The Utilization of C 14 O 2 in the Production of Acid Metabolites by Moniliformis dubius (Acanthocephala)

Abstract

The fixation of carbon dioxide (NaHCO0) by Moniliformis has been studied. This parasite, incubated in NaHC403 for periods ranging from 30 sec to 5 min, incorporated label into aspartic acid, alanine, serine, malate, succinate, fumarate, and lactate. Pyruvate, oxaloacetate, and a-ketoglutarate, identified as 2,4-dinitrophenylhydrazine derivatives, were found but only the two former compounds were labeled. The pattern of labeling indicated that fixation into pyruvate or phosphoenolpyruvate had occurred. Moniliformis dubius was found to produce no carbon dioxide during anaerobic metabolism (Laurie, 1957). Acids excreted during aerobic metabolism were tentatively identified as acetic, formic, and lactic acids (Laurie, 1959). The present author studied the distribution of label in organic and amino acids in M. dubius incubated in glucose-U-C14 (Graff, 1964). The pattern of labeling indicated that carbon dioxide fixation had occurred at a significant level. The above observations initiated the present study in which M. dubius was exposed to NaHC1403. MATERIALS AND METHODS Female worms were removed, after 42 days of infection, from male Sprague-Dawley rats, which were reared and infected as previously described (Graff and Allen, 1963). Approximately 1 g of female M. dubius was weighed in each case and preincubated for 15 min in buffered mammalian Ringer's solution [pH 7.5 with 0.03 M tris (hydroxymethyl) aminomethane]. Worms were removed, blotted, and transferred to flasks containing 5 ml buffered Ringer's and 10.0 gM NaHC03 (sp. activity 1.89 mc/mM). One gram of worms was Received for publication 20 May 1964. * From a dissertation submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy. This investigation was supported by National Institutes of Health Training Grant No. 2E-70. t Present address: Biology Department, Rice University, Houston 1, Texas. removed after 0.5, 1.5, and 5.0 min incubation, rinsed in five changes (500 ml) of buffered Ringer's, blotted on hard filter paper, and placed in 15 ml 80% ethanol. The worms were homogenized in a blendor for 3 min at 5 C. The homogenates were centrifuged for 15 min at 1,200 g and the supernatants were extracted with 20 ml water-saturated chloroform. After centrifugation at 1,200 g for 15 min the chloroform layer was removed, the water layer was evaporated to dryness, and the residue was redissolved in 1.0 ml demineralized water. Aliquots of all extracts were plated in uniform thickness on planchets and assayed with a Baird-Atomic gas flow counter with a counting efficiency of approximately 9%. The extracts were placed on columns of Dowex 50( H+) and separated into acid effluents and NH40H eluates. These fractions were evaporated to dryness and redissolved in 0.50 ml demineralized water. Aliquots were counted as described previously. Free amino and organic acids were separated and identified by ascending oneand two-dimensional chromatography as previously described (Gr ff, loc. cit.). High-voltage electrophoresis was also employed as another means of identification of amino acids. The radioactivity in individual amino acids and organic acids was estimated by counting the radioactivity in eluates of uniform spots cut from chromatograms. Spots were located by superimposing chromatograms on fixed radioautographs over transmitted light. The 2,4-dinitrophenylhydrazine derivatives of known keto acids (pyruvate, oxaloacetate, a-ketoglutarate) and of keto acids in the acid fractions were prepared by the method of El Hawary and Thompson (1953). The hydrazones were dissolved in 0.20 ml phosphate buffer (pH 7.2). Aliquots were spotted on Whatman No. 4 paper and developed with n-butanol: ethanol: 0.5 N NH4OH