TISSUE-SPECIFIC MOLECULAR DIVERSITY OF AMIDATING ENZYMES (PEPTIDYLGLYCINE ALPHA-HYDROXYLATING MONOOXYGENASE AND PEPTIDYLHYDROXYGLYCINE N-C LYASE) IN XENOPUS-LAEVIS

被引:11
作者
IWASAKI, Y [1 ]
SHIMOI, H [1 ]
SAIKI, H [1 ]
NISHIKAWA, Y [1 ]
机构
[1] CIBA GEIGY JAPAN LTD,INT RES LABS,BIOORGAN RES DEPT,POB 1,TAKARAZUKA 665,JAPAN
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 214卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1993.tb17984.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We investigated the molecular diversity of the paired enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase (PHL), involved in peptide C-terminal amidation. Three kinds of amidating enzyme (AE) cDNAs (AE-I, AE-II and AE-III) have previously been isolated from Xenopus laevis skin. While AE-I cDNA encodes only PHM, AE-III cDNA encodes a protein containing both PHM and PHL sequences and a transmembrane domain. On the other hand, the translated product of AE-II has not been detected yet. Endoproteolytic cleavage of the AE-III protein generates separated forms of PHM and PHL that are purified from X. laevis skin. Expression of AE-III in insect cells using a baculovirus expression vector system indicated that PHM and PHL exist as a membrane-associated, bifunctional enzyme without endoproteolysis in insect cells. Both PHM and PHL activities were detected in all the X. laevis tissues examined. Particularly, the highest levels of both activities were found in skin, brain and heart. We identified basically three types of enzymes in X. laevis; soluble PHM, soluble PHL and a membrane-associated, bifunctional enzyme that has both PHM and PHL domains. While the skin contained soluble types of PHM and PHL, the brain and heart predominantly contained the membrane-associated, bifunctional type. Analysis of mRNA levels by the reverse-transcript polymerase chain reaction method and Western blot analysis using PHM-specific antibody revealed that such molecular diversity of PHM and PHL among the tissues are produced by changing the ratio of AE-I mRNA/AE-III mRNA, and by endoproteolytic processing of the membrane-associated precursor protein.
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收藏
页码:811 / 818
页数:8
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