High-pressure liquid-chromatographic assay of nucleotide-pool concentrations during polysaccharide biosynthesis in four ascomycetes

Abstract

High-resolution liquid-chromatographic methods developed for analyzing nucleotide pools at the nanogram level in four representative species of ascomycetes ( Penicillium citrinum, Aspergillus niger, Fusarium moniliforme, and Cladosporium herbarum) were used to study polysaccharide biosynthesis. Nucleotides extracted from the mycelial mat were preseparated from interfering polysaccharides, glycoproteins, and nucleic acids on a column of Biogel P-2. Resolution of 18 nucleotides from each fungal species was accomplished on AS-Pellionex-SAX, pellicular anion-exchanger by using a high-pressure liquid chromatograph. Nucleotides were identified by comparing peak retention-times, by differential u.v. absorption with two detectors in series at selected wavelenghts, and by acid or enzymic hydrolysis with product identification by liquid chromatography. Pyrimidine bases exceeded purines by at least three fold, and uridine nucleotides often constituted 60-80 mole percent of the total nucleotides; extractable cytidine nucleotides were negligible. Uridine 5'-(2- acetamido-2-deoxy-α-D-glucopyranosyl disphosphate) is the preponderant nucleotide throughout the growth cycles of all four species, amounting to 30-60% of all nucleotides present. For all four fungal species, a burst of nucleotide formation was observed after the first 48 h (15-30 μmol/g tissue), with fluctuations that eventually fell to 0.1 μmmol/g on the tenth day.