Rabbit pulmonary angiotensin-converting enzyme: the NH2-terminal fragment with enzymatic activity and its formation from the native enzyme by NH4OH treatment.
The NH2-terminal sequence of 22 residues of rabbit lung angiotensin-converting enzyme has been determined as (NH2)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp-Phe-Ala -Ala-Asp-Asn-Ala-Gly-Ala-Arg-Leu-Phe-Ala-. In the course of purification of the enzyme for structural analysis a protein of Mr = 82,000 with angiotensin-converting activity was separated from the major fraction containing the native enzyme (Mr = 140,000). This low-molecular-weight enzyme catalyzed the hydrolysis of the synthetic substrate Hip-His-Leu at a rate 23% of that with the native enzyme, and exhibited a similar Km value as well as behaviors towards various effectors of angiotensin-converting enzyme. Edman degradation of both the native and the 82K enzymes revealed that they contain identical amino acid sequences from the NH2-termini. This result and those of peptide mapping and carbohydrate and amino acid analyses indicate that the 82K enzyme is a fragment derived from the NH2-terminal portion of the native enzyme, and hence contains its catalytic site. Evidence has been obtained indicating that the active fragment was formed from the native enzyme during its elution from the antibody-affinity column with NH4OH: on treatment of the native enzyme (140K Mr) with 1 N NH4OH at room temperature, a cleavage occurred and two proteins with Mr = 82K and Mr = 62K were obtained. The 82K Mr fragment was found to be enzymatically active and to contain the same NH2-terminal sequence as the native enzyme. The other fragment (62K Mr) was devoid of the activity and was shown to derive from the COOH-terminal portion of the native enzyme by the peptide mapping and terminal analyses. Cleavage of a peptide bond with NH4OH is unusual and appears to be specific for the native angiotensin-converting enzyme from rabbit lung.
D000596:Amino Acids / Q000032:analysis; D064753:Ammonium Hydroxide; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification; D006878:Hydroxides; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification*; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification*; D011817:Rabbits; D013329:Structure-Activity Relationship
D000596:Amino Acids; D004792:Enzyme Precursors; D006878:Hydroxides; D010446:Peptide Fragments; D064753:Ammonium Hydroxide; D007703:Peptidyl-Dipeptidase A