Primary structure of and studies on Acanthamoeba actophorin.
We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).
Quirk
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S
Department of Biophysics and Biophysical and Biophysical Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21205.
Maciver
S K
SK
Ampe
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C
Doberstein
S K
SK
Kaiser
D A
DA
VanDamme
J
J
Vandekerckhove
J S
JS
Pollard
T D
TD
D000048:Acanthamoeba / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D016054:DNA, Protozoan; D008840:Microfilament Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C047604:actophorin protein, Acanthamoeba