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Purine synthesis in suspensions of mucosa from the small intestine of the rat Paterson, Alan Robb Phillips

Abstract

Purine biosynthesis has been studied in vitro using surviving, whole cell suspensions of the mucosa from the small intestine of the rat. The preparation and use of such suspensions have been described as have been certain features of their metabolic behaviour. The techniques used to isolate and measure the radioactivity of the several purine fractions of mucosal suspensions containing 150-200 mg. of fresh tissue have been outlined. The de novo synthesis of purines by this tissue system was demonstrated by measuring the incorporation of several C¹⁴-labelled purine precursors into adenine and guanine of the acid-soluble (AS) nucleotides and the nucleic acids. Essential parts of this demonstration consisted of establishing the radiochemical purity of the isolated, purines, and of showing that the isotope incorporation was not attributable to bacteria present in the tissue suspension. Upon incubation of mucosal suspensions with formate-C¹⁴, adenine and guanine of the AS nucleotides displayed a rapid renewal, being approximately 40 and 14 times as radioactive, respectively, as the adenine and guanine of the mixed nucleic acids. The specific activity of AS adenine was approximately 6 times greater than that of AS guanine, and nucleic acid (NA) adenine was 2-3 times as active as NA guanine. Following incubation of the mucosal preparation, the suspending medium contained adenine, guanine and uric acid, but since these purines were only slightly radioactive they were regarded as breakdown products. Incorporation of formate-C¹⁴ by exchange reactions (as opposed to de novo synthesis) appeared to be excluded as a major process by the demonstration that two other purine precursors, glycine-1-C¹⁴ and bicarbonate-C¹⁴, also became incorporated into the purines. The theoretical incorporation of 2 molecules of formate for each glycine molecule used in the process of de novo purine synthesis was approached in these experiments by the observed incorporation of 2 - 4 molecules of formate per molecule of glycine. This is sufficiently close to the theoretical value to exclude a major incorporation by known exchange reactions. Time studies indicated that the renewal of AS adenine and guanine proceeded at an approximately constant rate with a decline in rate starting at 3 - 4 hours of incubation. In several experiments an initial lag in the rate of synthesis of NA purines was apparent. The rate of purine synthesis as measured by the incorporation of formate-C¹⁴ was not significantly affected by the addition of non-isotopic glycine or glutamine to the incubation medium. Similarly, glycinamide and 4-amino-5-imidazolecarboxamidine, both of which resemble known intermediates in purine biosynthesis, did not alter the incorporation of formate-C¹⁴ into the purines. Renewal of either AS or NA purines from formate-C¹⁴ did not take place in homogenates of intestinal mucosa. The incorporation of formate-C¹⁴ by the purines of the intestinal mucosa of the intact rat was measured 24 hours after administration of the isotope. In contrast to the in vitro experiments, the large differences between the specific activities of the acid-soluble and NA purines were absent in the intact animal, the levelling effect very probably being due to the 24 hour interval. A comparison of purine synthesis in the intact animal with that of the in vitro system suggested that guanine synthesis was suppressed in the latter. The use of intestinal mucosa for in vitro studies of purine metabolism has not hitherto been reported. In view of the demonstrated rapid synthesis of soluble purine nucleotides in this system, particularly of adenine nucleotides, it is felt that suspensions of mucosa may have useful applications in in vitro studies of purine biosynthesis.

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