Separation and quantitation of the polyamine biosynthesis inhibitor D,L-alpha-difluoromethylarginine and other guanidine-containing compounds by high-performance liquid chromatography.

The arginine decarboxylase inhibitor difluoromethylarginine (DFMA) is an important tool in the study of polyamine metabolism, particularly with respect to the human pathogen Trypanosoma cruzi. This paper demonstrates a unique method for the detection and quantitation of intracellular DFMA using the fluorogenic agent 9,10-phenanthrenequinone. After separation of cell extracts by HPLC, DFMA can be accurately and reproducibly quantified with a lower sensitivity limit of 0.1 nmol by this simple fluorometric method. This assay can also be used to detect other guanidine-containing compounds such as arginine, agmatine, creatinine, and hirudonine, but not substituted guanidines such as aminoguanidine and creatine, or the structurally related amidines such as benzamidine and pentamidine.

The histones of the insect trypanosomatid, Crithidia fasciculata.

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.

A comparison of the amino acid sequences of Crithidia fasciculata and Trypanosoma rhodesiense cytochromes c.

Apocytochrome c was isolated from procyclic trypomastigotes of Trypanosoma rhodesiense EATRO 1895 and purified on Amberlite IRC-50 ion-exchange resin. Tryptic peptides were generated from the purified apoprotein and a partial amino acid sequence was determined. A comparison of the amino acid sequence of Crithidia fasciculata with the partial amino acid sequence of T. rhodesiense reveals significant homology.

A simple procedure for detecting proteins that bind preferentially to kDNA networks.

A procedure is described in which a fractionated protein extract from the trypanosome Crithidia fasciculata was incubated either with deproteinized C. fasciculata kDNA networks or nuclear DNA. At least three proteins in the range of 45-65 kDa had a specific affinity for the networks. We suggest that these proteins may play an important role in the biology of the kinetoplast.

Isolation of the peptide inhibitor of H+-ATP synthase from Crithidia fasciculata and Trypanosoma cruzi.

An inhibitor of Crithidia fasciculata and Trypanosoma cruzi H+ -ATP synthase (ATPase) was isolated from these organims mitochondrial particles, either by (a) ammonium sulfate-cholate extraction followed by heat treatment and ethanol precipitation, or (b) gel-filtration on Sephadex G-50, followed by a similar purification procedure. Inactivation by trypsin supported the inhibitor peptide structure. Removal of the peptide inhibitor increased about three-fold the specific activity of the protozoan ATPases. The isolated peptides and a highly purified bovine heart ATPase inhibitor inhibited C. fasciculata ATPase as a function of the peptide concentration.

Immunocytochemical studies with antibodies to three proteins prominent in the isolated microtubule cytoskeleton of a trypanosomatid.

The cytoskeleton of Crithidia fasciculata consists of a corset of parallel microtubules enclosing the cell body and closely underlying the plasma membrane. Distinct sets of crosslinks appear to connect tubules to each other and to membrane. Our objective is to determine the composition of these crosslinks and to elucidate the basis of this spectacular example of membrane-microtubule interaction. We purified three proteins (designated COP-33, -41, and -61 by their subunit Mr), which were consistently abundant in highly purified cytoskeletons. All three bound strongly to microtubules in vitro, and the first two induced bundles through periodic crosslinking. Polyclonal antibodies against each have been used to try to localize these proteins in thin sections of cells or whole mounts of cytoskeletons. Antibodies to COP-41 bound specifically to glycosomes, organelles that encapsulate many glycolytic enzymes in these protozoa, and COP-41 has been identified as glyceraldehyde 3-P dehydrogenase.

Ultrastructural visualization of lipids in trypanosomatids.

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.

S-adenosylmethionine and transmethylation reactions in trichomonads.

S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues.

Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.

Fine structure and cytochemistry of the mitotic plaques of Trypanosoma cruzi and Crithidia fasciculata.

The mitotic plaques are double, electron-dense structures which are located at the equator of the nucleus during the equatorial (metaphase) stage of mitosis in Trypanosoma cruzi, Crithidia fasciculata and other trypanosomatids. Each part of the equatorial plaques separates from the other and becomes an hemiplaque at the beginning of nuclear elongation. Variations of size of the plaques in different species of Trypanosomatidae are restricted to a limited range (less than 30% of the average thickness). At least two different components are found in the plaques with cytochemical methods: a) a basic protein with a high affinity for ethanolic-phosphotungstic acid, which is located in a narrow band towards the cleavage plane in each hemiplaque; and b) an osmiophilic component (possibly a protein) with a low affinity for uranyl acetate and which is located throughout the body of the plaque. The affinity for uranyl acetate can be abolished by methylation and acetylation, but remains after extraction with cold perchloric acid. No cytochemical evidence for the presence of DNA in the plaques is found. However, electron microscopy and cytochemical observations show that the PTA-affine band of the plaques is associated at its sides with chromatin fibers. Thus plaques, as the outer layer of kinetochores in higher eukaryots, have a component with high affinity for phosphotungstic acid, strengthening the hypothesis that these structures are phylogenetically related.

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.

Presence of a bent helix in fragments of kinetoplast DNA minicircles from several trypanosomatid species.

Some restriction fragments of kinetoplast minicircles from several trypanosomatid species (Leishmania tarentolae, Trypanosoma brucei, T. equiperdum, Herpetomonas muscarum, Crithidia fasciculata, but not T. cruzi) migrate anomalously on polyacrylamide gels. This behavior is probably due to a natural curvature of the helix. Bent helices appear to be a common property of kinetoplast minicircles, and may be important for minicircle function. In the case of T. equiperdum, we present evidence that each minicircle has a single bent region which resides in or near the 'conserved sequence.'

Characterization of Crithidia fasciculata oligosaccharide-lipid and its elongation by bovine mammary microsomes.

It was recently shown that a Man7(GlcNAc)2-lipid species serves as the precursor for the biosynthesis of asparagine-linked glycoproteins in the trypanosomatid Crithidia fasciculata. Preliminary results indicated it to be similar to the intermediate in the major pathway for the biosynthesis of lipid-linked Glc3Man9(GlcNAc)2 in animal systems. To explore the potential of this glycolipid as an acceptor for studying the biosynthesis of mammary glycoproteins, we conducted a detailed structural analysis of the labelled Man7(GlcNAc)2-lipid isolated from exponentially growing cells of C. fasciculata. The results showed its structure to be Man alpha 1----2Man alpha 1----2Man alpha 1----3(Man alpha 1----2Man alpha 1----3Man alpha 1----6)Man beta----GlcNAc beta----GlcNAc, identical to the nonasaccharide synthesized by the animal systems. Incubation of the Man7(GlcNAc)2-lipid with bovine mammary microsomes along with GDP-mannose or mannosyl-phosphodolichol elongates it to give Man9(GlcNAc)2-lipid having the same structure as the corresponding intermediate in animal systems. Inhibition of the elongation reaction by EDTA or amphomycin indicates that the intermediary formation of mannosyl-phosphodolichol is required for the incorporation of mannose residues into the nonasaccharide-lipid. Mannosyl.phosphoretinol failed to serve as a mannosyl donor in this reaction.

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.

Subcellular distribution and partial characterization of the cyclic AMP-binding proteins of Trypanosoma brucei.

We assayed the cyclic AMP-binding activity of Trypanosoma brucei by two well established methods, such as the one of Gilman (Proc. Natl. Acad. Sci. U.S.A. 67, 305-312, 1970) and Ueland & Doskeland (Biochem. J., 157, 117-126, 1976). The results indicate that the former technique underestimated the total amount of cyclic AMP bound by T. brucei homogenates by up to 7.5 fold. Similar results were obtained with other Trypanosomatidae such as Leishmania tropica, and Crithidia luciliae. The bulk of the cyclic AMP-binding proteins of T. brucei appeared soluble after centrifugation of post-large-granule extracts in isopycnic sucrose-gradients. Upon fractionation by DEAE-Sephacel column chromatography, two peaks of activity eluted which were responsible for all the specific cyclic AMP-binding activity present in the cytosol of T. brucei. These two activities, which we denominated as Peak 'a' and Peak 'b' respectively, differed in a number of properties such as sensitivity to proteases, stability to storage at -20 degrees C, displacement of cyclic AMP bound by adenine analogues, and coefficients of sedimentation.

[Ubiquinone-9 of the flagellates Crithidia oncopelti and Astasia longa]. / Ubikhinon-9 flagelliat Crithidia oncopelti i Astasia longa.

The content of ubiquinones (Co Q) of the Astasia longa and Crithidia oncopelti protozoa was studied. The protozoa were grown on an artificial nutrient broth. The cells were separated, washed, freeze-dried, and refluxed with KOH and pyrogallol in ethanol media. The hydrolyzate was concentrated. The residue was stored at -20 degrees and filtered. An ubiquinone fraction was isolated from the filtrate by TLC on silica gel. Identification of the ubiquinone homologues was carried out by reverse phase TLC and mass spectrometry. Ubiquinones were quantitated with respect to the difference in the density between the oxidized and reduced forms of Co Q at 275 nm. The A. longa and C. oncopelti flagellates were shown to contain ubiquinone-9 (Co Q9) at a concentration of 0.48 and 1.14 mumole/g dry cells, respectively. The higher Co Q level in zooflagellates as compared to that in phytoflagellates is discussed.

Primary structures of four novel small ribosomal RNAs from Crithidia fasciculata.

We report here the complete primary structures of four novel small RNA species (designated e, f, g, and j) found in the large ribosomal subunit of Crithidia fasciculata, a trypanosomatid protozoan. These RNAs, which are distinct from Crithidia 5S (species h) and 5.8S (species i) rRNAs, do not have counterparts in the more conventional eukaryotic ribosomes characterized to date. The small RNAs are 212 (e), 183 (f), 135-136 (g), and 72-73 (j) nucleotides long, with g and j displaying 5'-terminal heterogeneity. All have unique sequences and all contain 5'-monophosphorylated and 3'-unphosphorylated termini. In their basic structural features, therefore, species e, f, g, and j are indistinguishable from other RNAs (including 5S and 5.8S) that are recognized components of eukaryotic ribosomes, although they are unrelated to 5S or 5.8S rRNA in sequence. Since previous work from this laboratory has ruled out the possibility that these small RNAs are generated by quantitative and highly specific (albeit artifactual) RNase cleavage of large rRNAs during isolation, we conclude that species e, f, g, and j are native components of the Crithidia ribosome. With the exception of e, which appears to contain a single pseudouridine residue, all of these novel RNA species are devoid of modified nucleosides. In connection with primary sequence analysis, we present a simple modification of the standard G-specific chemical sequencing reaction which in our hands yields reproducible and unambiguous results using commercially available dimethyl sulfate.