Search

This study analysed the suramin sensitivity of 29 stocks of Trypanosoma evansi isolated from Egypt, Sudan, and Indonesia and compared the results with the isoenzyme banding patterns of 20 soluble enzymes in these stocks of T. evansi. The results showed that the type VII banding pattern of malic enzyme was found only in T. evansi stocks which were highly resistant to suramin.

A serological comparison between British Theileria mutans and Japanese T sergenti using the indirect fluorescent antibody test, showed that the two parasites were indistinguishable. On the basis of this and previous findings it is suggested that the British parasite is identical with the Japanese one and that its name should therefore be changed to T sergenti.

Several pyroglutamylaminoacyl-tRNA's were prepared by T4 RNA ligase mediated condensation of synthetic pyroglutamylaminoacyl-pCpA's with tRNA's from which the last two nucleotides at the 3'-end had been removed. The derived pyroglutamylaminoacyl-tRNA's were incubated in the presence of calf liver pyroglutamate aminopeptidase, which effected their conversion to free aminoacyl-tRNA's. The lack of contaminating esterase activities in the pyroglutamate aminopeptidase was verified by direct assay for the presence of the aminoacyl moieties in the formed aminoacyl-tRNA's and by the use of the deblocked aminoacyl-tRNA's as acceptors in the peptidyltransferase reaction using an Escherichia coli ribosomal system. These findings provide the wherewithal for a detailed investigation of the substrate specificity of the peptidyltransferase center and for the elaboration of polypeptides containing modified amino acids at predetermined sites.

Uridine 5'-phosphate (UMP) synthase is multifunctional protein that contains the last two enzyme activities of the de novo pathway for UMP biosynthesis, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate (OMP) decarboxylase (EC 4.1.1.23). We have previously reported that UMP synthase from mouse Ehrlich ascites cells can exist in at least three distinct aggregation and/or conformation states, as measured by changes in sedimentation through sucrose gradients [Traut, T. W., & Jones, M. E. (1979) J. Biol. Chem, 254, 1143-1150]. The major sedimenting species were a 3.6S monomer, a 5.1S dimer, and a 5.6S species. The formation of the 5.1S dimer from the 3.6S monomer occurs in the presence of ligands that are competitive inhibitors at the OMP decarboxylase catalytic site. This paper presents evidence for a regulatory site, distinct from either of the two catalytic sites, which appears to mediate the conversion of the 5.1S dimer to the 5.6S form upon binding certain pyrimidine nucleotides (OMP, UMP, and 6-azaUMP). Since UMP synthase sediments predominantly as a dimer in the presence of substrates, regulation of the aggregation/conformation state of this multifunctional protein may be physiologically significant.

A population of Rhipicephalus appendiculatus ticks infected with buffalo-derived Theileria lawrencei was established in a paddock. Two infected buffalo had been introduced into this paddock which contained a small population of Theileria-free R. appendiculatus. At intervals, successive groups of Theileria-susceptible cattle were grazed with the buffalo. Transmission of T. lawrencei from buffalo to cattle was first noted 7 to 8 months after the introduction of the buffalo, this delay being accounted for by the slow buildup of the tick population and low infection rates in ticks. A T. lawrencei challenge lethal for cattle was finally achieved 10 months after the introduction of the buffalo. The paddock was then seeded with engorged R. appendiculatus female ticks and an increase in tick population occurred. The lethal challenge was observed for a further 3 years, death of exposed cattle becoming progressively more rapid as the tick population increased. The T. lawrencei challenge establsihed in the paddock may be suitable for testing the efficacy of various T. lawrencei-immunization procedures for cattle.

A comparison was made between the microplate enzyme linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) and complement fixation (CF) tests for the detection of antibodies in the serum of cattle experimentally infected with Babesia divergens and B major. Antibodies were detected using all three tests but they were detected earlier using the CF test. However CF titres were consistently lower than those obtained using the other tests. Although there was little to choose between the IFA and ELISA tests, it was suggested that the ELISA may be preferable since it is less subject to operator error and operator stress, and can be adapted readily to field use.

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.

Amblyomma cohaerens nymphs, which had been collected as engorged larvae from African buffalo (Syncerus caffer) in the Mara region of Kenya, transmitted a theilerial parasite to a steer. Macroschizonts were detected in the regional lymph node of this steer 12 days after tick application and were subsequently detected for 3 days at low levels of parasitosis. The macroschizonts were large and had numerous nuclei, being morphologically unlike any other theilerial schizonts described previously from cattle in Eastern and Southern Africa, but they resembled the macroschizont of T. barnetti Brocklesby 1965 from a buffalo also obtained in the Mara region. Piroplasms morphologically indistinguishable from T. mutans were first detected in the blood of the steer on the sixty-first day after tick application. The steer showed a significant rise in antibody titre to only T. mutans (Aitong) piroplasm antigen, using the indirect fluorescent antibody test. There was no evidence that this parasite was pathogenic. The intraerythrocytic piroplasms proved to be blood-transmissible to a splenectomised calf. Although the parasite showed affinities to T. barnetti and T. mutans (Aitong) definite conclusions regarding its identity must await further transmission experiments.

Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer.