Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.     

Purification and characterization of uracil phosphoribosyltransferase from Crithidia luciliae

1. Uracil phosphoribosyltransferase (UPRTase) was purified 370-fold from the protozoan parasite, Crithidia luciliae. 2. The enzyme was a dimer of mol. wt 80 000 and was highly specific for uracil. 3. GTP, which is an activator of UPRTase from E. coli had a slight inhibitory effect on the parasite enzyme. 4. The C. luciliae UPRTase demonstrated a broad specificity for activating divalent metal ions.     

Effects of inhibitor,competitors and temperature on transport of carbohydrate in Crithidia luciliae

Effects of KCN (10^?4 M), simultaneous presence of varying concentrations of D-glucose and L-sorbose, and temperature on transport of carbohydrate in C. luciliae have been studied. The rate of carbohydrate entrance is inhibited, in all sugars used, ranging from 19% to 70% inhibition at 0.5 mM external concentrations. However, this inhibitor does not affect transport from external concentrations of the order of 0.02 M. At 20 mM external concentration, the rate of L-sorbose entrance is greatly inhibited by the simultaneous presence of D-glucose, and the transport mechanism shows enormously greater affinity for glucose than for other monosaccharides. However, at 0.5 mM external concentration, the rate of sorbose entrance is not inhibited at all by the simultaneous presence of D-glucose. In the temperature interval 15°–25°C, the Q₁₀ for rate of entrance when the external concentration is 0.5 mM is 2.8 times larger than the Q₁₀ when the external concentration is 20 mM. These data are interpreted as strongly suggesting two mechanisms for carbohydrate entrance: (a) facilitated diffusion, of importance only at high external concentrations; (b) an active transport mechanism, active at low external concentrations and dependent upon a supply of metabolic energy. These results are compared with those reported in the literature for other types of cells.     

Orotate phosphoribosyltransferase and orotidylate decarboxylase from Crithidia luciliae: subcellular location of the enzymes and a study of substrate channeling

Orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) have been found to be particulate in the kinetoplastid protozoan, Crithidia luciliae. Sucrose density centrifugation indicated that these two enzymes are associated with the glycosome, a microbody which appears to be unique to the Kinetoplastida and which contains many of the glycolytic enzymes. The particulate location of OPRTase and ODCase was considered to be favorable for channeling of orotidine-5'-monophosphate (OMP), the product of the first enzyme and substrate for the second. The degree of channeling was determined by double radioactively labeled experiments designed to determine the relative efficiency of endogenous and exogenous OMP as substrates of ODCase. The efficiency of channeling was high, with an approximate 50-fold preference for endogenous OMP. By comparison, the degree of channeling for the yeast enzymes, which are soluble and unassociated, was less than 2-fold. The OPRTase-ODCase enzyme complex was solubilized using Triton X-100 in the presence of dimethyl sulfoxide, glycerol, and phosphoribosyldiphosphate. The percentage recovery of the overall enzyme activity was approximately 20%. The degree of channeling was reduced by approximately 10-fold for the solubilized complex. The Km for OMP changed from 7.5 (+/- 1.8) to 1.6 (+/- 0.3) microM in the ODCase reaction. There was no alteration in the Km for orotate in the OPRTase reaction.     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Fixation of various lectins at the surface of Crithidia luciliae]

Suspensions of Crithidia luciliae have been treated with 30 lectins: protozoans are agglutinated only by lectins inhibited with oses of structures I an II according to M?kel?, and by lectins the site of fixation of which are unknown. The use of 5 lectins conjugated to fluorescein corroborate that lectins in congruity with group I and II, contrarily to those of group III, fasten upon the membrane and the flagella of Crithidia luciliae.     

The structure of kinetoplast DNA. I. Properties of the intact multi-circular complex from Crithidia luciliae.

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.     

The purine-2-deoxyribonucleosidase from Crithidia luciliae. Purification and trans-N-deoxyribosylase activity

Crude extracts of Crithidia luciliae catalysed a deoxyribosyl transfer from purine deoxynucleosides to free purine bases. Fractionation of a 0-80% (NH4)2SO4 fraction from C. luciliae on DEAE-cellulose resulted in the separation of three nucleosidase activities. Two of these were ribonucleosidases, one specific for inosine, uridine and xanthosine and the other for inosine and guanosine, whereas the third activity was specific for purine deoxyribonucleosides. This pattern is similar to that found in Leishmania donovani. Significant deoxyribosyltransferase activity was, however, associated with the purine-2'-deoxyribonucleosidase from C. luciliae. The purine-2'-deoxyribonucleosidase was purified to homogeneity by a six-step procedure involving (NH4)2SO4 fractionation and chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75, and a chromatofocusing resin. The purified enzyme migrated as a single band of 17 kDa on SDS/polyacrylamide gel electrophoresis. The enzyme catalysed the hydrolysis of deoxyinosine, deoxyguanosine and deoxyadenosine with Km values of 80 +/- 10.5 microM, 20.7 +/- 3.2 microM and 17.3 +/- 5.3 microM, respectively, and V values for these substrates in the ratio 1:0.5:0.39. The pH optimum for deoxyribosyl transfer from deoxyinosine to guanine was at pH 7.7, while deoxyinosine hydrolysis in the presence of guanine was optimal in the range pH 6-7. During the synthesis of deoxyinosine from hypoxanthine and deoxyadenosine two products were formed. One of these coeluted with deoxyinosine on HPLC, while the second was tentatively identified as the positional isomer, 7-(beta-D-2'-deoxyribofuranosyl)hypoxanthine.