L-Methionine 4-nitroanilide

Microplate assays with 96 wells were optimized to screen proteolytic activities in mite homogenates. Whole-mite extracts of Acarus siro, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Tyroborus lini, Carpoglyphus lactis, Lepidoglyphus destructor, and Dermatophagoides farinae exhibited non-specific proteolytic activity in buffers from pH 2 to 12, and three peaks of highest activity at pH 3, 5-6, and 10 were distinguished. The reducing agent Tris(2-carboxyethyl)phosphine hydrochloride decreased general proteolytic activity on azocasein at pH 5 and 6. The results obtained on two non-specific substrates, azocasein and azoalbumin, showed highly different ranks of the species at pH 5 and 6. Proteolytic activities toward N(α)-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride, N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide, N-Succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide, Benzyloxycarbonyl-L-arginine-L-arginyl 4-nitroanilide, and N-Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine 4-nitroanilide (MAAPMpNA) were highest at alkaline pH, but the activity toward MAAPMpNA was also high at pH 5 and 6. In contrast, N-Succinyl-L-alanyl-L-alanyl-L-phenylalanine 4-nitroanilide (AAPpNA) and L-arginyl 4-nitroanilide (ArgpNA) had the highest activity recorded at pH 6. The high activities observed on AAPpNA, ArgpNA, and MAAPMpNA at digestive pH suggest that enzymes present in these extracts could have the majority of proteolysis in the mite gut. Evidence of the presence of proteolytic activities on all tested substrates and in all the tested mite homogenates suggests that the proteolytic activities may contribute to allergenicity. Poor or undetected hydrolytic activities of mite extracts toward substrates for keratin and collagen at digestive pH underline the importance of ecological interactions between mites and microorganisms in the utilization of such substrates.

A cysteine proteinase was detected in the penetration glands of the cercariae of Tylodelphys excavata and examined by means of biochemical and histochemical methods. The enzyme hydrolyzed azocoll, azocasein, azoalbumin, and N-benzoyl-l-arginine-4-nitroanilide at optimal pH 6.8, 6.4, 6.8, and 6.4, respectively, but was incapable of degrading elastin-orcein at the pH range of 6.0-10.0. Under histochemical conditions, the proteinase cleaved N-benzyloxycarbonyl-l-alanine-2-naphthyl ester and N-benzyloxycarbonyl-l-arginine-2-naphthylamide. A number of N-blocked synthetic substrates with l-methionine, l-leucine, glycine, dl- and l-phenylalanine at the P(1) position were not hydrolyzed. The metal cation complexane ethylenediamine tetraacetic acid (EDTA) and the reducing compound dithioerythritol stimulated the proteinase activity, whereas 0.1 mM zinc sulfate and the specific thiol-blocking reagent p-hydroxymercuribenzoate inhibited it. The proteinase activity was also sensitive to phenylmethylsulfonyl fluoride, leupeptin, tosyl-lysyl-chloromethylketone, and tosyl-phenylalanyl-chloromethylketone. An electrophoretic separation of extract proteins under non-denaturing conditions, at an operative pH of 3.5, in the presence of 5 mM EDTA and 5 mM 2-mercaptoethanol, revealed one main band of a relatively high gelatinolytic activity and two to three faint bands of low and very low activity, one of which was produced by a non-glandular intracellular cysteine proteinase related to cathepsin B. The other faint bands presumably were produced by partly autolysed molecules of the enzyme from the penetration glands.

Previous research has shown that the enzymatic activity of hepatic gamma-glutamyltransferase was increased in streptozotocin-induced diabetic rats with no increase in the expression of the protein. The current work has characterized the differences in the kinetic properties of hepatic gamma-glutamyltransferase from diabetic versus control rats. Hepatic gamma-glutamyltransferase was purified from control male and female rats and from rats made diabetic 30 days previously with streptozotocin. The maximal velocity and the Michaelis constant were determined for the purified enzyme with two separate donors (L-gamma-glutamyl-p-nitroanilide or L-gamma-glutamyl-(7-amido-4-methylcoumarin)) in the presence of one of eight acceptors (L-alanine-glycine, L-glycine-glycine, L-methionine, L-glutamate, L-alanine, L-glutamine, L-phenylalanine or L-aspartate). With both donors, hepatic gamma-glutamyltransferase from diabetic rats had a consistently higher kinetic efficiency than gamma-glutamyltransferase from controls. The kinetic efficiency percent increase of diabetic over control gamma-glutamyltransferase when averaged across all acceptors was higher in males than in females. With L-gamma-glutamyl-p-nitroanilide, the kinetic efficiency increase of diabetic over control gamma-glutamyltransferase was higher with poor acceptors than with highly efficient acceptors. These data indicate that there are differences in the physical properties of hepatic gamma-glutamyltransferase from diabetic versus control rats and from female versus male rats.