Identification of protein phosphatase 2A as the primary target for microcystin‐LR in rat liver homogenates

[摘要]

The liver-specific toxin microcystin-LR (MC-LR) is a potent inhibitor of type 1 (PP1) and type 2A (PP2A) protein phosphatases. A tritiated form of the toxin, [³H]dihydromicrocystin-LR ([³H]DMC-LR), was used to identify target proteins in cellular fractions prepared from rat liver homogenates. About 80% of the [³H]DMC-LR bound to proteins was in the cytosolic fraction, which contained essentially all of the PP2A. In contrast, much of the PP1 was found in particulate fractions, each with only a few percent of the total protein-bound [³]HDMC-LR. Protein-bound [³H]DMC-LR in the cytosol co-eluted with PP2A, but not with PP-1 from a DEAE-Sepharose column. Native forms of liver cytoplasmic PP2A and PP1 separated by aminohexyl-Sepharose adsorption showed similar sensitivity to inhibition by MC-LR, and bound [³H]DMC-LR proportional to the amount of phosphatase activity. The results indicate that [³H]DMC-LR can bind both PP2A and PP1 in the liver which must be important for microcystin-induced toxicity, but is recovered mainly bound to PP2A in the cytosol.

[学科分类] 生物化学/生物物理

[关键词] Protein phosphatase 1;Protein phosphatase 2A;Microcystin-LR;Protein phosphorylation;Hepatotoxin;Rat liver homogenate;EDTA;ethylenediaminetetraacetic acid;EGTA;ethyleneglycol-bis(α-amino-ethyl ether) N;N;N′;N′-tetraacetic acid;[3H]DMC-LR;[3H]dihydromicrocystin-LR;kDa;kilodalton;LD50;lethal dose;the concentration of chemical where 50% of test animals are killed;MC-LR;microcystin-LR;PPase;protein phosphatases;PP1;protein phosphatase 1;PP2A;protein phosphatase 2A;TCA;trichloroacetic acid