Conversion of 2-¹⁴C-Pteroyl-L-Monoglutamic Acid into Isoxanthopterin in Drosophila melanogaster

Abstract

2-¹⁴C-Pteroyl-L-monoglutamic acid (0.5 mc. per millimole, 1 mgm.) was incorporated into banana food medium (100 gm.), and adult flies of Drosophila melanogaster (Oregon, wild-type) were allowed to feed and lay eggs on this medium. Larvæ (six days old) were removed from this medium, transferred to moist filter paper and allowed to pupate. Pupæ (100; 86 mgm.; not less than 48 hr., not more than 56 hr. pupation) were crushed with sand and 0.5 N aqueous ammonia solution (0.5 ml.) and heated on a steambath (10 min.). Solids were centrifuged off and supernatant liquid spotted on Whatman 3 MM paper. The solids were washed again with 0.5 N aqueous ammonia solution (0.4 ml.), centrifuged off and supernatant liquid added to the first spot. The paper area was cut out and radioactivity measured by immersion in a solution of 0.3 per cent diphenyloxazole in toluene (2 ml.) and counting with a ‘Panax’ scintillation counter (background, 1.096 counts/sec; background + sample, 1.357 counts/sec; sample, 0.26 count/sec; standard error, ± 0.035 count/sec). The radioactive area of the paper was chromatographed on 3 MM paper with propanol/5 per cent aqueous acetic acid (2 : 1 vol. by vol.) as developing solvent. To improve spot resolution, the paper was developed three times. On viewing in 365-mµ light, a bright purple fluorescent area was seen. This was cut out and chromatographed on 3 MM paper with butanol/acetic acid/water (4 : 1 : 5 vol. by vol.) (triple development). After air-drying, the paper was viewed in 365 mµ light and the fluorescent spots of isoxanthopterin (purple) and 2-amino-4-hydroxy-pteridine-6-carboxylic acid (bright blue) cut out. The acid spot was measured for radioactivity (background, 0.65 count/sec; background + sample, 1.10 counts/ sec; sample, 0.45 count/sec; standard error, 0.033 count/sec). The isoxanthopterin spot was further purified by chromatography on 3 MM paper with tert.-butanol/pyridine/water (50 : 15 : 35 vol. by vol.). The isoxanthopterin spot was measured for radioactivity (background, 0.65 count/sec; background + sample, 0.76 count/sec; sample, 0.11 count/sec.: standard error, 0.030 count/sec).