Inactivation of protein disulfide isomerase by alkylators including alpha,beta-unsaturated aldehydes at low physiological pHs.
Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDIred) or oxidized form (PDIoxid) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38 degrees C, a remarkable inactivation (>90%) of PDIred was caused by iodoacetamide (IC50=8 microM). However, PDIoxid was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDIred was significantly inactivated by N-ethylmaleimide (NEM), but PDIoxid was not. When the inactivation by these alkylators was analyzed by pseudo-first order kinetics, NEM (k3=1.75x10(-2) s(-1); K(i)=124 microM) was observed to be more potent than iodoacetamide (k3=9.1x10(-3) s(-1); K(i)=311 microM). Interestingly, the inactivation of PDIred by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDIred or PDIoxid by iodoacetamide was mainly observed around pH 6.0. In addition, PDIred was found to be inactivated by acrolein (IC50=10 microM) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDIred is susceptible to inactivation by alkylators including endogenous alpha,beta-unsaturated aldehydes at low physiological pHs.
Liu
Xi-Wen
XW
College of Pharmacy, Chungnam National University, Taejon 305-764, Korea.
Sok
Dai-Eun
DE
D000447:Aldehydes / Q000494:pharmacology*; D000477:Alkylating Agents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors*; D013997:Time Factors
D000447:Aldehydes; D000477:Alkylating Agents; D019704:Protein Disulfide-Isomerases