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SYNTHESIS OF MOUSE RENIN AS A 2‐5‐33‐5 KILODALTON PRE‐PRO‐ TWO‐CHAIN MOLECULE AND USE OF ITS cDNA TO IDENTIFY THE HUMAN GENE
Author(s) -
Morris Brian J.,
Catanzaro Daniel F.,
Mullins John J.,
Hardman Judy,
Shine John
Publication year - 1983
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.1983.tb00200.x
Subject(s) - complementary dna , renin–angiotensin system , microbiology and biotechnology , biochemistry , messenger rna , biology , gene expression , pepstatin , chemistry , protease , gene , enzyme , endocrinology , blood pressure
SUMMARY 1. A DNA coding for mouse submandibular gland (MSG) renin was used in studies of the biosynthesis and processing of MSG renin and precursors and as a probe for identification of the human renin gene. 2. A 2 kilodalton signal peptide was demonstrated by cleavage of preprorenin (45 kilodaltons) in the presence of microsomal membranes from dog pancreas. Prior selection of renin mRNA by hybridization with its cDNA obviated the possibility that a protease translated from total MSG mRNA could be activated by the microsomal membranes and then act on the primary translation product. 3. In vitro labelling experiments with female MSG demonstrated that prorenin (43 kilodaltons) is rapidly converted to renin (38 kilodaltons) and that testosterone stimulated synthesis by increasing transcription of renin mRNA. 4. Electrophoresis under non‐reducing conditions demonstrated 38 kilodalton renin which when reduced ran as two bands of 33 and 5 kilodaltons. Thus, native MSG renin has two chains linked by disulphide bonding. Hydrolysis of the 38 kilodalton single chain occurred only slowly during in vitro labelling. 5. Prorenin bound only weakly to a pepstatin affinity column and could be activated by adding trypsin to column fractions. Both 38 kilodalton single and two chain renin bound strongly however, suggesting that both are active. 6. Base sequencing of MSG renin cDNA indicated an Arg‐Arg residue additional to the published amino acid sequence of the heavy chain. This may account for the two forms seen on isolectric focusing. 7. Mouse renin cDNA was used as a hybridization probe in screening a human genomic library in order to identify the human renin gene. The DNA in positive colonies had overlapping restriction maps and the coding region was found.