Abstract
Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases. Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s). However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734). To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein. These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity. Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily. The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones. Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.
Highlights
Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleu- in the degradation of HMG-CoA reductase and HMGal, we cinal (ALLN), a cell-permeable synthetic tripeptide attempted to isolate a cell line resistant to ALLN with the with an aldehyde at itsC terminus inhibits anticipation of obtaining cells expressing elevated levels of the activity of cysteine proteases
The aldo-keto reductases are NADPHdependent oxidoreductases that catalyze the reduction of aldehydes and ketones to corresponding alcohols [8].Elevated levels of aldo-keto reductase in SI100 cells can inactivate ALLN by reducing its active aldehyde group to corresponding alcohol [2]
A Cytosolic 35-kDa Protein Is Highly Overproduced iSnIlOO Cells-When proteins in total cell extracts are separated by Characterization of SI100 Cells-A cell lineresistant to SDS-PAGE, we found that the amount of a 35-kDa protein
Summary
0.08 0.11 0.06 for 10 s, and used as a “membrane fraction.” Both cytosolic and “ALLM is not soluble in aqueous solution at a concentration membrane fractions were dialyzed against 500 ml of 10 mM sodium higher than 500 p ~. Calpeptin is a dipeptide containing an aldehyde group at its C terminus [16, 17], and E-64-d is the cell-permeable inhibitor derived from E-64 and does notcontain an aldehyde group [2, 18]. These results were stained with Coomassie Brilliant Blue R-250. Protein was hydrolyzed in 6 N HC1, and amino acid composition analysis was performed on a Beckman inhibitors is specific for ALLN and ALLM. We isolated the ALLN-resistant cells in the presence of verapamil to prevent selection of the MDR phenotype, 7300 amino acid analyzer using a ninhydrin-based detector system.
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