Renal alpha-actinin-4: purification and puromycin aminonucleoside-binding property

Nephron Exp Nephrol. 2003 Jan;93(1):e27-35. doi: 10.1159/000066647.

Abstract

Mutations in the gene encoding nonmuscle alpha-actinin-4 (actinin-4), an actin cross-linking protein, lead to congenital nephrosis. This suggests that actinin-4 is an essential component of the glomerular filtration barrier. In the present study, we attempted to purify actinin-4 from the mammalian kidney. We also examined an interaction of the protein with puromycin aminonucleoside (PAN), which can induce nephrosis in animals. A 100-kD protein reactive with antibody against muscle alpha-actinin was purified from the Triton-insoluble cytoskeleton of porcine kidney, by MgCl2 treatment, ammonium sulfate fractionation, and subsequent DEAE-cellulose chromatography and hydroxyapatite chromatography. Its partial amino acid sequence was then determined. A filamentous actin (F-actin)-binding activity of the purified protein was examined by a cosedimentation assay. Interactions of the purified protein and its fragments with PAN were analyzed by an affinity assay using PAN-Sepharose. Determined 134 amino acid sequences of the purified porcine renal 100-kD protein were completely identical with those deduced from nucleotide sequence of the cDNA encoding human actinin-4. The purified protein possessed the known function of alpha-actinin, the F-actin-binding activity, and was tightly bound to PAN. The PAN-binding site was mapped within a central rod domain of the protein, which is a possible interaction site for various cytoskeletal and transmembrane proteins. We have established an efficient purification method for renal actinin-4. Moreover, our findings indicate that the central rod domain of actinin-4 has a high affinity to PAN. In the PAN nephrosis animal model, actinin-4 might be a target protein from PAN nephrotoxicity.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinin / chemistry
  • Actinin / isolation & purification*
  • Actinin / metabolism*
  • Actins / metabolism
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Blotting, Western
  • Cytoskeleton / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Kidney Glomerulus / chemistry
  • Microfilament Proteins*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / analysis
  • Peptide Fragments / metabolism
  • Peptide Mapping
  • Peptides / metabolism
  • Protein Binding / physiology
  • Protein Structure, Tertiary
  • Puromycin Aminonucleoside / metabolism*
  • Sequence Analysis, Protein
  • Swine

Substances

  • ACTN4 protein, human
  • Actins
  • Microfilament Proteins
  • Peptide Fragments
  • Peptides
  • Actinin
  • Puromycin Aminonucleoside