RAT TESTICULAR CARBOXYLESTERASE - CLONING, CELLULAR-LOCALIZATION, ANDRELATIONSHIP TO LIVER HYDROLASE-A

We recently purified from rat liver microsomes a carboxylesterase, des ignated hydrolase A, that catalyzes the hydrolysis of para-nitrophenyl acetate with high affinity (K-m similar to 25 mu M) and is very sensit ive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminal amino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cl oned from a rat liver cDNA library by Robbi ct al. (Biochem. J, 269, 4 51-458, 1990). A PMSF-sensitive esterase with high affinity toward par a-nitrophenylacetate is also present in testicular microsomes at level s that slightly exceed those in liver microsomes. Antibody against pur ified hydrolase A recognizes a 57-kDa protein in both liver and testic ular microsomes, suggesting that hydrolase A is expressed to a high de gree in both tissues, To determine whether the testicular carboxyleste rase is identical to hydrolase A, a rat testicular cDNA library was co nstructed and screened with antibody against hydrolase A, A 709-bp cDN A was isolated from immunopositive clones, Screening the same cDNA lib rary by polymerase chain reaction (PCR) with one primer based on the s equence of the 709-bp cDNA and one primer based on the sequence of the adjoining lambda gt11 arm yielded a 1,1-kb cDNA that overlapped with the 709 bp-sequence. Together these two cDNA fragments spanned a 1792- bp sequence with an opening reading frame encoding 518 amino acids, wh ich corresponds to similar to 95% of the C-terminal sequence of the li ver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide di fferences at positions 479, 855, 1335, and 1350, the sequence of the t esticular cDNA was identical to the cDNA sequence of the liver pi 6.1 esterase reported by Robbi ct al. None these changes results in an ami no acid substitution. However, these four base substitutions were not observed when a cDNA encoding hydrolase A was isolated from a rat live r cDNA library by PCR. These results establish that the same carboxyle sterase, namely, hydrolase A, is expressed in rat liver and testis. Th e levels of mRNA for hydrolase A in various rat tissues was estimated from Northern blots probed with the 709-bp cDNA isolated from the rat testicular cDNA library. A similar to 2-kb mRNA for hydrolase A was de tected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western i mmunoblotting. Immunocytochemical studies established that hydrolase A is localized in the centrilobular region of the liver, in the interst itial (Leydig) cells of the testis, and in the Clara cells of the lung . The expression of hydrolase A in the testis, but not the liver, was abolished in rats treated with ethane dimethane sulfonate, a selective Leydig cell toxicant, which confirmed that the high levels of hydrola se A in rat testis are confined to the Leydig cells, The presence of h ydrolase A in rat testis may be physiologically and toxicologically im portant. Testicular dysfunction in rats treated with tri-ortho-tosylph osphate (TOTP) is the result of damage to Sertoli cells, despite the f act that TOTP is activated to cresylbenzodioxaphosphorin oxide (CBDP) in Leydig cells, We previously hypothesized that Leydig cells are prot ected from the toxic effects of TOTP by extremely high levels of hydro lase A, which avidly binds to CBDP, The results of this study support this hypothesis. (C) 1995 Academic Press, Inc.