Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B).
The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Morgan
E W
EW
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66160-7417.
Yan
B
B
Greenway
D
D
Petersen
D R
DR
Parkinson
A
A
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D002415:Cats; D002729:Chloromercuribenzoates / Q000494:pharmacology; D006224:Cricetinae; D003902:Detergents; D004285:Dogs; D006023:Glycoproteins / Q000737:chemistry; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013045:Species Specificity; D013696:Temperature; D014018:Tissue Distribution; D014105:Tosyl Compounds / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D003902:Detergents; D006023:Glycoproteins; D009943:Organophosphorus Compounds; D014105:Tosyl Compounds; C003614:4-toluenesulfonyl fluoride; D020245:p-Chloromercuribenzoic Acid; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D000581:Amidohydrolases