Abstract
A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.
Highlights
EXPERIMENTAL PROCEDURESThe radiolabelwas released at cycle 3 and the amount Conuerting Enzyme-Bovine lung converting enzyme wasprepared recovered was equivalent to theamountofphenyl- by affinity chromatography (2,6).We routinely obtain about 3 mg of thiohydantoin-Glu detected on HPLC
16,000) was digested with trypsinandthelabeled peptide formed(T-2) was further degraded with thermolysin
The radiolabelwas released at cycle 3 and the amount Conuerting Enzyme-Bovine lung converting enzyme wasprepared recovered was equivalent to theamountofphenyl- by affinity chromatography (2,6).We routinely obtain about 3 mg of thiohydantoin-Glu detected on HPLC
Summary
The radiolabelwas released at cycle 3 and the amount Conuerting Enzyme-Bovine lung converting enzyme wasprepared recovered was equivalent to theamountofphenyl- by affinity chromatography (2,6).We routinely obtain about 3 mg of thiohydantoin-Glu detected on HPLC. The alkylatingagentsp-[NJV-bis(chloroethyl)amino]phen- High Performance Liquid Chromatography-HPLCwasused to ylbutyric acid (chlorambucil) antdhe chlorambucil derivative separate the cyanogen bromide and enzyme cleavagepeptides and to of L-proline (chorambucyl-L-proline) rapidly and irreversibly identify PTH-derivatives after each cycle of the Edman procedure. Inactive angiotensin I-converting enzyme (peptidyl dipeptide All separations were performed on Cls reverse-phase columns; the carboxyhydrolase, EC 3.4.15.1) (1). Both compounds behave as affinity labels andchlorambucyl-~-[U-'~C]prolinreeacts in cyanogen bromide and enzyme cleavagepeptides were resolved with a linear gradient of 0.1% trifluoroacetic acid to 0.1%trifluoroacetic acid, 50% acetonitrile (6, 10) and were detected at 220 nm
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