Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.
The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.
Maciejewski
J J
JJ
Department of Biological Sciences, University of Illinois at Chicago 60607-7060, USA.
Vacchiano
E J
EJ
McCutcheon
S M
SM
Buhse
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D018076:DNA, Complementary; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D018257:Helix-Loop-Helix Motifs; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016802:Oligohymenophorea / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D003285:Contractile Proteins; D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C026555:spasmin