Gly-Pro p-nitroanilide hydrochloride

Proteins are the most prominent bioactive component in deer antler velvet. The aim of the present study was to track the fate of protein of antler velvet by protein digestomics. The peptide profile identified by LC-MS/MS and the in vitro bioactivity of antler velvet aqueous extract (AAE) were investigated in simulated gastrointestinal digestion. A total of 23, 387 and 417 peptides in AAE, gastric and pancreatic digests were identified using LC-MS/MS, respectively. Collagens, the predominant proteins, released 34 peptides in gastric digests and 146 peptides in pancreatic digests. The gastric and pancreatic digests presented dipeptidyl peptidase IV (DPP-IV) and prolyl endopeptidase (PEP) inhibition activities. Four peptides from digests were proved to be DPP-IV and PEP inhibitory peptides. The results showed that the peptides released from antler velvet protein contributed to the bioactivity of antler velvet during digestion.

Objective: Polycystic ovary syndrome (PCOS) is a complex disease, the aetiology of which is not well understood. Alterations in potential candidate genes involved in the biosynthesis and metabolism of androgens, folliculogenesis and insulin and glucose metabolism have been suggested as possible aetiologies. Dipeptidyl peptidase-4 (DPP4) plays a key role in glucose homoeostasis and, thus, in the regulation of insulin secretion. The aim of our study was to analyse the DPP4 activity and concentrations in the serum of PCOS and non-PCOS patients and, additionally, study the activation of the DPP4 promoter by androgens in vitro. Design, patients and measurements: Serum samples were obtained from 288 female patients treated at the Center for Reproductive Medicine and Andrology (154 non-PCOS and 134 patients with PCOS). DPP4 activity was measured by the conversion of the DPP4 substrate Gly-Pro p-nitroanilide hydrochloride and DPP4 concentration with a commercial ELISA. Luciferase reporter assays, qPCR and Western Blot analyses were performed for the in vitro evaluation of the activation of the DPP4 promoter by androgens. Results: DPP4 serum activity was increased in women with PCOS, regardless of which Rotterdam criteria led to the PCOS diagnosis. Furthermore, DPP4 serum levels were strongly correlated with the anti-Müllerian hormone (AMH) serum level. In vitro, the DPP4 promoter was stimulated by androgens in luciferase reporter assays, and DPP4 mRNA expression was increased in KGN granulosa carcinoma cells after androgen treatment. Conclusions: The results suggested that a deregulation of DPP4 serum levels could be an additional characteristic of the metabolic imbalances associated with PCOS.

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.