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Titolo:
RAT TESTICULAR CARBOXYLESTERASE - CLONING, CELLULAR-LOCALIZATION, ANDRELATIONSHIP TO LIVER HYDROLASE-A
Autore:
YAN BF; YANG DF; BRADY M; PARKINSON A;
Indirizzi:
UNIV KANSAS,MED CTR,CTR ENVIRONM & OCCUPAT HLTH,DEPT PHARMACOL TOXICOL & THERAPEUT KANSAS CITY KS 66160 UNIV KANSAS,MED CTR,CTR ENVIRONM & OCCUPAT HLTH,DEPT PHARMACOL TOXICOL & THERAPEUT KANSAS CITY KS 66160
Titolo Testata:
Archives of biochemistry and biophysics
fascicolo: 2, volume: 316, anno: 1995,
pagine: 899 - 908
SICI:
0003-9861(1995)316:2<899:RTC-CC>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
PHENOL-CHLOROFORM EXTRACTION; SINGLE-STEP METHOD; ENDOPLASMIC-RETICULUM; MICROSOMAL CARBOXYESTERASE-E1; NUCLEOTIDE-SEQUENCE; BETA-GLUCURONIDASE; SERINE HYDROLASES; CRESYL PHOSPHATE; RNA ISOLATION; CELLS;
Keywords:
CARBOXYLESTERASE; CDNA CLONING FROM RAT TESTIS; ESTERASE; CDNA CLONING FROM RAT TESTIS; HYDROLASE A; CDNA CLONING; IMMUNOCYTOCHEMISTRY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
40
Recensione:
Indirizzi per estratti:
Citazione:
B.F. Yan et al., "RAT TESTICULAR CARBOXYLESTERASE - CLONING, CELLULAR-LOCALIZATION, ANDRELATIONSHIP TO LIVER HYDROLASE-A", Archives of biochemistry and biophysics, 316(2), 1995, pp. 899-908

Abstract

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase A, that catalyzes the hydrolysis of para-nitrophenylacetate with high affinity (K-m similar to 25 mu M) and is very sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminalamino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cloned from a rat liver cDNA library by Robbi ct al. (Biochem. J, 269, 451-458, 1990). A PMSF-sensitive esterase with high affinity toward para-nitrophenylacetate is also present in testicular microsomes at levels that slightly exceed those in liver microsomes. Antibody against purified hydrolase A recognizes a 57-kDa protein in both liver and testicular microsomes, suggesting that hydrolase A is expressed to a high degree in both tissues, To determine whether the testicular carboxylesterase is identical to hydrolase A, a rat testicular cDNA library was constructed and screened with antibody against hydrolase A, A 709-bp cDNA was isolated from immunopositive clones, Screening the same cDNA library by polymerase chain reaction (PCR) with one primer based on the sequence 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 withthe 709 bp-sequence. Together these two cDNA fragments spanned a 1792-bp sequence with an opening reading frame encoding 518 amino acids, which corresponds to similar to 95% of the C-terminal sequence of the liver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide differences at positions 479, 855, 1335, and 1350, the sequence of the testicular cDNA was identical to the cDNA sequence of the liver pi 6.1esterase reported by Robbi ct al. None these changes results in an amino acid substitution. However, these four base substitutions were notobserved when a cDNA encoding hydrolase A was isolated from a rat liver cDNA library by PCR. These results establish that the same carboxylesterase, namely, hydrolase A, is expressed in rat liver and testis. The levels of mRNA for hydrolase A in various rat tissues was estimatedfrom Northern blots probed with the 709-bp cDNA isolated from the rattesticular cDNA library. A similar to 2-kb mRNA for hydrolase A was detected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase A is localized in the centrilobular region of the liver, in the interstitial (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, wasabolished in rats treated with ethane dimethane sulfonate, a selective Leydig cell toxicant, which confirmed that the high levels of hydrolase A in rat testis are confined to the Leydig cells, The presence of hydrolase A in rat testis may be physiologically and toxicologically important. Testicular dysfunction in rats treated with tri-ortho-tosylphosphate (TOTP) is the result of damage to Sertoli cells, despite the fact that TOTP is activated to cresylbenzodioxaphosphorin oxide (CBDP)in Leydig cells, We previously hypothesized that Leydig cells are protected from the toxic effects of TOTP by extremely high levels of hydrolase A, which avidly binds to CBDP, The results of this study supportthis hypothesis. (C) 1995 Academic Press, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 30/10/20 alle ore 09:00:04