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

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.

• Authors: Hiroyuki Goto, Hideki Wakui, Atsushi Komatsuda, Hiroshi Ohtani, Hirokazu Imai, Ken-ichi Sawada, Ryoji Kobayashi
• Journal: Nephron. Experimental nephrology (Nephron Exp Nephrol) Vol. 93 Issue 1 Pg. e27-35 (Jan 2003) ISSN: 1660-2129 [Electronic] Switzerland
• PMID: 12411747 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2003 S. Karger AG, Basel

Chemical References

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

Topics

• Actinin (chemistry, isolation & purification, 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, metabolism)
• Peptide Mapping
• Peptides (metabolism)
• Protein Binding (physiology)
• Protein Structure, Tertiary
• Puromycin Aminonucleoside (metabolism)
• Sequence Analysis, Protein
• Swine