Gene_locus Report for: ratno-kmcxe

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)

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

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)

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.