Susumu Maruyama

Prolyl endopeptidase inhibitory activity of peptides in the repeated sequence of various proline-rich proteins

Abstract Peptides present in the repeating region in proline-rich proteins such a corn γ-zein, soybean cell wall protein (SbPRP1), carrot 33-kDa protein, collagen and mouse salivary gland protein were chemically synthesized. The prolyl endopeptidase (PEP, from Flavobacterium meningosepticum or bovine brain) inhibitory activity of these peptides was investigated. γ-Zein-related synthetic peptides such as His-Leu-Pro-Pro-Pro-Val and His-Leu-Pro-Pro-Pro-Val-His-Leu-Pro-Pro-Pro-Val inhibited PEP from both bacterial and mammalian sources. The IC 50 values of these peptides for F. meningosepticum PEP were 80 μM and 30 μM, respectively. Soybean cell wall protein-related synthetic peptides, Lys-Pro-Pro-Val and Lys-Pro-Pro-Ile also inhibited PEP to some degree. However, Pro-Pro-Pro-Pro-Gly-Gly-Pro-Gln-Pro-Arg-Pro-Pro-Glu-Gly (synthetic peptide fragment of salivary gland protein) and Gly-Pro-Hyp-Gly-Pro-Ala (fragment of collagen) showed hardly any inhibition. Therefore, we hydrolyzed γ-zein with subtilisin (Carlsberg) and confirmed the liberation of the native hexapeptide, His-Leu-Pro-Pro-Pro-Val.

Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting

Inhibitory effects of some enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting were investigated. The hydrolysate of porcine skin collagen with thermolysin or bacterial collagenase inhibited fibrinogen/thrombin clotting, but did not inhibit the activity of thrombin. Although the activity was not pronounced, the hydrolysate of collagen with such proteinases as trypsin and pepsin also inhibited the clotting. Gly-Pro-Arg, which is a known inhibitor of fibrinogen/thrombin clotting, was isolated from the bacterial collagenase hydrolysate of porcine collagen by HPLC. Collagen-related synthetic peptides such as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-Pro-Ala, Gly-Pro-Arg-Gly-Pro-Pro, and Gly-Pro-Arg-Pro-Pro also inhibited the clotting, but did not inhibit the activity of thrombin. The inhibitory activity of Gly-Pro-Arg-Gly-Pro-Pro and Gly-Pro-Arg-Pro-Pro was more marked than that of Gly-Pro-Arg. However, Gly-Pro-Lys, Gly-Ala-Arg, Gly-Pro-Hyp, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory effect on the clotting.