By using folic acid-2-14C in the growth medium as the sole source of pteridine it was found that the kinetoplastid flagellate, Crithidia fasciculata, produced a total of 87 mμg of labeled biopterin and 23 μg of 2-amino-4-hydroxy-6-hydroxymethylpteridine from 2 mg of folic acid in 2 liters of medium. Eighty percent of the biopterin and 70% of the 6-hydroxymethylpteridine was isolated from the spent medium, the remainder from the cells. These results and the results whereby the total pteridine was supplied as a folic acid analog make it obvious that this flagellate does convert folate to biopterin and is incapable of de novo pteridine synthesis.

The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.

Both adenosine and 2′-deoxyadenosine are inhibitory to the growth of Crithidia fasciculata. Half-maximal inhibition is caused by 0.3 mM adenosine. Effective reversal of the inhibition is brought about by uridine or cytidine; uracil has little effect. This difference may be attributed to inhibition of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase by adenosine nucleotides (especially ADP) formed inside the cell, thus interrupting pyrimidine nucleotide biosynthesis, either de novo or from exogenous uracil. Uridine, on the other hand, is converted to the ribonucleotide by uridine kinase and thus bypasses the block. The adenosine analogs tubercidin and cycloadenosine are the most potent inhibitors of the growth of C. fasciculata so far tested. This inhibition of growth is reversed by pyrimidine nucleosides and with adenosine.

SYNOPSIS The fatty acids lauric, myristic, and oleic, as well as long‐chain bases (LCBs) obtained from sphingolipids, and Tween 80 “spare” the requirement for folate by Tetrahymena pyriformis W. Since LCBs are metabolized by the ciliate to ethanolamine phosphate and fatty aldehydes which can be converted to either fatty acids or fatty alcohols, the latter compounds are used as precursors of phospho‐ and phosphonolipids and ether phospholipids. It is suggested that lipid biosynthesis is a rate‐limiting step in growth of the ciliate as is the folate concentration. Removal of one restraint on growth rate mimics the effect of increased folate concentration. Alternatively, if the enzymes responsible for lipid synthesis are repressible, the presence of exogenous fatty acids would make available more formylmethionyl‐tRNA for the initiation of synthesis of other proteins.

Adenine deaminase (adenine aminohydrolase, EC 3.5.4.2) has been found to occur in Crithidia fasciculata with a specific activity higher than that of the same enzymes of bacteria and yeasts. It is remarkable for its stability to heat, exhibiting no appreciable loss of activity after 60 min at 55 °C. It occurs in the soluble portion of cell extracts but can be released into the suspending medium by osmotic and/or cold shock.

The adenine analog 4-aminopyrazolo(3,4-d)pyrimidine inhibits the growth of the kinetoplastid (trypanosomatid) flagellate Crithidia fasciculata. This inhibition is partially overcome only by adenine (of a number of purines tested), with an inhibition index of 0.025. More effective reversal of inhibition is obtained with any of a number of naturally occurring pyrimidine compounds, up to a concentration of 0.18 mM. Higher concentrations of pyrimidines or addition of guanine, as well as adenine and uracil, to the medium increases inhibition. The analog (presumably as the ribonucleotide) was found not to be inhibitory to any enzyme of the pyrimidine biosynthetic pathway that could be tested. It is suggested that the analog competes with adenine for adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), is converted to a ribonucleotide, and is incorporated into nucleic acid.

A study of the enzymes of the orotate biosynthetic pathway in the kinetoplasid flagellate Crithidia fasciculata has revealed a number of differences between them and those of other organisms, either prokaryotic or eukaryotic. Carbamyl phosphate synthesis could not be demonstrated in cell-free extracts. However, the incorporation of both CO2 and the ureide carbon of citrulline into pyrimidines occurs in growing cells, the latter predominating over the former. The aspartate transcarbamylase of the flagellate has properties which are similar to those of this enzyme as it occurs in mammals rather than other microorganisms. Two enzymes, dihydroorotate synthetase and dihydroorotate hydrolase, are present, the former being responsible for the conversion of carbamylasparate to dihydroorotate. Dihydroorotate hydroxylase, a soluble enzyme requiring a reduced pteridine as a cofactor, converts dihydroorotate to orotate. The hydroxylase is inhibited by orotate, but not by pyrimidine or purine ribonucleotides. Thus orotate serves to control its own biosynthesis.

1.1. The growth of the ciliate, Tetrahymena pyriformis, is inhibited by a number of dipeptides in the presence of suboptimal concentrations of folate.2.2. Its growth is also sensitive to chloramphenicol and does not occur in the absence of folate. The reverse is true of the kinetoplastid flagellate, Crithidia fasciculata.3.3. It is suggested that the initiation of protein synthesis in the kinetoplast-mitochondrion does not require a formylated species of aminoacyl-tRNA or that the proteins of the organelle may be cytoplasmic in origin.

A series of pteridines having varying degrees of diuretic activity were tested for their effects on the growth of the pteridine-requiring flagellate, Crithidia fasciculata . Inhibition of growth was correlated with diuretic activity insofar as compounds devoid of diuretic effects were uninhibitory and active inhibitors were also good to fair diuretics. Reversal of the growth inhibition was obtained with 2-amino-4-hydroxy-6- l - erythro -1',2',3'-trihydroxy-propylpteridine in a competitive manner, while folate showed a logarithmic relationship to the drug concentration for 50 per cent inhibition.

SYNOPSIS Reversal of the growth inhibition of Crithidia fasciculata by allopurinol requires both a purine and a pyrimidine. Hypoxanthine is the most effective purine in the reversal. Cell‐free extracts were prepared which were capable of the decarboxylation of orotidine 5′‐phosphate. Other enzyme preparations carried out the phosphoribosylation of allopurinol. By the use of [4‐ 14 C] orotidine 5′‐phosphate (enzymatically prepared), it was shown that allopurinol ribotide (enzymatically prepared), but not the free base, inhibits orotidine 5′‐phosphate decarboxylase.

An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl2, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.

SYNOPSIS. By the use of 14C‐labeled substrates it has been shown in Tetrahymena that proline is rapidly and completely oxidized to carbon dioxide and glutamate (65–70%), plus small amounts of aspartate and alanine (20%), the remainder being incorporated into macromolecular cell components. In comparison, acetate, glucose and glutamate are oxidized to a lesser extent (55%, 37% and 16%, respectively). Glucose and acetate are extensively incorporated into cell components (53% and 36%, respectively), while glutamate remains in the medium (76%). Thus proline is a source of readily available energy.

SYNOPSIS. Crithidia fasciculata can synthesize threonine but it lacks an aspartokinase. The carbons of threonine may be derived from methionine when it is present in the medium. However, methionine can be synthesized by the organism provided organic sulfur is present. When both methionine and threonine are omitted from the medium, growth will occur if cysteine and high levels of folate are present. The compound common to both methionine and threonine under these conditions, α‐keto‐γ‐hydroxy‐butyrate, is derived from phosphoenolpyruvate (PEP) and the β‐carbon of serine. High levels of folate are required for this coupling reaction, which is carried out by what may be called phosphoenolpyruvate‐tetrahydrofolate hydroxymethyltransferase. By the use of radioactive tracers, both in growth experiments and in cell‐free preparations, virtually all of the intermediates in these two series of reactions were identified.

This chapter describes the assay of unconjugated pteridines. The only organisms to have a dietary requirement for an unconjugated pteridine are members of the flagellate order Kinetoplastida (trypanosomids). Stock cultures are maintained by weekly transfer in a peptone medium and are used for the depletion of the organisms of folate. The medium used for the depletion of the organisms of folate is tabulated. Growth in this medium reaches only about half of that obtained in a similar medium containing folate, but it can be transplanted indefinitely. For depletion of the organisms of all pteridines, biopterin is omitted from the medium. All work with pteridines must be done in red or very dim light to avoid photodecomposition. The cultures must be incubated in the dark. It is convenient to make up the ingredients of the experimental media in the form of several solutions that are mixed in the appropriate proportions to give the final medium. The preparation of the inoculums and standards are also discussed.

This chapter describes the synthesis of pteridine in tetrahymena. The ciliated protozoan tetrahymena pyriformis does not synthesize biopterin or any unconjugated pteridine from folic acid. It synthesizes the major pteridine, ciliapterin, entirely from the carbons and nitrogens of guanosine. The procedures for isolating and characterizing the pteridine and demonstrating its precursors are discussed. Fluorescent fractions containing biologically active pteridine are combined and placed on a longer Sephadex G-25 column and eluted. The gel filtrations are very advantageous because the pteridines are concentrated about 4-fold in a single passage and are separated from the bulk of the ultraviolet absorbing materials in the original extract. The fluorescent fractions containing biologically active material are combined, dried by lyophilization, and again taken up in 8–10 ml of water. This material, nearly pure ciliapterin, is chromatographed on an anion-exchange column. The spectrum obtained is compared with the spectra of authentic formaldehyde and acetaldehyde hydrazones under identical conditions. The structure of ciliapterin is, therefore, 2-amino-4-hydroxy-6-pteridine, or threobiopterin. The biological activity indicates that it is probably of the L-configuration.

Abstract— Unconjugated pteridines are associated with the photosynthetic systems of several organisms. Inhibition of the development of the photosynthetic system of Rhodospirillum rubrum with the pteridine inhibitor 4‐phenoxy‐2,6‐diamino pyridine (PDAP) is reversed by biopterin, a natural pteridine. Evidence is presented which indicates an electron transport function for pteridines. Specific interaction of reduced pteridines with isolated pigment protein complexes, leading to red‐shifted absorption bands, has suggested a mechanism for photochemical energy trapping.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAntiprotozoal 4-aryloxy-2-aminoquinolines and related compoundsDiether G. Markees, Virginia C. Dewey, and George W. KidderCite this: J. Med. Chem. 1970, 13, 2, 324–326Publication Date (Print):March 1, 1970Publication History Published online1 May 2002Published inissue 1 March 1970https://pubs.acs.org/doi/10.1021/jm00296a048https://doi.org/10.1021/jm00296a048research-articleACS PublicationsRequest reuse permissionsArticle Views206Altmetric-Citations91LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

AbstractDurch Curtius‐Abbau entsprechender Ester entstehen die Hydrazide sowie Amine (I) und (II).

The growth of the flagellate, Crithidia fasciculata , is inhibited by 2, 4-diamino-5, 6, 7, 8-tetrahydroquinazoline. This inhibition is reversed by biopterin. Folic acid does not reverse the inhibition but increases the effectiveness of biopterin in reversing the inhibition at low concentrations of the inhibitor.

Ciliapterin, a new unconjugated pteridine, has been isolated from the ciliated protozoan, Tetrahymena pyriformis. It has been determined by alkaline permanganate oxidation to be a 2-amino-4-hydroxy-6-substituted pteridine and by acid hydrolytic treatment to possess no labile bonds (such as glycosidic or phosphoester). The substitution at position 6 is dihydroxypropyl, as shown by the identification of acetaldehyde as one of the products of periodate oxidation. Although its absorbance spectrum is identical with that of biopterin, its fluorescence and activation spectra are similar, but not identical, with those of biopterin. Mobilities on paper with a number of solvent systems and its elution patterns from cation and anion exchange columns together with its biological activity in the Crithidia system indicate that it is 2-amino-4-hydroxy-6-(threo-dihydroxypropyl)pteridine. Its biological activity suggests that it may be the l-threo compound. The origin of this pteridine has been shown, by the use of both guanine-2-14C and uniformly labeled guanosine monophosphate-14C in the culture medium, to be entirely from guanosine. No hydroxymethylpteridine could be detected, indicating the inability of this organism to produce the pteridine precursor of folic acid.