28 research outputs found
Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes
Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK
Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi
Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27 kDa, 31 kDa, and 53 kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines
Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax
AbstractSalivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48–67kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended
Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi
The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides.
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En esta revisión se describirán los avances obtenidos en la dilucidación de las principales funciones del sistema olfatorio, incluyendo la recepción y discriminación de los olores, así como los mecanismos que se encargan de transmitirlos
Identification of Casein Kinase 1, Casein Kinase 2, and cAMPdependent Protein Kinase-like Activities in Trypanosoma evansi
Trypanosoma evansi contains protein kinases capable of phosphorylating
endogenous substrates with apparent molecular masses in the range
between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was
predominantly localized in the particulate fraction. Anti-α and
anti-β tubulin monoclonal antibodies recognized pp55 by Western
blot analyses, suggesting that this band corresponds to phosphorylated
tubulin. Inhibition experiments in the presence of emodin, heparin, and
2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was
a casein kinase 2 (CK2)-like activity. GTP, which can be utilized
instead of ATP by CK2, stimulated rather than inactivated the
phosphorylation of tubulin in the parasite homogenate and particulate
fraction. However, GTP inhibited the cytosolic CK2 responsible for
phosphorylating soluble tubulin and other soluble substrates. Casein
and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for
casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as
substrates in T. evansi. While the enzymes present in the soluble
fraction predominantly phosphorylated P1, P2 was preferentially labeled
in the particulate fractions. These results demonstrated the existence
of CK1-like and CK2-like activities primarily located in the parasite
cytosolic and membranous fractions, respectively. Histone II-A and
kemptide (LRRASVA) also behaved as suitable substrates, implying the
existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only
increased the phosphorylation of histone II-A and kemptide in the
cytosol, demonstrating the existence of soluble cAMP-dependent protein
kinase-like activities in T. evansi. However, no endogenous substrates
for this enzyme were identified in this fraction. Further evidences
were obtained by using PKI (6-22), a reported inhibitor of the
catalytic subunit of mammalian cAMP-dependent protein kinases, which
specifically hindered the cAMP-dependent phosphorylation of histone
II-A and kemptide in the parasite soluble fraction. Since the sum of
the values obtained in the parasite cytosolic and particulate fractions
were always higher than the values observed in the total T. evansi
lysate, the kinase activities examined here appeared to be inhibited in
the original extract