Identification of a Tripeptidyl Aminopeptidase in the Anterior Pituitary Gland: Effect on the Chemical and Biological Properties of Rat and Bovine Growth Hormones*

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Abstract
The NH2-terminal heterogeneity which is generated in bovine [b] GH [growth hormone] during its extraction from mildly acidified pituitary homogenates is attributable to a newly identified peptidase. The .beta.-naphthylamide of Phe-Pro-Ala, modeled after the NH2-terminal tripeptide sequence of the phenylalanyl monomer of bGH, was cleaved by the peptidase into the tripeptide and .beta.-naphthylamine and served as a substrate for assay of enzyme. The .beta.-naphthylamide of Ala-Phe-Pro, modeled after the NH2-terminal tripeptide sequence of the alanyl monomer, was not cleaved. In harmony with this specificity, the peptidase cleaved 11 tripeptides sequentially from the NH2-terminus of the phenylalanyl monomer of bGH but none from the alanyl monomer. Six of the tripeptides nearest the NH2-terminus were unequivocally identified and their sequences were consistent with the NH2-terminal octadecapeptide sequence of the phenylalanyl monomer of bGH. Five additional peptides were by composition consistent with their being tripeptides derived from residues 19-33. Because of the apparent specificity for the hydrolytic release of tripeptides and inability to cleave substituted tripeptidyl derivatives, the enzyme is considered to be a tripeptidyl aminopeptidase. In its hydrolysis of phenylalanyl monomers of rat GH, a similar number of tripeptides was released, associated with which there was a 70% loss of biological activity but no reduction in immunological activity. The enzyme could be solubilized by extraction with 1% Triton X-100 at pH 3.0, precipitated between 2 and 3 M (NH4)2SO4, and further purified by gel filtration on [Sephadex] G-75 in 10 M acetic acid. The enzyme has a MW of 57,000 and is optimally active at pH 4. It can be differentiated from cathepsin D by its insensitivity to inhibition by pepstatin.