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

T.W. DOEBBER and A. R. DIVOR

Biomedical Research Division, Ames Research Center, National Aeronautics and Space Administration, Moffett Field, California 94035

Address reprint requests to: S. Ellis, Biomedical Research Division, Ames Research Center, NASA, Moffett Field, CA 94035

Abstract

The NH₂-terminal heterogeneity which is generated in bovine GH during its extraction from mildly acidified pituitary homogenates is attributable to a newly identified peptidase. The β-naphthylamide of Phe-Pro-Ala, modeled after the NH₂-terminal tripeptide sequence of the phenylalanyl monomer of bovine growth hormone, was cleaved by the peptidase into the tripeptide and /8-naphthylamine and served as a substrate for assay of the enzyme. However, the β-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 bovine GH 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 monomerf bovine GH. 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 growth hormone, 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 G- 75 in M/10 acetic acid. The enzyme has a mol wt of 57,000 and is optimally active at pH 4. It can be differentiated from cathepsin D by its insensitivity to inhibition by pepstatin

Footnotes

^* This work was conducted during the tenure of an NRC-NASA Resident Research Associateship.

Current address: Department of Physiology and Biophysics, Washington University School of Medicine, St.Louis, MO.

Received February 8, 1978.

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