = 2.4, Purines = 0 52 uridylic acid pyrimidines ’ ’ These figures are difficult to explain, particularly the relatively large amount of incorporation into cytidylic acid. Perhaps this latter result occurs because of HCOs- recycling, or it may merely reflect an artifact in methodology. Uric acid and cytidylic acid have different R, values in the chromatographic system used, but there is always the possibility that not all the purine was metabolized and that some overlapping occurred. In any case, the carbon labelling for both nucleic acids would appear to have been derived from the catabolism of uric acid to labelled CO, and thence to carbamyl phosphate since this appears to be the most probable way by which the amino acids were labelled. That 14C0, was formed was shown in the 14C0, capture experiment: 14C0, accounted for 1.5 per cent of the injected radioactivity at the end of 2 hr. Amino acids Nineteen amino acids could be detected with certainty with the gas chromatographic method used (Table 1). Also, a number of the minor peaks found may represent other amino acids, but we could not identify them unequivocally. All those identified were labelled. In the fraction containing free amino acids the isoleucine, leucine, greatest specific activity was shown in valine, threonine, methiordne, tryptophan, cysteine, and serine. The first six of these acids are
NORMAN~UITLIN ANDGLENN WIYGUL
1572
labelled.
essential
for the weevil (VANDERZANT, 1965) as are a number of the others that were Thus, showing again (MITLIN and WIYGUL, 1969) these acids are endo-
genously
synthesized.
about
In all, the fraction
1.2 per cent of the injected
containing
free amino acid accounted
for
radioactivity.
TABLE ~-QUANTITIES OF AMINOACID( + S.E.) PRESENTIN MALEBOLL WEEVILSANDAMOUNTS OF URIC ACID CONVERTEDTO EACHAMINOACID (PER AMOUNTOF AMINOACID) Free amino acid
Lipoprotein amino acid
Protein amino acids
Amino acid
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Alanine Valine Glycine Isoleucine Leucine Proline Threonine Serine Cysteine Methionine Phenylalanine Aspartic acid Glutamic acid Tyrosine Lysine Tryptophan Arginine Histidine Cystine
37*2+ 3.7 14.7 f 2.2 29.5 + 3.0 6.2+ 0.8 10.7 + 2.8 44.8 + 0.9 5.6+ 1.0 6.3 + l-2 0.9* 0.2 2.5 -e 0.5 9.3* 1.4 4.5 + 0.9 82.2 + 16.7 30.4+ 5.3 8.0&- l-7 2.7+ 2.2 3.9+ 0.7 5.0* 0.4 Trace
7.6 12.9 6.7 22.2 16.1 2.4 13.7 11.4 46.5 10.3 4.3 8.2 1.1 1.1 3.6 9.1 1.6 1.7
304.4 f 45.6
180.5
Totals
The
differences
and in an earlier either
strain
in quantities
study (MITLIN
or differences
92.8 + 16.0 74.2 f 14.3 157.0 + 26.9 36.8 + 7.8 104.7 + 22.4 74.8 + 11.4 19.1 + 2.4 20.0 f 3.8 4.3 f 0.1 1.9f 0.2 46.0f 8.9 65.9+ 7.1 167.5 f 19.7 90-O +_20.1 18.3 + 2.9 -
0.02 0.39 0.10 0.28 0.15 0.05 0.12 0.20 0.79 1.50 0.05 0.04 0.02 0.04 0.20 -
320.0 f 38.5 645.1 + 126.9 1001.8 + 83.1 339.7 _+ 52.8 831.6 f 145.1 527.3 f 69.3 192.5 f 43.7 125.7 f 10.0 60.0 + 0.3 154.2 + 27.2 247.9 f 32.4 516.3 + 71.0 259.5 f 42.1 90.5 It: 5.5 281 .O+ 69.2 -
0.21 0.11 0.06 0.15 0.06 0.10 0.26 0.04 1.23 040 0.29 0.11 0.28 0.77 0.25 -
18.3 f 2.9 23.7 + 4.5 -
0.09 0.09 -
63.7 &- 9.5 55*2+ 7.3 31*4* 4.7
0.67 1.06 1.48
1015.3 + 172.0
of free amino
4.13
acids found
5743.4 k 1185.1
in the present
and WIYGUL, 1970) can probably
in diet rather
than methodology.
7.53
study
be attributed
In the earlier
to
study,
we employed an ebony mutant; in the present study, we used an ebony-pearl eyed strain. CHEN (1966) d emonstrated that differences in free amino acids occur in different mutants. Also, more recent work in our laboratory (unpublished data) has shown that amounts of free amino acids in boll weevils vary with diet (the ingredients
of the standard
diet, which
the years between the two studies). The lipoprotein fraction accounted
is essentially
empirical,
for only a small part (0.14
have changed
in
per cent) of the
radioactivity attributable to uric acid (Table 1) despite the fact that the total amount of amino acids was greater than the total amount of free amino acids.
URIC ACID IN NUCLEIC ANDAMINOACIDSYNTHESISIN THE BOLLWEEVIL
1573
This finding may be attributable to the fact that the lipoamino acids are precursors of the proteins (MITLIN et al., 1968) and that activity is therefore lost quickly. The greatest amount of labelling was found in 7 amino acids: cysteine, valine, threonine, leucine, and isoleucine, serine, and methionine. Again, 5 of those labelled are essential amino acids. Among the protein amino acids, the basic and two sulphur amino acids, cyteine and cystine, had the highest specific activity (Table 1). In all, protein labelling accounted for 083 per cent of the administered radioactivity. Generally, the greatest amount of radioactivity was found in the fraction containing the free amino acids, possibly because of the timing of the test. Perhaps more incorporation would have been found if the weevils had been killed at a later time after the administration of the uric acid. However, we have no explanation for the high activity of the sulphur amino acids throughout all three fractions. At 2 hr after administration of the labelled uric acid, 3.8 per cent could be accounted for as nucleic acids and amino acids; 1.5 per cent more was found as 14C0,. If one assumes that the nitrogen accompanies the carbon, the purine has a potentially large role as a source of nitrogen for the boll weevil. Earlier we showed (MITL.[N and MAULDIN, 1966) that the weevil accumulates relatively large amounts of uric acid in its tissues during the larval and pupal periods and that the quantity drops precipitously at the first day after eclosion. Some of this drop is, of course, attributable to the loss in the meconium, but the increase in the total level of nitrogen probably means that the weevil utilizes at least some of the reserve of uric acid as a source of nitrogen. We accounted for only a relatively small amount of the injected uric acid present in the treated weevils after 2 hr. However, we did not consider the free nucleotides nucleoaides, and bases and the other nitrogenous metabolites to which the uric acid undoubtedly contributed. Neither do we know with any degree of certainty Although we have not yet found an active uricase how the purine is catabolized. in the boll weevil, we suspect that one exists since we have found labelled allantoin in the insect after injection with labelled uric acid (unpublished data). The possibility exists, of course, that in the boll weevil, as in the cockroach, Blatta ( = Periplaneta) orient& (KELLER, 1950), symbiotic bacteria are responsible for the metabolism of uric acid. Such has not been demonstrated in this laboratory, and there have been no reports of intracellular organisms in the boll weevil. REFERENCES CHEN P. S.
(1966) Amino acid and protein development
in insect development.
Adv. Insect
Physiol. 3, 53-132. GAST R.. T. (1966) 3. eton. Ent. 59, HILLIAF:DS. D. and and uric acid in
Oviposition
and fecundity
173-176. BUTZ A. (1969) the hemolymph
71-74. KELLER H. (1950) Die Kultur 2. Natwf. 5B, 269-273.
of boll weevils
in mass-rearing
cultures.
Daily fluctuations in the concentration of total sugar of Periplaneta americana. Ann. ent. Sot. Am. 62,
der intrazellularen
Symbionten
von Periplaneta
orientalis.
1574
NORMANMITLIN ANDGLENN WIYCUL
KLEINSCHMIDTW. J. and MANTHEYJ. A. (1958) Q uantitative determination of nucleic acid in whole tissue by paper chromatography. Archs Biochem. Biophys. 73, 5244. MCENROE W. (1966) In viva preferential oxidation of ureide carbon No. 2 of uric acid by Periplaneta americana. Ann. ent. Sot. Am. 59, 1011. MITLIN N. and MAULDINJ. K. (1966) Uric acid in nitrogen metabolism of the boll weevil: a preliminary study. Ann. ent. Sot. Am. 59, 6.51-653. MITLIN N. and WIYGUL G. (1969) Incorporation and metabolism of ‘%-labeled tryptophan3 in the boll weevil, Anthonomusgrandis Boheman. Comp. Biochem. Physiol. 30, 375-381. MITLIN N. and WIYGUL G. (1970) Effect of y irradiation on utilization of glycine carbons in biosynthesis of RNA and amino acids in the boll weevil. r. Insect Physiol. 16,2271-2279. MITLIN N., WIYGUL G., and LUSK G. J. (1968) Incorporation of lysine 6-l% into the protein of the adult boll weevil, Anthonomus grandis. J. Insect Physiol. 14, 1277-1283. SCHMIDT G. and THANNHAUSER S. J. (1945) A method for the determination of desoxyribonucleic acid, ribonucleic acid and phospho-protein in animal tissues. J. biol. Chem. 161, 83-89. VANDERZANT,E. (1965) Axenic rearing of the boll weevil on defined diets: amino acid, carbohydrates, and mineral requirements. J. Insect Physiol. 11, 659-670. WYATT G. R. (1951) The purine and pyrimidine composition of deoxypentose nucleic acids. Biochem. J. 48, 584-590. ZIJMWALTR. W., ROACHD., and GEHRKEC. W. (1970) Gas-liquid chromatography of amino acids in biological substances. J. Chromat. 53, 171-193.
1569 to 1574. Pergamon Press. Printed in Great Britain
URIC ACID IN NUCLEIC AND AMINO ACID SYNTHESIS IN THE BOLL WEEVIL, ANTHONOMUS GRANDIS NORMAN Roll Weevil
Research
MITLIN
and
GLENN
WIYGUL
Laboratory, Agr. Res. Serv., U.S.D.A., Mississippi 39762, U.S.A.
(Received 19January
Mississippi
State,
1973)
fibstract-When
uric acid-2-i% was injected into the boll weevil, Anthonomus to RNA, DNA, amino acid, and i4C0, at the end of ;I hr. The free amino acids, lipoamino acids, and protein amino acids were all labelled with the free amino acids showing the highest specific activity. Incorporation in DNA was slight, but it was extensive in RNA ; cytidylic acid showed the greatest amount of incorporation.
grandis, it was metabolized
INTRODUCTION IN A preliminary study, MITLIN and MAULDIN (1966) reported that uric acid in the boll weevil, Anthonomusgrandis Boheman, was in a dynamic state and served as a nutritional source. This participation of uric acid in the metabolic processes is not unique to this insect since HILLIARD and BUTZ (1969) reported a daily fluctuation in the concentration of uric acid in the haemolymph of the American cockroach, Peripltzneta ame-ricuna, and MCENROE (1966) reported that labelled uric acid was metabolized to 14C0, in the same insect. However, the earlier study was made with less precise thin-layer chromatography. It was therein reported that after the injiections of uric acid-2-14C, 6 free amino acids were consistently found to be radioactive. That study was therefore expanded, and more accurate gas chromatographic methods were used to get quantitative data for the free amino acids and also for the amino acids of the lipoprotein and protein fractions of the weevil. Additionally, we were interested to see to what extent uric acid contributed to nucleic acid synthesis within the conditions of the experiment.
MATERIALS
AND
METHODS
The insects used were newly emerged adult ebony male boll weevils derived from t.he laboratory colony and reared by the method of GAST (1966). The uric acid-2-14C used in the test was purchased from Amersham-Searle, Arlington Heights, Ill., U.S.A. It had a specific activity of 52.0 mCi/m-mole. Radiopurity was in excess of 97% and was determined by paper chromatography in four systems: (1) n-butanol-ethoxyethanol-acetic acid-water (70 : 70 : 20 : 40), (2) ethanol-ammonia-water (80 : 4 : 16), (3) methanol-pyridine-water (160 : 8 : 80), (4) tervt-butanol-ethyl methyl ketone-formic acid-water (40 : 30 :15 : 15). 1569
1570
NORMANMITLIN ANDGLENN WIYGUL
Experimental procedure For each experiment, 80 to 100 weevils were each injected with 0.7 ~1 of solution of labelled uric acid dissolved in 0.1 N NaOH (preliminary tests showed the dose was nontoxic) with a Dutky-Fest microinjector, held for 2 hr, and then frozen. The frozen weevils were subsequently ground in a glass homogenizer with 0.25 N perchloric acid and centrifuged. The supernatants contained the free amino acids. The residue was extracted with ethanol and ethanol-ether for lipoprotein and then with 5% trichloroacetic acid to obtain the nucleic acids. The nucleic acid fraction was discarded, and the lipoprotein and the final protein residue were retained for hydrolysis. Alternatively, to obtain RNA and DNA separately as hydrolysates, we extracted the free amino acids and lipoproteins from other groups of weevils as before. Then tissue residue was treated with 1.0 N KOH and held for 3 days at 37°C (SCHMIDT and THANNHAUSER, 1945). After the RNA hydrolysate was removed, the DNA residue was hydrolysed with trifluoroacetic acid to free the constituent bases (KLEINSCHMIDT and MANTHEY, 1958). Also, the RNA hydrolysate (nucleotides) was further hydrolysed with hydrochloric acid to obtain the purine bases and pyrimidine nucleotides. The lipoprotein fractions obtained as noted were taken to dryness. Then these and the protein fractions were hydrolysed to free amino acids by adding to them an excess of 6 N HCl in ampoules which were then filled with nitrogen and sealed. After the ampoules were heated for 18 hr at llO”C, they were chilled and opened, and the humin was filtered off. The lipoprotein hydrolysates were partitioned with ethyl ether to remove the fatty acids, and the amino acid fraction thereof and the protein hydrolysates were evaporated to dryness on a rotary evaporator, washed with water, and evaporated; the amino acids were finally taken up in 0.1 N HCl. Chromatography The nucleic acid bases and nucleotides were chromatographed by WYATT’S (1951) procedure on paper. The resultant spots (located by shortwave ultraviolet light) were cut out, and each was rolled in a cylinder, placed in a counting vial containing scintillation fluid, and counted. The amino acids as IV-trifluoroacetyl n butyl ester derivatives were chromatographed by gas-liquid chromatography after preparation by the method of ZUMWALT et al. (1970). All three fractions, the free amino acids and the lipoprotein and protein hydrolysates, were cleaned by passing them through a column containing Dowex 50@ (lOO/ZOO mesh) anion exchange resin as set forth in the method. A Micro-Tek@ Model GC 2000 R dual hydrogen flame gas chromatograph was used, first with a 6 ft x $ in. coiled glass column and later, when experience showed that better resolution could be achieved, with an 8 ft x & in. glass column packed with Tabsorb @. The second column was a 6 ft x t in. coiled glass column packed with SO/l00 mesh OV 17 (1%) on chromosorb G. The chromatograph was equipped with a stream splitter, which allowed us to capture the radioactive
URIC ACID IN NUCLEIC
fractions directly then be counted.
AND AMINO ACID SYNTHESIS
into a vial containing
a toluene-based
IN THE BOLL WEEVIL
scintillation
1571
fluid that could
Measurement of 14C0, Individual weevils were injected with labelled uric acid and housed in a CO, collection apparatus. The expired 14C0, was captured in a methyl cellosolveethano!amine (7 : 3) mixture. Aliquots of the fluid were incorporated into scintillation fluid and counted. RESULTS
AND DISCUSSION
Nucleic acids Both the RNA and DNA fractions from injected weevils were radioactive though so little activity was found in the DNA fraction that we could not obtain precise figures for the percentages of each base. This result was not unexpected since we know from earlier studies that little DNA is synthesized within 2 hr (MITLIN and WIYGUL, 1969). The RNA, however, was highly labelled, the amount of incorporation representing 0.2-0.4 per cent of the injected dose. On an average, 0.02 per cent of the injected radioactivity of uric acid was found as DNA and 1.6 per cent as RNA. The average percentages of radioactivity in the constituent bases and in nucleotides of RNA were: guanine 11.5, adenine 22.8, cytidylic acid 45.8, and uridylic acid 19% The ratios of incorporation were . . cytidyhc acid = 2.4, Purines = 0 52 uridylic acid pyrimidines ’ ’ These figures are difficult to explain, particularly the relatively large amount of incorporation into cytidylic acid. Perhaps this latter result occurs because of HCOs- recycling, or it may merely reflect an artifact in methodology. Uric acid and cytidylic acid have different R, values in the chromatographic system used, but there is always the possibility that not all the purine was metabolized and that some overlapping occurred. In any case, the carbon labelling for both nucleic acids would appear to have been derived from the catabolism of uric acid to labelled CO, and thence to carbamyl phosphate since this appears to be the most probable way by which the amino acids were labelled. That 14C0, was formed was shown in the 14C0, capture experiment: 14C0, accounted for 1.5 per cent of the injected radioactivity at the end of 2 hr. Amino acids Nineteen amino acids could be detected with certainty with the gas chromatographic method used (Table 1). Also, a number of the minor peaks found may represent other amino acids, but we could not identify them unequivocally. All those identified were labelled. In the fraction containing free amino acids the isoleucine, leucine, greatest specific activity was shown in valine, threonine, methiordne, tryptophan, cysteine, and serine. The first six of these acids are
NORMAN~UITLIN ANDGLENN WIYGUL
1572
labelled.
essential
for the weevil (VANDERZANT, 1965) as are a number of the others that were Thus, showing again (MITLIN and WIYGUL, 1969) these acids are endo-
genously
synthesized.
about
In all, the fraction
1.2 per cent of the injected
containing
free amino acid accounted
for
radioactivity.
TABLE ~-QUANTITIES OF AMINOACID( + S.E.) PRESENTIN MALEBOLL WEEVILSANDAMOUNTS OF URIC ACID CONVERTEDTO EACHAMINOACID (PER AMOUNTOF AMINOACID) Free amino acid
Lipoprotein amino acid
Protein amino acids
Amino acid
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Pg nM amino ‘uric acid acid/insect equiv.‘/nM
Alanine Valine Glycine Isoleucine Leucine Proline Threonine Serine Cysteine Methionine Phenylalanine Aspartic acid Glutamic acid Tyrosine Lysine Tryptophan Arginine Histidine Cystine
37*2+ 3.7 14.7 f 2.2 29.5 + 3.0 6.2+ 0.8 10.7 + 2.8 44.8 + 0.9 5.6+ 1.0 6.3 + l-2 0.9* 0.2 2.5 -e 0.5 9.3* 1.4 4.5 + 0.9 82.2 + 16.7 30.4+ 5.3 8.0&- l-7 2.7+ 2.2 3.9+ 0.7 5.0* 0.4 Trace
7.6 12.9 6.7 22.2 16.1 2.4 13.7 11.4 46.5 10.3 4.3 8.2 1.1 1.1 3.6 9.1 1.6 1.7
304.4 f 45.6
180.5
Totals
The
differences
and in an earlier either
strain
in quantities
study (MITLIN
or differences
92.8 + 16.0 74.2 f 14.3 157.0 + 26.9 36.8 + 7.8 104.7 + 22.4 74.8 + 11.4 19.1 + 2.4 20.0 f 3.8 4.3 f 0.1 1.9f 0.2 46.0f 8.9 65.9+ 7.1 167.5 f 19.7 90-O +_20.1 18.3 + 2.9 -
0.02 0.39 0.10 0.28 0.15 0.05 0.12 0.20 0.79 1.50 0.05 0.04 0.02 0.04 0.20 -
320.0 f 38.5 645.1 + 126.9 1001.8 + 83.1 339.7 _+ 52.8 831.6 f 145.1 527.3 f 69.3 192.5 f 43.7 125.7 f 10.0 60.0 + 0.3 154.2 + 27.2 247.9 f 32.4 516.3 + 71.0 259.5 f 42.1 90.5 It: 5.5 281 .O+ 69.2 -
0.21 0.11 0.06 0.15 0.06 0.10 0.26 0.04 1.23 040 0.29 0.11 0.28 0.77 0.25 -
18.3 f 2.9 23.7 + 4.5 -
0.09 0.09 -
63.7 &- 9.5 55*2+ 7.3 31*4* 4.7
0.67 1.06 1.48
1015.3 + 172.0
of free amino
4.13
acids found
5743.4 k 1185.1
in the present
and WIYGUL, 1970) can probably
in diet rather
than methodology.
7.53
study
be attributed
In the earlier
to
study,
we employed an ebony mutant; in the present study, we used an ebony-pearl eyed strain. CHEN (1966) d emonstrated that differences in free amino acids occur in different mutants. Also, more recent work in our laboratory (unpublished data) has shown that amounts of free amino acids in boll weevils vary with diet (the ingredients
of the standard
diet, which
the years between the two studies). The lipoprotein fraction accounted
is essentially
empirical,
for only a small part (0.14
have changed
in
per cent) of the
radioactivity attributable to uric acid (Table 1) despite the fact that the total amount of amino acids was greater than the total amount of free amino acids.
URIC ACID IN NUCLEIC ANDAMINOACIDSYNTHESISIN THE BOLLWEEVIL
1573
This finding may be attributable to the fact that the lipoamino acids are precursors of the proteins (MITLIN et al., 1968) and that activity is therefore lost quickly. The greatest amount of labelling was found in 7 amino acids: cysteine, valine, threonine, leucine, and isoleucine, serine, and methionine. Again, 5 of those labelled are essential amino acids. Among the protein amino acids, the basic and two sulphur amino acids, cyteine and cystine, had the highest specific activity (Table 1). In all, protein labelling accounted for 083 per cent of the administered radioactivity. Generally, the greatest amount of radioactivity was found in the fraction containing the free amino acids, possibly because of the timing of the test. Perhaps more incorporation would have been found if the weevils had been killed at a later time after the administration of the uric acid. However, we have no explanation for the high activity of the sulphur amino acids throughout all three fractions. At 2 hr after administration of the labelled uric acid, 3.8 per cent could be accounted for as nucleic acids and amino acids; 1.5 per cent more was found as 14C0,. If one assumes that the nitrogen accompanies the carbon, the purine has a potentially large role as a source of nitrogen for the boll weevil. Earlier we showed (MITL.[N and MAULDIN, 1966) that the weevil accumulates relatively large amounts of uric acid in its tissues during the larval and pupal periods and that the quantity drops precipitously at the first day after eclosion. Some of this drop is, of course, attributable to the loss in the meconium, but the increase in the total level of nitrogen probably means that the weevil utilizes at least some of the reserve of uric acid as a source of nitrogen. We accounted for only a relatively small amount of the injected uric acid present in the treated weevils after 2 hr. However, we did not consider the free nucleotides nucleoaides, and bases and the other nitrogenous metabolites to which the uric acid undoubtedly contributed. Neither do we know with any degree of certainty Although we have not yet found an active uricase how the purine is catabolized. in the boll weevil, we suspect that one exists since we have found labelled allantoin in the insect after injection with labelled uric acid (unpublished data). The possibility exists, of course, that in the boll weevil, as in the cockroach, Blatta ( = Periplaneta) orient& (KELLER, 1950), symbiotic bacteria are responsible for the metabolism of uric acid. Such has not been demonstrated in this laboratory, and there have been no reports of intracellular organisms in the boll weevil. REFERENCES CHEN P. S.
(1966) Amino acid and protein development
in insect development.
Adv. Insect
Physiol. 3, 53-132. GAST R.. T. (1966) 3. eton. Ent. 59, HILLIAF:DS. D. and and uric acid in
Oviposition
and fecundity
173-176. BUTZ A. (1969) the hemolymph
71-74. KELLER H. (1950) Die Kultur 2. Natwf. 5B, 269-273.
of boll weevils
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cultures.
Daily fluctuations in the concentration of total sugar of Periplaneta americana. Ann. ent. Sot. Am. 62,
der intrazellularen
Symbionten
von Periplaneta
orientalis.
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NORMANMITLIN ANDGLENN WIYCUL
KLEINSCHMIDTW. J. and MANTHEYJ. A. (1958) Q uantitative determination of nucleic acid in whole tissue by paper chromatography. Archs Biochem. Biophys. 73, 5244. MCENROE W. (1966) In viva preferential oxidation of ureide carbon No. 2 of uric acid by Periplaneta americana. Ann. ent. Sot. Am. 59, 1011. MITLIN N. and MAULDINJ. K. (1966) Uric acid in nitrogen metabolism of the boll weevil: a preliminary study. Ann. ent. Sot. Am. 59, 6.51-653. MITLIN N. and WIYGUL G. (1969) Incorporation and metabolism of ‘%-labeled tryptophan3 in the boll weevil, Anthonomusgrandis Boheman. Comp. Biochem. Physiol. 30, 375-381. MITLIN N. and WIYGUL G. (1970) Effect of y irradiation on utilization of glycine carbons in biosynthesis of RNA and amino acids in the boll weevil. r. Insect Physiol. 16,2271-2279. MITLIN N., WIYGUL G., and LUSK G. J. (1968) Incorporation of lysine 6-l% into the protein of the adult boll weevil, Anthonomus grandis. J. Insect Physiol. 14, 1277-1283. SCHMIDT G. and THANNHAUSER S. J. (1945) A method for the determination of desoxyribonucleic acid, ribonucleic acid and phospho-protein in animal tissues. J. biol. Chem. 161, 83-89. VANDERZANT,E. (1965) Axenic rearing of the boll weevil on defined diets: amino acid, carbohydrates, and mineral requirements. J. Insect Physiol. 11, 659-670. WYATT G. R. (1951) The purine and pyrimidine composition of deoxypentose nucleic acids. Biochem. J. 48, 584-590. ZIJMWALTR. W., ROACHD., and GEHRKEC. W. (1970) Gas-liquid chromatography of amino acids in biological substances. J. Chromat. 53, 171-193.