The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15. 1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxylterminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.Peptidyl-dipeptidase A (EC 3.4.15.1) plays an important role in blood pressure homeostasis by hydrolyzing angiotensin I, the inactive peptide released after cleavage of angiotensin by renin, into angiotensin II (1). Accordingly, this Zn metallopeptidase is designated angiotensin I-converting enzyme (ACE), although being the same enzyme as kininase II, it is also able to hydrolyze bradykinin and various other peptides (2, 3). This enzyme is a widely distributed peptidase, occurring, for example, as a membrane-bound ectoenzyme on the surface of vascular endothelial cells and renal epithelial cells and as a circulating enzyme in plasma (3-5). We report here the amino acid sequence of ACE as deduced from the nucleotide sequence of DNA complementary to the ACE mRNA.t MATERIALS AND METHODSPurification and Sequencing of ACE and Preparation of Oligodeoxyribonucleotide Probe. The cortex offresh postmortem human kidneys (600 g) was homogenized (54:100, wt/vol) in 20 mM potassium phosphate buffer (pH 8) containing 250 mM sucrose and a mixture of protease inhibitors, cells debris was discarded, and the particulate fraction was sedimented by centrifugation at 105,000 x g for 1 hr. The pellet was resuspended in 200 ml of 150 mM potassium phosphate buffer (pH 8; buffer I) and treated for 18 hr with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 8 mM; Serva). The supernatant obtained after centrifugation at 105,000 x g for 1 hr was dialyzed extensively against b...
In earlier studies we identified in a human genomic library a gene (human relaxin gene HI) coding for a relaxin-related peptide. We now have evidence that the human genome possesses an additional relaxin-related gene (designated human relaxin gene H2) which appears to be selectively expressed in the ovary during pregnancy. Nucleotide sequence analysis revealed striking differences in the predicted structures of relaxin encoded by these two genes. Chemical synthesis of biologically active relaxin based on the sequence obtained from ovarian cDNA clones confirmed that the expressed gene (H2) encodes an authentic human relaxin. The expressed gene appears to be transcribed into two different sized mRNAs and preliminary evidence suggests that the mRNA transcripts possess different 3'-untranslated regions. There was no evidence for the expression of human relaxin gene HI in the ovary and so far it is unclear whether gene Hl is expressed in another tissue or whether it represents a pseudogene. From the sequence data presented here it will now be possible to construct oligonucleotide probes and raise antibodies against synthetic peptides which could then be used to identify sites of relaxin biosynthesis and specifically quantitate the expression from either the Hl or H2 relaxin genes.
Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.
The ammo terminal sequence of the myosin light cham (Mr = 20 000) Isolated from chicken gizzards was found to be acetyl-Ser-Ser-Lys-Ag-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser(P)-Asn-Val-Phe. This sequence assignment differs from that reported by Msuta et al. [(1981) European J. Biochem. 117, 4171 in the order of the tryptic peptides. The revised amino acid sequence exhibits greater homology with the phosphorylation site sequences of the regulatory light chains from cardiac and skeletal muscle. Moreover it is now apparent why synthetic peptides corresponding to the previously reported sequence were very poor substrates for the myosin light chain kinase.
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