Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae.
ABSTRACT Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.
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ABSTRACT: The synthesis and properties of l-lysine p-nitroanilide dihydrobromide and benzoyl dl-arginine p-nitroanilide hydrochloride are described. Both compounds are hydrolyzed by trypsin, the latter being a substrate at least as sensitive as benzoyl l-argininamide. Their hydrolysis can be followed conveniently by colorimetric procedures, since one of the products, p-nitroaniline, is yellow. Values of Km and k3 for their reaction with trypsin were determined, as well as the K4 of benzoyl d-arginine p-nitroanilide, which was isolated from a tryptic digest of the dl isomer. The pH-activity curves were also determined, that of l-LPA being in a more alkaline region than normally found for trypsin substrates. The possible significance of this shift is discussed.Preliminary studies indicate that benzoyl dl-arginine p-nitroanilide hydrochloride is also hydrolyzed by papain.Archives of Biochemistry and Biophysics 12/1961; · 3.37 Impact Factor
- Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology - COMP BIOCHEM PHYSIOL B BIOCHEM MOL BIOL. 01/1994; 109(1):1-62.
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ABSTRACT: The major proteases of the grass grub (Costelytra zealandica) larval midgut have been identified, partially purified and characterized. Identification was made initially on the basis of hydrolysis of synthetic substrates (blocked and partially blocked esters and amides of specific amino acids), thus classifying the activities into different classes of endo- and exopeptidases. A range of inhibitors specific to different classes of proteases were used to confirm the presence of trypsin, chymotrypsin, elastase, leucine aminopeptidase and carboxypeptidases A and B and to establish the absence of thiol- and metallo-endopeptidases. The dominant endopeptidase in the midgut is trypsin, which is present in four forms, distinguishable by net charge, but indistinguishable either in terms of Michaelis-Menten parameters (Km and kcat) or in molecular weight (23,000). The pH optimum lies between pH 9–10. Leucine aminopeptidase has a molecular weight of 91,000 and a pH optimum at pH 8.0. Carboxypeptidase A has a molecular weight of 43,000 and a pH optimum at pH 8.5. All enzymes retained substantial activity at pH 7.0–7.1, the pH of the midgut lumen, where the bulk of the activity was located. Protease levels in the hindgut (or fermentation sac) were 1–5% of those in the midgut. The range of enzymes appears sufficient for complete breakdown of ingested protein.Insect Biochemistry. 01/1989;