CHARACTERIZATION OF EHCABP, A CALCIUM-BINDING PROTEIN OF ENTAMOEBA-HISTOLYTICA AND ITS BINDING-PROTEINS

parasite Entamoeba histolytica. In order to decipher the function of t his protein, a few basic properties were investigated and compared wit h the ubiquitous Ca2+-signal transducing protein calmodulin (CaM). Ind irect immunofluorescence and immunoprecipitation analyses using specif ic antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [S-35]methionine-labelled prot eins of E. histolytica trophozoites using EhCaBP-sepharose column show ed Ca2+ -dependent binding of a group of proteins. Radiolabelled prote ins from the same extract also bound to CaM-sepharose. However, the pr oteins bound to the two columns were different as revealed by sodium d odecyl sulphate polyacrylamide gel electrophoresis. Al least one of th e EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshk ovskii (which is found in the human gut but is not pathogenic), two sp ecies in which EhCaBP-like protein has not been found. Two distinct Ca 2+-dependent protein kinases; which get activated by EhCaBP and CaM re spectively, were detected in E. histolytica. These kinases require dif ferent levels of Ca2+ for their maximal activities. Affinity chromatog raphy also showed the binding of protein kinase(s) to EhCaBP in a Ca2-dependent manner. Our data suggest that there may be a novel Ca2+-sig nal transduction pathway in E. histolytica mediated by EhCaBP. Copyrig ht (C) 1997 Elsevier Science B.V.