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Primary structure of and studies on Acanthamoeba actophorin

Stephen Quirk, Sutherland K Maciver, Christophe Ampe UGent, Stephen K Doberstein, Donald A Kaiser, Jozef Van Damme UGent, Joël Vandekerckhove and Thomas D Pollard (1993) BIOCHEMISTRY. 32(33). p.8525-8533
abstract
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 actophorin 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).
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
ACTIN DEPOLYMERIZING FACTOR, AMINO-ACID SEQUENCE, NUCLEOTIDE-SEQUENCE, PORCINE BRAIN, COFILIN CDNA, MONOCLONAL-ANTIBODIES, MYOSIN-II, PROTEIN, PURIFICATION, FILAMENTS
journal title
BIOCHEMISTRY
Biochemistry
volume
32
issue
33
pages
8525 - 8533
Web of Science type
Article
ISSN
0006-2960
DOI
10.1021/bi00084a019
language
English
UGent publication?
yes
classification
A1
id
202985
handle
http://hdl.handle.net/1854/LU-202985
date created
2004-01-14 13:42:00
date last changed
2018-01-04 12:50:28
@article{202985,
  abstract     = {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 actophorin 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).},
  author       = {Quirk, Stephen and Maciver, Sutherland K and Ampe, Christophe and Doberstein, Stephen K and Kaiser, Donald A and Van Damme, Jozef and Vandekerckhove, Jo{\"e}l and Pollard, Thomas D},
  issn         = {0006-2960},
  journal      = {BIOCHEMISTRY},
  keyword      = {ACTIN DEPOLYMERIZING FACTOR,AMINO-ACID SEQUENCE,NUCLEOTIDE-SEQUENCE,PORCINE BRAIN,COFILIN CDNA,MONOCLONAL-ANTIBODIES,MYOSIN-II,PROTEIN,PURIFICATION,FILAMENTS},
  language     = {eng},
  number       = {33},
  pages        = {8525--8533},
  title        = {Primary structure of and studies on Acanthamoeba actophorin},
  url          = {http://dx.doi.org/10.1021/bi00084a019},
  volume       = {32},
  year         = {1993},
}

Chicago
Quirk, Stephen, Sutherland K Maciver, Christophe Ampe, Stephen K Doberstein, Donald A Kaiser, Jozef Van Damme, Joël Vandekerckhove, and Thomas D Pollard. 1993. “Primary Structure of and Studies on Acanthamoeba Actophorin.” Biochemistry 32 (33): 8525–8533.
APA
Quirk, S., Maciver, S. K., Ampe, C., Doberstein, S. K., Kaiser, D. A., Van Damme, J., Vandekerckhove, J., et al. (1993). Primary structure of and studies on Acanthamoeba actophorin. BIOCHEMISTRY, 32(33), 8525–8533.
Vancouver
1.
Quirk S, Maciver SK, Ampe C, Doberstein SK, Kaiser DA, Van Damme J, et al. Primary structure of and studies on Acanthamoeba actophorin. BIOCHEMISTRY. 1993;32(33):8525–33.
MLA
Quirk, Stephen, Sutherland K Maciver, Christophe Ampe, et al. “Primary Structure of and Studies on Acanthamoeba Actophorin.” BIOCHEMISTRY 32.33 (1993): 8525–8533. Print.