Trypanosoma cruzi adenylate cyclase activity. Purification and characterization

Abstract

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.

Cyclic-nucleotide signalling in protozoa

“…Functional characterization of three T. cruzi AC genes confirms the sequence‐based prediction, as forskolin had no effect on T. cruzi AC activity (Taylor et al ., 1999; D'Angelo et al ., 2002). These results agreed with an early report on the activity of T. cruzi AC purified with the aid of monoclonal antibodies that also failed to see an effect from forskolin (Torruella et al ., 1986). Previous work reporting the effects of forskolin on trypanosomes will need to be reappraised in the light that in these parasites, forskolin‐mediated effects may be due to non‐AC or non‐cAMP signalling effects.…”

Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes

“…Evidence indicates that such fusion procedures yield amounts of cell hybrids that are several orders of magnitude higher than those obtained with methods using chemical fusogenic agents. Cells T. cruzi epimastigotes from the Tulahuen 0 strain and lymphoma S49 cyc-cells were cultured and collected as indicated elsewhere (Torruella et al, 1986;. In order to inactivate the catalytic component of T. cruzi adenylate cyclase, a cell suspension (109 cells/ml) in 0.25 M-sucrose containing 5 mM-KCl was treated for 30 min at 37°C with 1 mM-N-ethylmaleimide, followed by a further 10 min incubation under the same conditions in the presence of 5 mM-dithiothreitol.…”

Cyclic nucleotide phosphodiesterase activity in Neurospora crassa Purification by immunoaffinity chromatography and characterization

“…The sources of materials used in this work have been given in previous papers [1, 3,5,6]. Immunoglobulins were obtained from Cappel Laboratories, West Chester, PA, USA or from Zymed Laboratories, San Francisco, CA, USA.…”

“…Transference to nitrocellulose membranes was performed according to the procedure of Yowbin et al [11]. Modifications of this method as well as blockage of the nitrocellulose strips and antibody reactions were as described by Torruella et al [6]. Proteins were stained with India ink [12].…”

“…The procedure followed has been described elsewhere [5,6]. Clones were selected for the capacity of culture media to inhibit cyclic AMP phosphodiesterase activity.…”