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(éfl~“\\\ L Place in book return to name charge from circulation records 15'?'\...“uru .‘ u” - y CARBON DIOXIDE ASSIMILATION BY ENTAMOEBA HISTOLYTICA BY Marlyn Ann Hamborg A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1980 ABSTRACT CARBON DIOXIDE ASSIMILATION BY ENTAMOEBA HISTOLYTICA BY Marlyn Ann Hamborg Entamoeba histolytica incorporated 14 Cd sodium bicarbonate into the cold trichloroacetic acid (TCA) soluble, the ethanol ether soluble, the hot TCA soluble, and the hot TCA insoluble fractions.v Two dimensional thin layer chromotgraphy (TLC) of the formic acid hydrol- ysate of the hot TCA soluble fraction showed that the isotope W33 incorPérated into adenine, guanine, cytosine and uracil, but not thymine. The failure of detecting thymine was probably due to the low concentration of thymine in the cell. Three unidentified substances were located on the thin layer plates. Only one of these spots (unknown #1) had an absorbtion maxima between 240- 350 nm. Entamoeba showed stimulated growth with the addi- tion of 1.25 umoles of the nitrogenous bases, nucleosides, or nucleotides in the presence of 10 percent C02. Adenosine-S'-phosphate (AMP), improved growth the best Marlyn Ann Hamborg in the absence of CO2 over the control, followed by adenine, guanosine-5'-phosphate (GMP), cytidine-5'- phosphate (CMP), and uridine-5'-phosphate (UMP). To Bill ii - ACKNOWLEDGMENTS This research was made possible through the generous support of Dr. R. Neal Band. His cOOperation, guidance, and encouragement were necessary for the completion of this thesis project. Thank you, Dr. Band, for your continual support. I would also like to thank Dr. John Breznak and Dr. Hironobu Ozaki for their interest, cooperation, helpful suggestions, and support during this research. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O 0 LIST OF FIGURES . . . . . . . . ABBREVIATIONS INTRODUCTION . . . . . . . . MATERIALS AND METHODS . . . . . . l4C-SodiumBicarbonate Uptake . . TCA Fractionation Procedure . . Nucleic Acid Hydrolysis . . . TLC of the Nitrogenous Bases of E. histolytica . . . . . . Liquid Scintillation Counting . . Spectrophotometry of Nitrogenous Bases Growth Experiments . . . . . Statistics 0 O O O O O O 0 RESULTS 0 O O O O O O O O O 90... Sodium Bicarbonate Uptake in E. histolytica Thin Layer Chromatography and Ultraviolet Absorbance Spectra of E. histolytica' s Nitrogenous Bases . . . . . fibeorbance of Nitrogenous Bases . C-Sodium Bicarbonate Incorporation into E. histolytica' s Nitrogenous Bases C02 Sparing Experiments . . . . DISCUSSION 0 O C O O O O C . SUMRY I C O O O O O C O 0 LIST OF REFERENCES . . . . . . . iv 'Page vii viii Pomona \IUIoB oh I-' h‘ re r4 n) no 18 33 38 43 54 S7 Table 10. 11. LIST OF TABLES l4C-SodiumBicarbonate Uptake into Growing Cells of E; histolytica in a 5 Min Pulse . . . . . . . . l4C-SodiumBicarbonate Uptake into Growing Cells of E; histolytica in a 15 Min Pulse . . . . . . . . 14C-Sodium.Bicarbonate Uptake Into Growing Cells of L. histolytica in a 30 Min Pulse_ . . . . . . . . Rf Values of Pure Nitrogenous Bases Developed in Isopropanol:HC1:Hzo (65:16.7:18.3, v/v) . . . . . . . Rf Values of L. histolytica' s Nitrogenous Bases Developed in Isopropanol: HCl: 320 (65:16. 7:18. 3, v/v) . . . . . . Rf Values of Pure Nitrogenous Bases Developed in n-Butanol:820 (86:14, v/V) o o o o _ o o o o o Rf Values of E. histol tica' s Nitrogenous Bases Developed 1n n-Butanol: .320 (86:14, v/V) o. o o o o o o o o Rf Values of Parallel Migration of L. histolytica' s Nitrogenous Bases and Purified Samples in Isopropanol: 8C1: H20 (65:16. 7: 18. 3, v/v) . . . . Absorbance Maxima of Nitrogenous Bases . Uptake of 14C-SodiumBicarbonate into the Nitrogenous Bases of E. histolytica in a 5 Min PuIEe . . . . Uptake of l4C-Sodium Bicarbonate Into the Nitrogenous Bases of E. histolytica in a 15 Min PEIse . . . . V Page 13 15 16 20 21 22 23 25 26 34 36 Table Page 12. Uptake of l4C-Sodium Bicarbonate Into the Nitrogenous Bases of E; histolytica in a 3 0 Min Pulse 0 O O O O O O O 0 37 13. Growth of g; histolxtica in the Presence of 10 Percent CO2 . . . . . . . . 39 14. Growth of g; histolxtica in the Absence of 10 Percent CO2 . . . . . . . . 40 15. Two Way Analysis of Variance of CO2 Sparing Experiments . . . . . . . 41 vi LIST OF FIGURES Figure ~ Page 1. Two dimensional thin layer chromotography of E; histolytica's nitrogenous bases . 19 2. Ultraviolet absorption spectra of L. histolytica' s adenine . . . . . . . 27 . 3. Ultraviolet absorption spectra of L. histolytica' 3 guanine . . . . . . . 28 4. Ultraviolet absorption spectra of L. histolytica' s cytosine . . . . . . 29 5. Ultraviolet absorption spectra of L. histolytica' s uracil . . . . . . . 30 6. Ultraviolet absorption spectra of L. histolytica' 3 unknown #1 . . . . . . 32 vii ABBREVIATIONS Abbreviations used are:. TLC, thin layer chromotography; TCA, trichloracetic acid; R-S-P, ribose-S'-phosphate; AMP, adenosine-5'-phosphate; GMP, guanosine-sf-phosphate; UMP, uridine-5'-phosphate: CMP, cytidine-s'-phosphate. viii INTRODUCTION Carbon dioxide is a common requirement for gut dwelling microorganisms (Dehority, 1971; Buhtanen, Carleton and Roberts, 1954), which is also the case for E; histolytica (Band and Cirrito, 1979). In bacteria, yeast extract can spare this requirement (Lwoff and Monod, 1947). In Entamoeba, although the addition of yeast extract to a culture improves the growth it does not replace the C02 requirement (Band and Cirrito, 1979). Reeves and West (1980) reported that adenosine was effective in stimulating the growth of Entamoeba when cultivated in Diamond's TP-S-l medium which was made flavin deficient. These authors also noted that the four of the five 5'-mononucleotides also produced very good growth. Nakamnra and Baker (1956) tested a large variety of compounds for their ability to improve axenic growth of Entamoeba histolytica, and found that the compounds involved in nucleic acid metabolism gave a good response. The combination of all five nitrogenous bases gave the best response, while good growth was also seen with them individually. Methylthioadenosine was also found to be 1 a potent growth factor (Nakamura, 1957). Many purine and pyrimidine analogs inhibited growth and multiplica- tion of E; histolytica, especially 2-aminopyrimidine, 2 dichloroacetamidopyrimidine, 4-dichloroacetamido-2, 6-dimethypyrimidine and 8-azaquanine. With some of the analogs the inhibition could be reversed by the addition of certain purines or pyrimidines (Nakamura and Jonsson, 1957). Wittner (1968) developed a semi-defined medium for the axenic growth of Entamoeba which included the five nitrogenous bases as essential ingredients. The rate of incorporation of purines, pyrimidines, and their derivatives by Entamoeba histolytica varies. Adenine and adenosine are taken up to the highest degree, followed by guanosine and guanine. All three nucleosides are incorporated more easily than the respective free base. The amebae preferred cytidine over uridine, and least thymidine (Booden, Boonlayangoor and Albach, 1976). It is known that Entamoeba takes up adenine, adenosine, and guanosine, in part, by a "carrier”- mediated system, and guanine, hypoxanthine, and inosine by diffusion. There is also some evidence to indicate that additional transport sites for adenine, adenosine, and guanosine are present (Boonlayangoor, Albach, Stern and Booden, 1978). It was the objective of this study to determine the importance of CO2 to the growth of this ameba, and the fate of €02 incorporation during growth. Particular attention was directed to incorporation of C02 into purines and pyrimidines. It is known that CO2 is used as a carbon source for the C2 of pyrimidines, and the C6 l4C-sodium bicarbonate of purines (Lehniger, 1975). was added to growing cultures of Entamoeba, which were then fractionated by TCA precipitation. The hot trichloroacetic acid soluble extract was separated by two dimensional thin layer chromotography, and the purines and pyrimidines were examined for incorporation of the isotope. The five basic nitrogenous bases, selected nucleosides and nucleotides were added exogenously to unmodified Diamond's TP-S-l medium to study their ability to spare C02. MATERIALS AND METHODS 14C-SodiumBicarbonate Uptake Entamoeba histolytica, strain H-K9, was cultured axenically by the method of Band and Cirrito (1979). The cells were incubated in sealed 200 x 25 mm test- tubes (Kimax) in the modified TP-S-l medium for 72 hrs at 35.50C. All test-tubes were initially inoculated with 2 x 104 organisms/m1. The amebae were chilled for 5 min in an ice-water bath and harvested by centrifuga- tion in an International centrifuge, universal model, at 300 x g for ten min at 25°C. The cell pellets were pooled to achieve a total count of 7 x 107 cells and then were washed twice with 50 ml of buffered saline, pH 7.0 (Serrano and Reeves, 1975). The composition of the buffer was 0.1 M NaCl, 20m1k HPO 2 4' 0.1mM Ca(NO3)2. The final pellet of amebae, approxi- 0.5 mM MgCl2 and mately 1 ml, was resuspended in 9.5 m1 of complete TP-S-l medium, and transferred to a 50 ml Micro- Pernbach flask (Bellco Glass Inc., New Jersey) which was sealed with a modified rubber stopper (Band and Cirrito, 1979). The flask was flushed for 2 mins with a mixture consisting of 10 percent CO2 in argon. This particular gas mixture provided optimum growth conditions (Band and Cirrito, 1979). The amebae were allowed to adjust to growing conditions in a 35.5°C water-bath for 15 mins 1 before addition of the 4C-isotope. 14 A 500 uCi aliquot of C-sodium bicarbonate . ('10'5M) with a specific activity of 52 mCi/m mole (New England Nuclear) was added through the inlet valve of the rubber stopper. The cells were pulse-labeled for 5, 15, and 30 mins, with occasional shaking. At the end of labeling the flasks were immediately submerged into an ice-water bath, and gassed with 100 percent argon for 2 mins. The gas leaving the flask was directly passed through two 15 m1 aliquots of 0.25N NaOH in series to trap any radioactive carbon dioxide that was in.the gas phase. Entamoeba was harvested by centrifugation for 10 mins at 300 x g at 25°C in an International clinical centrifuge. The pellet of cells was washed twice with buffer, pH 7.0 before TCA fractionation. TCA Fractionation Procedure The TCA fractionation procedure can be seen in the flow diagram below (Roberts, Abelson, Cowie, Bolton, and Britten, 1955). One ml aliquots of each step throughout the TCA fractionation were placed in scintil- lation vials containing 10 m1 of Aquasol (New England 14 Nuclear) to determine the amount of C-sodium.bicarbonate TCA Fractionation Procedure 1 ml washed packed amebea add 60 ml 5‘ TCA incubate 30 min, 4°C .1 centrifugation, 300 x g (10 mi /\ cold TCA soluble fraction cold TCA insoluble fraction ~14 wash 2x. cold 58 TCA (60 m1) 1 add so ml 75; ethanol: ether (1:1 v/v) i incubate 15 min, «to-50°C l centrifugation, 300 x g ethanoloether soluble \‘ fraction ethanol-ether insoluble fraction add 60 m1 5! TCA 1' incubate 30 min, 100°C J; centrifugation, 300 x 9 hot TCA soluble fraction hot TCA insoluble fraction incorporated. The hot TCA soluble fraction was the main point of interest in this study. Nucleic Acid Hydrolysis Before hydrolysis TCA was removed from the hot TCA soluble fraction by extraction with ether (1:1 v/v), 4 times. An absorbance reading on a Gilford spectro- photometer, model 2400-2, was taken at 260 nm to determine the amount of nucleic acid present (Warburg and Christian, 1942). This was necessary in order to know the con- centration of nucleic-acid added to TLC plates. 5 m1 aliquots cf the extracted hot TCA soluble fraction were evaporated to dryness under reduced pressure at 25°C. The residue was then dissolved in 0.25 ml of 88 percent formic acid (Fisher Scientific Co., New Jersey) and vacuum sealed according to the methods described by wyatt and Cohen (1953). After hydrolysis, at 175°C, for 30 mins the sample was evaporated to dryness under reduced pressure at 25°C. The residue was redissolved in 100 ul of 1N HCl. . TLC of the Nitrogenous Bases o E. Histolytica The plates were prerun in the first solvent of isopropanol:HCl:820 (65:16.7:18.3, v/v) to improve separation of the spots (Grippo, Iccarion, Rossi and Scarano, 1965), and then spotted with a 25 ul (0.15 mg) aliquot of the hydrolyzedhot TCA soluble fraction dissolved in 1N HCl. The plates were developed at room temperature until the solvent front reach 13 cm, and dried overnight at 20°C or 10 min in a ventilated oven at 40°C. N-butanol:H 0 (86:14, v/v) (Grippo, Iaccarion, 2 Rossi and Scarano, 1965) was the solvent used to develop the plates in the second direction. The solvent was allowed to migrate for 11 cm at 25°C. The pure nitrogenous bases, for standardization, uracil, cytosine, and guanine were obtained from California Biochemical Research: and thymine, adenine, xanthine, l-methyl adenine, and inosine were purchased from Sigma. The position.of the various nitrogenous bases on the TLC plates was determined by viewing with ultra- violet illumination. Liquid Scintillation Counting Each nitrogenous base was scraped from TLC plates, and directly placed in a scintillation vial, or dissolved first in 0.1N KOH or 0.1N HCl and then counted. Either method gave comparable results. A 25 ul aliquot (0.15 mg) of the hydrolyzed hot TCA solu- ble residue was placed in a vial for counting. Scintillation counting was performed in a Packard liquid scintillation spectrometer, model 3320. Quench corrections for each sample were determined by using the external standard method (Kobayash and Maudsley, 1974). Counting efficiency for the various samples ranged from 22 to 55_percent. Spectrophotometry of Nitrogenous Bases Each nitrogenous base scraped from the Avicel cellulose plate was dissolved in 1 ml of 0.1N KOH or 0.1 N HCl, and the solution was centrifuged for 5 min to remove the Avicel cellulose particles. The samples were then scanned continuously between the wavelengths of 240 and 350 nm by using a Gilford spectrophotometer, model 2400-2. Purified nitrogenous bases were also chromoto- graphed on Avicel cellulose TLC plates, scraped and dissolved in 0.1N ROB or 0.1N HCl, to obtain an absor- bance reading of known samples. ' To determine the concentration of the hydrolyzed, hot TCA soluble residue a 25 ul aliquot was dissolved in 1 m1 of 0.1N HCL and its absorbance read at 260 nm. Growth Experiments 'Amebae, 2 x 104, were added to 10 m1 of complete TT-S-l medium with or without the addition of nucleo- tides, nucleosides or nitrogen bases. 10 The nitrogenous bases: adenine, guanine, cytosine, uracil, and thymine: the nucleosides: adenosine and guanosine: and the nucleotides: adenosine-5'-phosphate (AMP), guanosine-5'-phosphate (GMP), cytidine-5'-phosphate (CMP), and uridine-5'- phosphate (UMP) were made up separately at a concentra- tion of 83 uM. These were sterilized by filtration (0.45 um membrane filter, Millipor Corp.), and 0.15 ml of each compound was added to the complete TP-S-l medium.for a final concentration of 1.25 umoles. It was necessary to dissolve guanine in a small volume of 1N BCl initially, before adjusting to the final concen- tration of 83 uM. The small volume of 1N HCl did not affect the growth of the amebae. Guanosine, AMP, and CMP were purchased from California Biochemical Research, and UMP, and GMP were bought from Sigma. Trophozoites in the complete medium with or without the added nucleosides, nucleotides, or nitro— genous bases were transferred to 50 m1 Micro-Fernback flasks which were sealed with the modified rubber stopper. The flasks were then gassed with 100 percent argon or a mixture of 10 percent CO2 and 90 percent argon for 2 min, twice daily. The cells were harvested after 72 hr at 35.5°C and counted with the aid of a hemocytometer. 11 Statistics Means and standard deviations were calculated on the C02 sparing experiments, Rf valves, and the dpm of the TCA fractions, and chromatographed nitrogeneous bases. Two way Analysis of Variance of the CO2 sparing experiments was also calculated. This was done on a Hewlett Packard calculator, model 9825A. The Bone- ferroni t-test was computed for the COz-sparing experiments in order to determine whether the addition of the nucleotides, nucleosides, or nitrogenous bases had a stimulatory effect on the growth of the amebae over the control. This was analyzed according to the method presented in Gill (1979). RESULTS Sodium Bicarbonate Uptake in in E. histolytica l4C-sodium bicarbonate The incorporation of (500 uCi) was studied at 5, 15, and 30 min. With experimentation it was found that in order to detect the level of radioactivity taken up by the cells into the nitrogenous bases, with the methods described in Materials and Methods, it was necessary to pulse for at least 5 min with a concentration of 500 uCi (0.9 mg/ 10 m1) of bicarbonate. The amebae looked healthy and attached to the surface of the flask at the termination of each experiment. The incorporation of the isotope into the amebae was studied by using TCA fractionation of cells. Table l expresses the distribution of the isotope into the four cell fractions (cold TCA soluble, ethanol-ether soluble, hot TCA soluble, and hot TCA insoluble) with a 5 min pulse of l4C-sodiumbicarbonate. The highest incorpora- tion was E a 62.5 percent, which was located in the cold TCA soluble fraction. Because this fraction contains a multitude of small molecules, it is not surprising with such a short pulse that the incorporation of the isotope was concentrated here. This fraction also had the 12 13 .c«s\amp uo acouumcueuouon n no ommuo>m so one mosam> omogsa OCH OCH OCH. x m.m OOH OOH x Add o>onm mo mamuoa 0.." #0..” x m.m H OOH x «.6 H OOH x .10 H OOH x N.o OMAR—“8.5.. ‘09 no: o.m~ @OH x N.N H mod on o.v hm OOH x «in mm OCH x m.m mange-”Om <09 Ho: m.m mod” on v.5 H 00H N m...“ m 00H x 5.0 m 00H on QJ GHQ-Ham Usualdocgum m.No . mo." x N.v H 00H x h.m we won on shm Ho mad as of: OHQQHOm g H.400 a «3:28 ~38. .. .3560 138. a cum: .o.m H com: co«uomwm «Us «dam seam .033 5: m a ca moauaaoun«£.dm no mHHoU neurone oucq cacao: ousconusowm anatomic II.~ names VH 14 highest level of activity in the 15 and 30 min pulses (Tables 2 and 3). 0f the other three fractions, the hot TCA soluble fraction had the next greatest level of incor- poration (i a 28 percent). This fraction is specific for the DNA and RNA molecules, and a small amount of pro- tein (Roberts, Abelson, Cowie, Bolton, and Britten, 1955). Because of this fraction's specificity and its level of incorporation of the isotope, this author turned her attention to the incorporation of the isotope into it. The ethanol-ether soluble fraction, which extracts phospholipids and lipoproteins from the cell (Roberts, Abelson, Cowie, Bolton, and Britten, 1955), had an average uptake of 1.3 x 106 dpm/min. Finally, the hot TCA insoluble residue, with an incorporation of l4C-sodium 1 percent, had the lowest uptake of bicarbonate. This residue contains proteins, peptides, and peptide fragments (Roberts, Abelson, Cowie, Bolton, and Britten, 1955). l4C-sodium bicarbonate The incorporation of the into the various fractions showed a slightly different distribution with a 15 min incubation. The level of incorporation into the cold TCA soluble fraction has dropped from i a 64 percent to i a 56.3 percent, and an increase can be seen in the ethanol-ether soluble, hot TCA soluble, and hot TCA insoluble fractions (Table 2). 15 TABLE 2.--14C-Sodium Bicarbonate Uptake into Growing Cells of L histolytica in a 15 Min Pulse. Exp 5* TCA Fraction ‘ Total Counts - 8 Cold TCA soluble , - 4.9 x‘ 106 56-3 Ethanol-ether soluble .. 1.1 x" 106' 12.2 Hot TCA soluble , ' 2.4 x 105 ' 23.0 Hot TCA insoluble 0.3. x 1.06 3.5 7 8 1 x 105 TOTALS of above . ' ,. 100.0 L *These values are an average of 3 determinations of dpm/min. 16 .cdaxamp mo occaumcweuouov n no oumuo>m so one mo=~m> omega. o.ooa o.ooa com a 0.0 o.ooH woe x o.m o>onm mo manage van mg a m.~ a m3 a To Tm 63 x m5 o4 o2 x To 0338:: <2. no: m.mw moa x H.m H ooa x N.v m.~v moa x o.v A.mv 00H x v.v mansaom €99 no: H.HH moa x m.n H 00H x H.H m.m won x m.o o.ha 00H x o.H mansdom Honuouaocmnum h.Hv mod x o.m H boa x o.v m.hv mod 3 m.v m.mn mod x e.N mansaom «Us mdoo a .o.m H com: a S3550 ~38. a «3550 138. con—03m con. 53: «dam none .omdsm ca! On m c« moauxnoum«:.dm no mdaoo ocfizouo oucw oxeum: ousconueoam sowmomao In.n mqm\> .m.ms.p.e~“mm. owe “Homnaocemoumomu :« oomo~o>oo nomem msocomouuaz onom mo mooamb umn|.e mnmdb 21 .o:0auouomoo oanmoumouosouno ucowouuam can one a use a omxu+ .oumconuaoan aseeomuov no omega sea me o as m axe... H .ouoconuoown sawtoosuvd mo ooasm sea on o sown one Q mam one n damn. .ouocoauooan soaoooau no cease cue m e lose one N mxm use a mum. vs we. a on. as. we. so. a». «o. as. we. as. mm. mm. m. czocxcs me. a mo. on. we. on. .66. me. me. no. no. an. we. a. usages: mo. « on. an. on. co. as. am. on. .mm.. on. em. mm. as ascexca no. a an. en. en. me. «e. mm. mm. an. no. me. an. enema: no. a «a. on. me. on. me. on. we. on. on. BA. ma. assuage mo. « an. on. an. no. mm. mm. mm. on. me. on. on. oceuouso no. a mu. Ha. em. on. em. mu. pd. . on. ma. _ an.h- mm. oedema: m a m a m a a a a a .o.m H coo: .WIIIIIMVI. mrllllmrl. mrllllmrl. mrllllwrl. + + oooem osocououuaz seem mam see man een mum on mxm ed mxm n ..>\;unn.o~.s.od.mo. o =.Hu=.aocemoumoou ca oomoao>oo ooeem osonomonudz.e.eo«ua~ouo«£ .m no oon~e> unla.m manta 22 cos. a as. as. as. . us. assume» Hanuoe-H «a. n ma. . me. me. as. ocamocn no. «.mn. an. . an. an. Hanan: mo. . Hm. m4. em.. mm. massage «a. a co. co. . be. ac. message no. H me. an. . 5H. NH. undocumo Na. « me. as. A . me. me. «engage .o.m H coo: m axe N man . .. A oxm oooom noocooouuwz .A>\> «canon. _ Cumuaocounmlc ca oomo~o>oo mooom osocomouuwz ouom mo mosao> «all.» mamas 23 .ocoHuouoooo oaomoumouoeonso umouomuao osu ouo m mom 4 moxm+ .ouoconuooan enamoonUe .ouocoouooan enacoonu A va «a mango see me as» as m axe... no onusm nae on on» ouo v one m omxmaa .ouoconuooan snaoOouoed mo ooasm :«e m on» own N can H odxm. mo. « as. aw. He. on. me. hm. Nn. .wm. mm. Nm. Nm. mm :3ocxcs ououmwa u.:oooo N. csocxca we. « oN. me. an. «a. mm. «a. we. mm. AN. NH. 44. a. nausea: Ho. « Nn. RN. av. mm. mm. on. mN. an. on. AN. 9N. Haven: so. u as. me. we. ac. me. no. so. on. «a. Na. mo. message No. a ho. ho. ca. he. no. me. me. no. new «a. we. ocwnouhu ma. . on. on. «a. as. we.. en. es. en. «a. as. me. cassava .o.m « coo: +m +4 +m +4 +m +4 +m +4 +m +4 oooom economouuaz seem mam «av mam eemxm «N mum 0H mum «docousmac ca oomodo>oo oooom moonomouudz .namp .eauoc. .o N: c.804usflouoaa .n no uo=Hu> manu.e mamas 24 in the Rf values could be attributed to small changes in temperature or different batches of thin layer plates. Parallel migration in Isopropanol:HCl:H20 (65:16.7:18.3 v/v) of spots believed to be adenine, cytosine, guanine, and uracil were compared against purified samples, and the results can be seen in Table 8. A spectrophotometric analysis of each spot was also done. Spots from g; histolytica's hot TCA soluble fraction believed to be adenine, cytosine, guanine, and uracil were also identified by determining the absor- bance maxima of each between the wavelengths of 240 and 350 nm in 0.1N HCl, and 0.1N KOH (Table 9 and Figures 2-5). The combination of two dimensional chromoto- graphic separation and spectrophotometric analysis gives strong evidence that the spots believed to be adenine, cytosine, guanine, and uracil are indeed so. Two spots separated by TLC were seen while viewing under the ultraviolet light which did not cor- respond to any of the known nitrogenous bases (uracil, cytosine, thymine, guanine, and adenine), minor bases (1 methyl-adenine) or precursors (xanthine and inosine) checked. One of the spots was unknown #1 which had Rf values of i I .56 and i a .20 in the first and second direction of development, respectively. This particular 25 mm. mm. HHONHD ea. we. ocficoou mm. mm. ocwoouao 5N. . hN. ocwcoo4 powmqusm o.o0fiu>~oao«: 4m ooom onocomOHufiz ..>\> .m.en.e.oeumoc.omzneoznaoooo IoumomMicwhooHQEom oowmwnsm moo oomom moocomouuwz o.oofiumaouowz .m mo coduouma: Hoaaouom mo oosao> mmu|.m mqm4a 26 _TABLE 9.--Absorbance Maxima of Nitrogenous Bases. Compound .lN HCl hm .lN KOH hm Adenine 274 269 Guanine 249 275 Cytosine 278 282 Uracil 258 284 Thymine 265 291 Inosine 260 254 Xanthine 261 247 1-methy1 adenine 259 271 Unknown #1 261 262 27 o O h’ l absorbency I T J“ ! 240 233 320 wavelength in am Pigure'2.--Ultraviolet absorption spectra of g; histolytica's adenine. ( l and 2 are the pure sample's and Entamoeba's base in 0.1N NOR, and 3 and 4 are the pure samples and Entamoeba’s base in 0.1N HCl. 28 0.04_ 0.03__ >0 " O " g . 0 .0 H 8 - ‘l .0 0.02 \ o ‘- 4 0.01 . . l 240 280 320 wavelength in nm Figure 3.--U1traviolet absorption spectra of g; histolytica's guanine. (l and 2 are the pure samplé's and Entamoeba's base in 0.1N KOH, and 3 and 4 are the pure sample's and Entamoeba's base in 0.1N HCl.) 29 0.2s__, 8 4 I 3' 2 c 0.15 o ‘1 ,g n o m .o e i t \ 0,05__+ f’ l 240 280 320 wavelength in nm Figure 4.'-U1traviolet absorption spedtra of g; histolytica's cytosine. (1 and 2 are the pure sample's and Entamoeba‘s base in 0.1N KOH, and 3 and 4 are the pure sample's and Entamoeba's base in 0.1N HCl.) 30 I 0e;2— ' | 2' 3 E 0.03 ‘3 " . 3 .1 C i 4 1 _ 0.04 e .- wwn“ ---..O--~“m ' " (i 240 200 3&0 wunduwr.intm -Figure 5.--Ultraviolet absorption spectra of E1 ”histolytica’s uracil. ( 1 and 2 are the pure sample's and Entamoeba's base in 0.1N KOH, and 3 and 4 are the pure sample's and Entamoeba's base in 0.1N HCl.) ‘ 31 spot has an absorbance maxima of 262 nm in 0.1N KOH and 261 nm in 0.1N HCl (Figure 6). This spot has been checked against such knowns as xathine, thymine, inosine, and l-methyl adenine, but the absorbance spectra and Rf values do not coincide. Unknown #2, the second spot, absorbed ultra- violet light strongly, but did not give an absorbance maxima between 240 nm.and 350 nm. Its Rf value in the first solvent was i s .68, and it did not migrate in the second solvent of TLC. A third spot, unknown #3, was removed from the general vicinity of thymine with an Rf value of i a .76 in the first direction of development, and i a .46 in butanol - HCl. This spot, also, did not produce any discernible peak between 240 and 350 nm. The amount of thymine in the cell is very small and difficult to recover (Gelderman, Bartgis, Keister, and Diamond, 1971); therefore, the concentration may have been too low for detection spectrophotometrically. However, there is no evidence at this time to support the fact that the spot is thymine. Bolton et a1. (1952) doing similar work with §;.gg;; were also unable to detect thymine. The identity of these unknown spots is not suspected to be a nucleoside or nucleotide because of the severity of the hydrolysis (wyatt and Cohen, 1953). 32 0.04 . 0.03 O O O N absorbency 0.01 I l 240 280 320 ‘wavelength in nm .Figure 6."U1traviolet absorption spectra of g; ~histolytica's unknown #1. ( 1 and 2 are Entamoeba's unknown base in 0.1N KOH and 0.1N HCl, respectively.) 33 Absorbance of Nitrogenous Bases An absorbance scan of all spots was completed 14C-sodiumbicarbonate, with the 5, 15, 30 min pulses of but only the results of one is given. All sets of absorbance spectra were comparable. 14C-Sodium Bicarbonate Incorporation into ETIhIStBI tica's NiEEogenous Eases After formdc acid hydrolysis of the hot TCA soluble fraction, the nitrogenous bases and possible minor bases were separated on Avicel cellulose plates to detect the level of incorporation into each. Table 10 presents the distribution of the 14C-sodiumbicarbonate into the various bases when given a 5 min pulse. Of the known bases, the greatest incorporation was in cytosine (3i - 277.5) and the other pyrimidine, uracil (2': a 229). The three unknown spots had an incorporation of the isotope higher than any of the known nitrogenous bases. One might suspect.that these spots may be minor bases, but in high concentration in E1 histolytica, because the hot TCA soluble fractions only contains polymeric molecules of DNA and RNA. The identity of these spots could not be determined at this time, so it is difficult to assess the importance or changes in the incorporation into these spots. 34 ..o2 mH.. eonuooum oansnoo «as no: couscouosn mo Bonuses as m~.. .cHoQEmo no ocONuocNBHouoo m mo omouo>m co one oocao> ooocat H.n¢v.m.n . o.mmo.n~ o.oom.ma a.-~.om .ruodo Honda m.ma~ « n.mmo a.~on N...@ m. ceases: e.om n N.Hmm e.mno n.eom a. excess: o n o.no~ o.no~ o.no~ do ascent: o.nos . o.m- o.~ma n.mom deco“: H.MH n.m.~e~ ~.on~ m.mo~ oseoouao ~.em n m.~ms N.oma e.om~ condone n.n « o.od N.ao H.~o~ monsoo4 .c.m « coo: «N arm. «A 95 ouem mcocomouuHZ. l... .onacm ca: m 4 cu coauaaouodc . -mfio oooom ococomouuaz ecu ouca ouoconwooam savanna: mo oxoudollda mama. 35 By 15 min (Table 11) the incorporation of the isotope is beginning to increase in adenine, and con- tinues to increase with a pulse of 30 min (Table 12). Incorporation into all other samples has dropped. Whether this drop in incorporation is an accurate repre- sentation of incorporation is uncertain because this experiment was performed only once. When one looks at the distribution of the isotope into the nitrogenous bases and possible minor bases through 30 mdn, there is a definite increase in all spots. Of the four nitrogenous bases, adenine has the highest level of inCorporation. ATP is required for many biosynthetic reactions which would correlate with a higher synthesis of adenine. The fact that adenine has the highest level of incorporation fits nicely with the CO2 sparing experi— ments presented in this paper. The results indicate that AMP allows the greatest level of growth in the absence of gaseous CO2 over the control. Adenine being a precursor to AMP. Reeves and West (1979), using dif- ferent media conditions, also found that adenosine stimulated amebae growth the best. At this time it is not known why the 25 ul aliquot of the hydrolyzed hot TCA soluble fraction added to the TLC plates has such a large number of counts as compared to the sum of the separated spots. 36 TABLE ll.--Uptake of l4C-Sodium Bicarbonate Into the Nitrogenous Bases of;§;_histolytica in a 15 Min Pulse. Nitrogenous Base. ' ‘ EXP 5* Adenine ' . 144.9 Guanine 80.0 cytosine . . 137.1 Uracil 109.1 unknown #1 . 181.2 unknown #2 , 351.4 Unknown #3 108.3 Total spot** . 23,810.0 *These values are an average of 3 determinations of dpm/min. **25 ul aliquot of hydrolyzed hot TCA soluble fraction (0.15mg), 37 .AmE mH.. caduceuu adamaOo 408 30: wonwdouwan mo Hosvfido Ho mNae ”fiancee no. mcowuecwsuouoo n no omeuobe ce one nooae> omocn... n.0mo.oN DI e.no . n.omo.o~ o.aoo.m~ ..uodo Hones H.nn . m.~on m.omn ~.ooo me neonate c.5oo « n.Noe o.nmn o.e~e N. ceases: a.mo~ . o.ome N.omo e.on~ He needed: m.m~ . o.oea H.ovc H.Hnn Heats: H.m « m.nna o.om~ e.mna onenouso o.no~ . «.mum m.on o.oom odesoso o.nom . m.m~m o.oo o.mmm odesoee .o.m « coo: «v oxm on mam ooem ncocomouuwz in .onasd on: on n on nanoseconan .m mo.oooem ococomouuaz on» oucH oueconueowm Ecaoomlo «a no oxoudouuma onm4e 38 The 1N HCl which the sample was dissolved in and the solvents used to chromatographically separate the spots did not produce a level of radioactivity above back- ground. Also area of the plate which did not contain a spot showed no significant level of radioactivity above background. This point of interest must be pursued further. C02 Sparing Experiments The growth of E; histolytica in 50 m1 Micro-, Fernback flasks, which were gassed twice daily with 100 percent argon, or 90 percent argon and 10 percent CO2 (Tables 13 and 14), in the presence of added nitrogenous bases, nucleosides, or nucleotides, supported the con- clusion of Band and Cirrito (1979) that there is a significant difference in amebae growth in the presence of CO2 in the gas phase. The statistical evidence for this can be seen in Table 15. This suggests that E; histolytica has a CD2 requirement. The next point of interest is the possible sparing effect of the CO2 by nitrogenous bases, nucleo- sides or nucleotides. The growth response of the amebea over a three day period in the presence of the different substances can be seen in Tables 13 and 14. With the addition of each compound at a concentration of 1.25 umole, it was found that AMP allowed the best 39 TABLE 13.--Growth of E. histolytica in the Presence of 10 Percent—5 02 . Sample Exp 2 Exp 2 Exp g Exp 2 Mean 1 §.D. (x 10 ) (x 10 ) (x 10 ) (x 10 ) (x 10 ) Adenine 3.3 2.7 3.2 2.8 2.9 t 0.24 Cytosine 2.6 3.0 2.9 2.8 2.8 i 0.17 Guanine 4.5 3.8 3.9 3.4 3.9 i 0.45 Uracil 1.5 1.7 2.9 3.2 2.3 i 0.85 Thymine 3.0 2.6 3.4 3.7 3.2 t 0.49 Adenosine 2.4 2.2 3.2 2.7 2.7 i 0.46 Guanosine 3.6 2.0 4.0 3.3 3.2 i 0.86 AMP 3.2 2.2 2.3 2.1 2.4 i 0.51 CMP 2.0 2-0 1.8 1.2 1.7 i 0.38 GMP 2.8 3.0 3.2 4.1 3.3 2 0.57 UMP 5.0 2.5 3.7 2.1 3.3 i 1.31 A11 bases 4.4 0.4 4.2 3.7 4.1 i 1.87 All Naps 3.0 2.8 3.5 4.7 3.5 f 1.72 None 3.2 3.6 2.4 1.6 2.7 i 0.89 NOTES: Above values were calculated by sub- tracting t a 0 hr. from t a 72 hr. the number of amebae in 10 ml. Above values are 40 TABLE 14.--Growth of E; histolytica in the Absence of 10 Percent C02. Sample' Exp 2 Exp 2 Exp g Exp 2 Mean i :.D. (x 10 ) (x 10 ) (x 10 ) (x 10 ) (x 10 ) Adenine 0.9 3.2 1.4 1.0 1.6 t 1.09 Cytosine 3.0 3.4 1.9 0.7 2.2 t 1.27 Guanine 2.7 2.1 2.9 2.5 2.5 t 0.34 Uracil 2.5 1.7 2.0 1.1 .1.8 i 0.57 Thymine 1.7 0.8 3.2 2.1 1.9 t 1.01 Adenosine 0.1 1.8 1.4 2.5 1.4 i: 1.05 Guanine 1.8 3.5 3.1 0.7 2.3 t 1.28 AMP 3.3 2.1 2.4 2.1 2.5 t 0.57 CMP 1.8 2.1 0.9 1.6 1.6 t 0.52 GMP 3.3 - 3.6 3.0 3.2 3.3 t 0.25 UMP 2.4 3.1 3.7 2.1 2.8 t 0.72 All bases 2.5 2.4 1.9 3.9 2.7 1 0.32 All NMPs 3.4 2.5 2.8 2.5 2.8 t 0.42 NOne 1.3 1.7 0.4 2.7 1.5 x 0.96 NOTES: Above values were calculated by sub- tracting the t a 0 hr. from t t 72 hr. Above values are the number of amebae in 10 ml. 41 TABLE 15.--Two Way Analysis of Variance of C02 Sparing Experiments. Source D.P. Mean Square 13‘ Treatment 1 1.26 x 10 Interaction 13 5.54 x 1011 Error '84 7.68 x 1011 F ratio _ l 84 42 replacement of CO2 over the control (p = ~.86), where p - (1 - C.D.F.). AMP was followed by adenine (p = ~.80) > GMP (p a ~.80), > CMP (p a ~.76), > UMP (p - ~.68). Even though the Boneferroni t-statistic for the above experiments is only moderately strong, there is some evidence that a difference between the treated and the control does exist. Stimulation by the nucleotides might be more evident if the flavin-deficient mediwm employed by Reeves (1980) was used instead of the standard TP-S-l. The other nitrogenous bases (uracil, cytosine, thymine, and guanine), nucleosides (adenosine or guanosine), or the combination of all the nucleotides did not show a significant stimulation in growth over the control. DISCUSSION The entire spectrum.of possible nutrient requirements of E; histolytica has been studied by a number of authors (Nakamura and Baker, 1956; Nakamura, 1957: Latour and Reeves, 1965: Wittner, 1968; Boonlayangoor, Albach, Stern and Booden, 1978; Band and Cirrito, 1979; Lo and Reeves, 1979; Reeves and West, 1980). Until 1968, when L. 5' Diamond developed a ‘medium.for axenic cultivation, it was difficult to evaluate the older studies (Nakamura and Baker, 1956; Nakamura, 1957; Latour and Reeves, 1965; Wittner, 1968) which dealt with the nutritional needs of Entamoeba. The reason being it was necessary to cultivate this ameba in the presence of bacteria. Therefore, whether E; histolytiba obtained its nutritional needs from the bacterial metabolites or the complex media was unclear. This paper looked into carbon dioxide and its possible interaction with nitrogeneous bases and their deriva- tives as growth stimulating substances. With the addition of 1.25 umoles of any of the nitrogenous bases, nucleosides, or nucleotides to the TP-S-l medimm, this author supported the conclusion of Band and Cirrito (1979) that the presence of C02 in the 43 44 gas phase above the medium.improved growth of Entamoeba histolytica. Carbon dioxide is a common requirement of gut dwelling microorganisms. Dehority (1971) studied the CO2 requirement of 32 strains of rumen bacteria. His results indicated that the major C02 requirement was a biosynthetic one, in which C92 was required for cell growth and multiplication. It should be emphasized that many species have an absolute C02 requirement even in complex culture media (Dehority, 1971). For orange-colored Streptococcus 29335 CO2 is important in the synthesis of amino acids and fatty acids (Hayashi and Kitahara, 1960; Prescott and Stutts, 1957; Prescott, Ragland and Stutts, 1957). It has been found that the CO2 requirement can be replaced by a mixture of amino acids, or a solution of some unsaturated fatty acids and oleate-containing compounds. Eh.22l£,i3 another microorganism which utilizes C02 in a biosynthetic mode. Carbon dioxide is incor- porated into the synthesis of proteins and nucleic acids. Sixty-two percent of NaHMCO3 was found to be incorporated into aspartic acid, glutamic acid, arginine, lysine, proline, and threonine (Abelson, Bolton and Aldous, 1952). The importance of CO2 in the synthesis of lipids, in E; histolytica, was not pursued in this 45 paper. But an incorporation of 14 C-sodium.bicarbonate in the ethanol-ether extract reached a level of 11 percent in 30 mins. So whether Entamoeba incorporates CO2 into malonyl-COA formation in the synthesis of long chain fatty acids (Lehninger, 1975) is not known. Because this biosynthetic reaction is common to some cell types, it would be worthwhile to investigate the presence of this reaction in Entamoeba, and any possible nutritional implications. Incorporation of 3 percent of Nat-114cc3 into proteins of gh.histolytica was noted over a 30 min period (Table 3). This hot TCA insoluble residue was hydrolyzed with 6N HCL for 24-48 hours and separated on two dimensional chromotography according to the methods of Detterbeck and Lillevik (1971). However, the level of incorporation of the isotope into the individual amino acids was too low for detection with the methods used. An attempt was also made to see if a sparing effect of CO2 could be achieved by an exogenous addition of amino acids. Using 2 mg or 0.1 mg of amino acid per m1 .of media depending on its solubility, the initial data seemed to indicate a possible stimulatory effect on growth. Further manipulation of the concentrations will be necessary to pinpoint specific amino acids, if any, and obtain statistically stronger evidence. Because the 46 14C-sodium bicarbonate into the protein incorporation of fractions was so low, the possibility remains that the amino acids present in the TP-S-l medium, supplied by panmede liver extract and trypticase,are sufficient to meet the nutritional needs of Entamoeba. The amino acids in the media may also inhibit or reduce the incor- poration of the isotope into the proteins (Abelson, Bolton, and Aldous, 1952). Wittner (1968) developed a semi-defined medium which included eighteen amino acids. The increase in cell number over time was not as great as cells grown in Diamond's TP-S-l media, but the shapes of the growth curves were identical. This would seem to indicate that the amebae are showing a normal growth response. Perhaps small adjustments of the concentrations or chemicals in the semi-defined media would produce a better growth response. As for the incorporation of NaHl4CO3 into the nucleic acid fraction (hot TCA soluble) of Entamoeba is concerned, about a 28 percent uptake was seen with a 5 min and 15 min pulse, which increased to 44 percent after 30 mins (Tables 1, 2, and 3). As in E; 391; (Bolton, Abelson and Aldous, 1952) the isotope was located in both purines and pyrimidines except-thymine (Tables 10, 11 and 12). E. T. Bolton et al. also noted the low recovery of thymine. As in the case of E; coli 47 quantitative uptake analysis of this pyrimidine could not be done here either. Geldermar et a1. (1971) found it necessary to use amebae in numbers in far excess than expected in order to determine the amount of DNA per cell (4 x 10-13 gm) by the standard CSCl gradient procedure. The fact that DNA isolation from the cell is difficult could explain the absence or very low concentration of thymine in the hot TCA soluble fraction. 4 It is known in the synthesis of pyrimidines that CO2 is incorporated into the second carbon of the pyrimidine ring via the formation of carbamoyl- phosphatic acid by carbamoyl-phosphate synthase, and into the sixth carbon of the purine ring structure via the intermidate, S'phosphoribosyl-S aminoimidazole-4- carboxylic acid (Lehninger, 1975). In analyzing a 0.15 mg sample of hydrolyzed hot TCA soluble over time, the highest incorporation of CO2 is into adenine (315.5 dpm/min). In Entamoeba the incorporation of CO2 into guanine followed adenine, cytosine being third, and uracil last. .The large loss of the isotope when the 0.15 mg sample is separated into its perspective come pounds by thin layer chromotography cannot be satis- factorily explained by checking the obvious places of loss (solvents, thin layer plates or chemiluminescence). Further investigation of this point is greatly needed. 48 During the separation of the hydrolyzed TCA soluble fraction three spots which did not correspond to any of the five basic nitrogenous bases were noticed. Unknown #3 was one which migrated on the TLC plates very similar to thymine. However, an absorbance peak between 240 and 350 nm could not be obtained. Therefore, its identity as thymine could not be confirmed. Unknown #2, which strongly absorbed under an ultraviolet light also did not produce any discernible peak when scanned spectrophotometrically between 240- 350 nmw The absorbance of this spot or unknown #3 by ultraviolet light may simply be due to contaminating material. This could explain the inability to get an ultraviolet spectrum, but not its uptake of the isotOpe. Analysis of these two samples by other means such as gas chromatography or mass spectrophotometry may determine their identity. An ultraviolet absorbance peak of 261 nm in 0.1N 8C1 and 262 nm in 0.1N KOH was achieved for unknown #1. Because it underwent 88 percent formic acid hydrolysis one would expect it to contain the ring structure of a purine or pyrimidine with no surviving sugar or phosphate group. Obvious possibilities for the identity of this spot would be one of the minor bases, perhaps in higher concentration in this ameba. It was checked against such knowns as xanthine, inosine, and 49 l-methyl adenine in this lab, but the Rf values or absorbance spectra did not coincide. Unknown #1 was also compared with all the minor base spectra presented in Venkstern and Baev (1975) and none of these appeared to be the correct known. However, an extensive compari- son of all minor bases will have to be made in this laboratory. The incorporation of the isotope is very high in all three unknowns, in fact, higher than any of the known bases, but the importance of this cannot be evaluated at this time. An extensive study to define the many growth factors necessary to grow Entamoeba axenically was attempted by Nakamura and Baker (1956). One group of substances they added to the modified Boeck-Drbohlau egg slant medium overlaid with horse-serum-Ringer's solution was components of nucleic acid metabolism. With this medium it was found that the combination of all five nitrogenous bases stimulated growth the best followed by RPS-P + ATP > cytOsine > thymine > R-S—P > guanine adenine > adenylic acid, and finally ATP. Later, methylthioadenosine was found to have a greater stimulatory effect than ATP or R-S-P when added to the basal medium. The addition of adenosine to the methylthioadensine containing medium did not increase the growth of this ameba; therefore, its stimulatory 50 activity is not due to the release of adenosine (Nakamura, 1957). Nakamura and Jonsson (1957) proceeded to look at the effects of analogsof purines and pyrimi- dines'on growth.- Many analogs had a negative effect on growth. Analogs such as 1-dichloroacetamido-Z-nitro- 4-methoxybenzene, 8-azaguanine, and 3,7-dimethylxanthine resulted in an inhibition which could not be reversed. Reversal could be seen with.2-dichloracetamido-2,6- dimethylpyrimidine, 2-aminopyrimidine, and uracil . Secarboxylic acid, as a few examples. Reeves and West (1979) studied the needs of nucleic acid precursors by Entamoeba grown axenically in Diamond's TP-S-l medium which was made flavin deficient. Their results indicated that the addition.of adenosine showed the best growth. .Adenosine being followed by adenine > AMP > AMP + CMP > AMP + GMP + UMP + CMP. The results of the present study, using unmodi— fied TP-S-l medium were slightly different from.the two reported above. AMP was found to be the one mononucleo- tide which stimulated growth the best. In order of decreasing stimulatory effect adenine > GMP > CMP > UMP followed. All three studies indicated that adenine or an adenine derivative produced a good growth response. This is not too surprising because of the high demand of any cell for ATP. The fact that there was some 51 discrepancy as to the order in which any of these mole- cules improves growth may just be the result of the different experimental procedures. From the data pre- sented in this study, one possible reason for the need of these compounds in the media is a replacement for the gaseous C02. Normally when E; histolytica is cultivated no concern for adjusting the gas concentration in the air phase above the media is made. -Band and Cirrito (1979) discovered that amebae grown in their 15 ml rubber sealed capped test-tubes containing 10 ml of medium will produce CO2 at a concentration of 3 percent within 6 hours, in the air phase. 'Since this CO2 is not removed during standard cultivation, it could easily satisfy any gas requirement. Therefore, its importance in any interactions with nucleic acid, protein, or lipid biosynthesis could be overlooked. Booden et a1. (1976) looked at the incorporation of tritiumelabeled purines and pyrimidines into g; histolytica over a 24 hour period. The results indicate that both purines and pyrimidines gained access inside the cell. Peak time of incorporation, and the concen- tration taken up by the cell varied with the nitro- geneous base. Adenine had an incorporation of 4.3 x S cpm/lo6 amebae, followed by adenosine (3.8 x 6 10 S 10 'cmp/lo amebae); but there is no significant 52 preference of either one by the cell. Guanosine is the next preferred nucleic acid precursor to be taken up by the cell. .Its incorporation is 2.5 fold greater than guanine. Purines enter the cell at higher concentration than pyrimidines. Cytidine (2.4 x 105 cpm/106 amebae) is the most extensively incorporated pyrimidine deriva- tive, followed by uridine > uracil > thymidine > cytosine > thymine. The study of the uptake mechanism of purine bases and their nucleosides was studied by Boonlayangoor et a1. (1978). Based on saturation kinetics, competi- tive homologs and analogs, diffusion and inhibitable components, and 010 values > 2, they found that adenine,- adenosine, and guanosine were taken up, in part, by a "carrier-mediated“ systun. Guanine, hypoxanthine and inosine enter the cell via diffusion. Individual trans- port sites for adenine-adenosine and adenosine-guanosine are supported by the inhibitor studies. The "non- productive” binding experiments-involving guanine hypoxanthine, and inosine gave evidence of additional sites of transport of adenine, adenosine, and guanosine. Also uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N—ethylmaleimide. In view of the fact thatthe rate of incorpora- tion of the various nucleosides, nucleotides, and 53 nitrogenous bases is different, this would certainly affect their ability to spare COé. Also the rate of incorporation of adenosine, guanosine, cytidine, and uridine is in the same order as their phosphorylated forms stimulate growth in the absence of C02. SUMMARY Entamoeba histolytica's growth improved when grown axenically in Diamond's TP-S-l medium with the addition of exogenous nitrogenous bases, nucleosides or nucleotides in the presence of 10 percent carbon dioxide in the air phase above the medium, It was shown that AMP stimulates growth the best, when added at a final concentration of 1.25 umoles in the absence of C02. Adenine, GMP, CMP and UMP also spared CO but to a 2 lesser degree. The rate of uptake of the various purines and pyrimidines varies. Part of this difference is due to the different types of transport systems used by Entamoeba to get these molecules into the cell. The ability of any of these molecules to spare CO2 is identical to its preference of uptake by the cell. Adenine and adenosine are taken up in the highest con- centrations by the ameba, and it is adenine and its derivatives which stimulate growth the best. Because the CO2 molecule is known to be incor- porated into the ring structure of purines and pyrimi- dines of many cells, the incorporation of this molecule was studied here in Entamoeba. Both the purines and the 54 55 pyrimidines incorporated C02, except thymine. There may also be incorporation into thymine, but, because the concentration of thymine in the cell is so small, the 14C-sodium bicarbonate into thymine incorporation of could not be quantified by the methods used. The highest uptake of the isotope was concentrated in adenine. Three spots which were identified with two dimensional TLC were noticed. Only one spot, unknown #1, produced a peak around 260 nm.. Its properties were compared with several minor bases, and various inter- mediates in the synthesis of purines and pyrimidines, but no identity was made. These three spots incorporated the isotope to a higher degree than any of the other nitrogenous bases. 14C-sodium.bicarbonate was also incorporated into the ethanol-ether soluble, and the hot TCA in soluble fractions. An attempt was made to identify the various amino acids which took up the isotope, but the level of actiVity was too low for detection under these authors' conditions. The ethanol-ether soluble fraction also showed some incorporation of sodium bicarbonate, but the importance of this incorporation into lipids was not pursued at this time. Many other rumen microorganisms have a carbon dioxide requirement. It seems only logical that Entamoeba, also found in the gut, would also have the 56 same requirement. It is known that §;_ggli and Streptococcus 22315, as two examples, need CO2 in the synthesis of proteins, nucleic acids and lipids, and this appears to be the case in the synthesis of nucleic acids by Entamoeba. LIST OF REFERENCES Abelson, P. H., Bolton, E. T. and Aldous, E. 1952. Utilization of carbon dioxide in the synthesis of proteins by Escherichia cLli, I. J. Biol. Chem. 198.165-177. Abelson, P. H., Bolton, E. T. and Aldous, E. 1952. Utilization of carbon dioxide in the synthesis of proteins by Escherichia cLli, II. _J. Biol. Chem. 198:173-178. Band, R. N. and Cirrito, H. 1979. Growth response of axenic Entamoeba histolytica to hydrogen, carbon dioxide, and oxygen. J. Protozool. 26: Bolton, E. T., Abelson, P. H. and Aldous, E. 1952. Utilization of carbon dioxide in the synthesis of nucleic acid by Escherichia cLli. J. Biol. Chem. 198: 179-185. Booden, T., Boonlayangoor, P. and Albach, R. A. 1976. 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