Renal alpha-actinin-4: purification and puromycin aminonucleoside-binding property.
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.
Goto
Hiroyuki
H
Third Department of Internal Medicine, Akita University School of Medicine, Akita, Japan.
Wakui
Hideki
H
Komatsuda
Atsushi
A
Ohtani
Hiroshi
H
Imai
Hirokazu
H
Sawada
Ken-ichi
K
Kobayashi
Ryoji
R
D000185:Actinin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D003599:Cytoskeleton / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007678:Kidney Glomerulus / Q000737:chemistry; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D011692:Puromycin Aminonucleoside / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013552:Swine
C111453:ACTN4 protein, human; D000199:Actins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010455:Peptides; D000185:Actinin; D011692:Puromycin Aminonucleoside