Studies pertaining to the purification and properties of thymine 7-hydroxylase

McCroskey, R.P.; Griswold, Wait R.; Sokoloff, R.L.; Sevier, E. Dale; Lin, Shih-Fan; Liu, Chen Kao; Shaffer, Patricia M.; Palmatier, R.D.; Parker, Thomas S.; Abbott, Mitchel T.

doi:10.1016/0005-2744(71)90059-3

Cited by 27 publications

(13 citation statements)

References 23 publications

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“…The sources of the radioactive comp described previously (19,22,26). The sp5 ties (curies per mole) of the compounds follows: 3.0 for [2-14C]thymine, 2.0 for [7-' carboxylic acid, and 0.50 for a-[1-'4C]kel All other fine chemicals were obtained mercial sources as previously described Growth of mycelium for enzymatic nidia were obtained from cultures that for 10 days (at 28 C in constant light) Fries minimal medium, solidified with 125-ml Erlenmeyer flasks.…”

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“…The nonaerated growth ofNeurospora carboy half-filled with medium and cli rubber stopper, containing a greased; through which a J-shaped glass stirrini tended, has been described (22). This apparatus was URIDINE modified so that several portions of an 8-liter culture could be removed while the gradual change of the a atmosphere in the carboy was preserved.…”

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“…After breaking toglutarate. the frozen pads into small pieces, 300 mg (moist I from com-weight) was combined with 4 ml of the (22,26). tris(hydroxymethyl)aminomethane buffer and 3 g of assays.…”

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“…tris(hydroxymethyl)aminomethane buffer and 3 g of assays. Coacid-washed glass beads (22) in the chamber (5-ml were grown capacity) of the Micro-homogenizer attachment to on 50 ml of the Sorvall Omni-Mixer and homogenized for 11 min 2% agar, in at 5,000 rpm while the chamber was immersed in an a were sus-ice-brine mixture at -2 C. The length of the homog--entration of enizing period was varied in experiments similar to solution was those previously described (22) to show that no more aid medium. protein could be extracted by prolonging the grindin a 10-liter ing and that the enzymatic activity was stable to the osed with a procedure.…”

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confidence: 99%

“…The assays used for the cell-free studies were based on the rate of formation of radioactive C02 from carboxy-labeled a-ketoglutarate. The standard incubation mixture (0.225 ml) was 10 mM in sodium phosphate, pH 7.5, and contained 0.1 ml of the enzyme preparation, 1 mM a-_1-14C]ketoglutarate, 1 mM ferrous sulfate, 1 mM ascorbate, 0.75 mM thymine or deoxyuridine, and 0.4 mg of catalase per ml (19,22,26). Since crude extracts could decarboxylate a-ketoglutarate in the absence of exogenous thymine and deoxyuridine, corrections were made in the calculation of enzyme activities with the values obtained from such control incubations.…”

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Regulation of thymidine metabolism in Neurospora crassa

Griswold¹,

Madrid²,

Shaffer³

et al. 1976

J Bacteriol

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The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'hydroxylase. Qualitatively similar effects on these enzymes were brought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis. Recent studies (34) have indicated that in order for Neurospora crassa to utilize pyrimidine deoxyribonucleosides they must be initially metabolized as depicted in Fig. 1. This report describes several factors that may be pertinent to the regulation of the pathway. The first indication of one ofthese factors came from attempts to facilitate purification of thymine 7hydroxylase by growing liquid cultures under forced aeration. Although the yield of mycelia was increased over that obtained from shallow liquid cultures, the 7-hydroxylase activity was markedly decreased and, furthermore, the incorporation of thymidine into ribonucleic acid was inhibited (22). However, larger nonaerated cultures with considerable 7-hydroxylase activity could be obtained if the Neurospora were grown with stirring in a sealed carboy halffilled with medium (22). Since the atmosphere inside of the carboy became anaerobic during growth, it was suspected that an increase in the 7-hydroxylase activity might, somehow, be initiated by this change in the gas composition.Another factor that may play a regulatory role is the ammonium ion content of the growth medium. The Westergaard-Mitchell medium permits the efficient utilization of intermediates in the pathway shown in Fig. 1 (39). The data in the present article indicate that mycelia grown in this medium contain higher activities

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