Dipeptidyl peptidase II of bovine dental pulp. Initial demonstration and characterization as a fibroblastic, lysosomal peptidase of the serine class active on collagen-related peptides.

ABSTRACT

Dipeptidyl peptidase II (dipeptidylpeptide hydrolase, EC 3.4.14.2), previously known as dipeptidyl aminopeptidase II, was shown to be present in relatively high concentrations in bovine dental pulp. Based on the DNA content of tissue homogenates, the fibroblasts of this connective tissue appeared to contain more dipeptidyl peptidase II than the cells of lysosome-rich tissues such as bovine spleen and rat liver. The newly-recognized properties of dipeptidyl peptidase II, from both pulp and pituitary sources, included a marked propensity for hydrolyzing prolyl bonds at acidic pH. Lys-Pro-2-NNap and Lys-Pro-2(4-methoxy)naphthylamide (designed for cytochemical application) were hydrolyzed at rates equal to that of Lys-Ala-2-NNap. The impure pulp enzyme and the authentic pituitary enzyme showed comparable relative rates of hydrolysis on a variety of fluorogenic substrates with the general structure X-Pro-2-NNap (X = Lys, Arg, Phe, Ala or Gly), and on a variety of collagen-related tripeptides represented by X-Pro-Ala (X = Gly, Ala or Lys). The highest rates wee obtained on Lys-Ala-Ala and Lys-Ala-Pro. The pH optima for the hydrolysis of the 2-naphthylamide derivatives varied from 5.0 to 5.7, and for tripeptides from 4.2 to 5.3. In all cases the N-terminal dipeptide was released intact. Although previously unrecognized as a serine protease, dipeptidyl peptidase II (of pulp and pituitary origin) was strongly inhibited by (1 mM) diisopropyl phosphorofluoridate, p-nitrophenyl-p'-guanidinobenzoate, and phenylmethylsulfonyl fluoride. The enzyme, from both sources, was fully inhibited by 0.1 mM Lys-Ala chloromethyl ketone, a newly-designed, active-site-directed inhibitor. The numerous properties shared by the putative dipeptidyl peptidase II of bovine dental pulp and an authentic preparation of the bovine pituitary enzyme provided strong support for their having a common identity.