Trypanosoma cruzi calmodulin: cloning, expression and characterization.
We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the alpha-helix content upon binding with Ca(2+), when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca(2+) pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.
Garcia-Marchan
Yael
Y
Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados (IDEA), Universidad Central de Venezuela, Caracas 1080, Bolivarian Republic of Venezuela.
Sojo
Felipe
F
Rodriguez
Evelyn
E
Zerpa
Noraida
N
Malave
Caridad
C
Galindo-Castro
Ivan
I
Salerno
Milena
M
Benaim
Gustavo
G
D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002645:Chickens; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000201:enzymology; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000913:Antibodies, Protozoan; D002147:Calmodulin; C016512:IgY; D007136:Immunoglobulins; D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases