High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein.
The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.
Katoh
M
M
Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony.
Aono
S
S
Keino
H
H
Kashiwamata
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002104:Cadmium / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000032:analysis*; D008297:Male; D008668:Metallothionein / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; C004203:cadmium-binding protein; D002104:Cadmium; D008668:Metallothionein