Nucleoside Metabolism in Brain Extracts From Rat, Rabbit and Guinea Pig*

Abstract

ALTHOUGH the action of nucleoside phosphorylase has been clearly elucidated (KALCKAR, 1947(KALCKAR, , 1950 and its wide distribution in various tissues and organisms has been documented (LEVENE and MEDIGRECEANU, 1911;KLEIN, 1935;STERN et al., 1952; SABLE, 1950; MANSON and LAMPEN, 1951), very little information on the detailed fate of both purine and pentose moieties in brain has been reported. A study, initiated to investigate the products of in citro nucleoside metabolism by brain preparations from rats, rabbits, and guinea pigs, showed that soluble extracts of brain metabolized purine ribosides to form phosphate esters and lactic acid from the pentose moiety, whereas the liberated purines were only deaminated, the ring structure remaining intact. E X P E R I M E N T A LBrain preparations. Whole brain from rat, rabbit, or guinea pig was excised after the animal was bled following ether anaesthesia or stunning by a blow on the back of the neck. The brain was packed in ice, weighed, minced, and suspended in Tris-KCI solution (1 part of M-Tris buffer, pH 7.4, plus 9 parts of isotonic KCI) in proportions of 1 part of tissue by weight to 2 parts of Tris-KCI by volume. In some experiments this proportion was changed to 1 part tissue (wt) and 4 parts Tris-KCI (vol). The tissue was homogenized in the cold by using a Potter-Elvehjem homogenizer with a Teflon pestle (Arthur H. Thomas Co., Cat. No. 4288-B), and the homogenate was centrifuged in the cold at 20,000 g for 15 min. The clear supernatant liquid was decanted from the residue and this extract was subsequently used for incubations. In some experiments the whole homogenate was used for incubation.Inarbation and analysis. Incubation mixtures contained 1 ml of brain extract (or homogenate) and 5 /moles of nucIeoside or pentose phosphate, 10pmoles of ["PI KH,PO, (containing 0.2 mc of ['zP] phosphate per 5 ml of 0,050 M-phosphate) at pH 7.4, and 1 Pmole MgCI, in 0 0 5 M-Tris buffer, pH 7.4, in a total volume of 2 ml. Incubations were carried out under air at 37" in a Dubnoff Metabolic Shaker. The reaction was terminated by cooling the incubation mixture and then adding cold 25 7; trichloroacetic acid (TCA) to a final concentration of 8 7;. The supernatant fluid was separated from the insoluble residue, and portions were removed for pentose analysis by the MEJBAUM (1939) orcinol procedure and for paper chromatographic separation of phosphate ester by the RAPPOPORT and CHEN (1960) procedure. Lactic acid was determined by the method of BARKER and SUMMERSON (1941), and proteins were estimated by the colorimetric biuret procedure (GoA, 1953). Absorption spectra of purines and purine nucleosides were measured by using a Beckman recording DK-2 spectrophotometer.the Army (SGO).Jersey.

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

“…The catabolites of hypoxanthine in chopped mouse cerebrum included xanthine and urate ( Fig. 6), although on the basis of previous studies xanthine oxidase has been thought to be absent from crude extracts of brain from a number of animals (see Al-Khalidi & Chalglassian, 1965;Rappoport et al, 1960;Rosenbloom et al, 1967;Santos et al, 1968).…”