Mammalian 60-kDa stress protein (chaperonin homolog). Identification, biochemical properties, and localization.
Mammalian chaperonin homolog (HSP60) was purified from porcine livers cytosol using a tandem ATP-Sepharose column and Mono Q column chromatography. A partial amino acid sequence (96 amino acid residues) of this protein was determined and coincided with those of human HSP60 with 96.9% homology, which was deduced from the nucleotide sequence of the cDNA. The sequence of the NH2 termini of the purified protein (5 amino acid residues) coincided with the signal sequence of HSP60. These facts led to the identification of the 60-kDa liver protein with the chaperonin homolog. Dihydrofolate reductase was able to form a stable complex with the liver chaperonin homolog. The liver chaperonin homolog was detected by at least five spots around pI = 5.6 on two-dimensional gel electrophoresis. Immunoblotting studies using an antibody against chaperonin homolog showed that the chaperonin homolog was localized in the cytosol, mitochondrial, and nuclear fractions of porcine liver. The chaperonin homolog was localized both in the mitochondria and cytoplasm of rat kidneys at the electron microscopic level. The chaperonin homolog in the cytosol, but not in the other subcellular fractions, was cross-reacted with an antibody against the synthetic peptide corresponding to the signal peptide of HSP60 as well as the purified chaperonin homolog on immunoblotting. These results suggested that the functional chaperonin homolog in the cytosol may be transported into the mitochondria and the protein may be processed to mitochondrial HSP60 in the organella.
Itoh
H
H
Department of Biochemistry, Akita University School of Medicine, Japan.
Kobayashi
R
R
Wakui
H
H
Komatsuda
A
A
Ohtani
H
H
Miura
A B
AB
Otaka
M
M
Masamune
O
O
Andoh
H
H
Koyama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D018834:Chaperonin 60 / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D018834:Chaperonin 60; D013762:Tetrahydrofolate Dehydrogenase