The active transport and metabolism of purines in the yeast, candida utilis

Abstract

In a study of the assimilation of a number of nucleic acid derivatives by Candida (Torulopsis) utilis, Di Carlo, Schultz, and McManus ('51) found that the nitrogen of all of the naturally occurring purines that were tested was utilized for growth. It was suggested that C . utilis contains the enzymes adenase, guanase, hypoxanthine oxidase, xanthine oxidase, and uricase. Following this suggestion, Roush ('54) found that adenine induces the formation of a specific adenase in C . utilis and noted that during the process of enzyme induction in an adenine medium the yeast accumulated dialyzable compounds with a high absorbancy at 260 mv. Subsequently, Roush and Domnas ('56) studied the uptake of uric acid by C . utilis, found an intracellular accumulation of uric acid by an active transport process, and demonstrated the induced biosynthesis of uricase in this yeast. In the above work, the purine served as the sole nitrogen source; in contrast, Cowie and Bolton ('57) have reported on the accumulation of adenine and guanine by exponentially growing yeast in a medium containing ammonium ion as the nitrogen source.The present work is a further characterization of the transport system involved in the accumulation of purines by the yeast cell and a study of the metabolism of those purines that are concentrated by C. utilis. A total of 22 purines and related compounds have been studied. MATERIALS AND METHODSCandida utilis (strain ATCC 9950) was grown with aeration in the buffer and salt medium of Schultz and Atkin ('47) containing 0.4% ammonium sulfate as the nitrogen source and 5% glucose as the carbon source. The yeast was harvested by centrifugation, washed once with distilled water, and stored near 4°C until used. In experiments with the purines, 1 gm (wet weight) of the yeast was aerated in 100 ml of a medium of the same composition except that the nitrogen source was 0.1 mg/ml of the purine.Disappearance of the purine from the medium was followed by optical density measurements in the ultraviolet at a wavelength of maximum absorption for the purine. One-milliliter samples of the culture were diluted with 10 ml of distilled water, the cells were centrifuged down, and the optical density of the supernatant fluid was measured. To estimate the intracellular purine content, the sedimented yeast cells were suspended in 10 ml of water, heated in a boiling water bath for 10 minutes, the resulting mixture centrifuged, and the optical density of the supernatant fluid measured at appropriate wavelengths. It was found that there was no extraction of the intracellular purines when the cells were washed with 10 ml of water or with 10 ml of 0.1 M, pH 7.0 phosphate buffer without heating. The intracellular concentrations of individual purines were determined on buffered extracts obtained in a similar manner. Specific spectrophotometric assays as given in the following references were used: guanine-rat brain guanase, Roush and Norris ('50); hypoxanthine and xanthine-milk xanthine oxidase, Krebs and Norris ('49);

<b><i>Arxula adeninivorans</i></b> Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content

“…This is consistent with Roush et al [1959] who observed an increase in enzymatic guanine deaminase activity of Candida utilis in the presence of purines. In another investigation, the promoter of guanine deaminase from B. subtilis was fused to a reporter gene [Nygaard et al, 2000].…”

A history of research on yeasts 13. Active transport and the uptake of various metabolites1

“…Like S. cerevisiae , transport of glucose into K. lactis has been shown to be by facilitated diffusion 319, 320: the gene HGT1 19 encodes a constitutive high‐affinity carrier41 with K m = 1 m M 396 and RAG1 20 encodes a low‐affinity glucose carrier which is induced by glucose127, K m = 20–50 m M 396. Anja Diezemann and Eckhard Boles speculate that Hgt1p may be proton‐coupled, as is the fructose carrier Frt1p, which they described [95, p. 287].…”

Observations on 6-mercaptopurine activity in Saccharomyces cerevisiae

“…No growth inhibition occurred during treatment in YCM broth, but assessment of the internal 6MP concentration (Fig. 2) according to the method of Roush et al [10] indicates that this does not result from the exclusion of 6MP from cells.…”

“…6MP-uptake by yeast was estimated by measurement of the intracellular drug concentration [10]. Log phase cells were inoculated into 25 ml YCM broth in the presence of 40.0 /zg/ml 6MP and incubated at 28°C with shaking.…”