Caracterização cinética da inibição da calicreína tecidual do rato, isolada da glândula submandibular, com a aprotinina

Abstract

Kallikreins are a group of serine proteases that play an important role in many physiologic processes depending on the tissue end conditions of expression. They are found in pancreas, salivary glands, intestine, kidneys, pituitary gland, plasma and other tissues. Many kallikreins seem to be related in many pathologic processes, including in central nervous system (CNR), such as epilepsy and Alzheimer, and cancer, being, therefore, future biomarkers for tumours of different organs. The aim of this study was to stablish the kinetic model of the inhibition of rat tissue kallikrein, isolated from submandibulary glands, by aprotinin and tocompare the results with data from literature. Lyophilized powder was obtained from three hundred submandibulary glands extracts of adults Wistar rats and rK1 was purified by DEAE-Sepharose, Sepharose-aprotinin and gel filtration Superose 12 HR column chromatographies. The protein concentration was determined byBradford method (1976) and the rK1 amidasic activity was determinated with Bz- Arg-Nan as substrate. The purification factor was 32 with 5% recovery. Electrophoresis was performed using 5-15% gradient polyacrilamide gel and 0,1% SDS. Mass spectrometry was also accomplished. Both methods showed a pure rK1 corresponding to Mr 28000 approximately. Purified rK1 was titrated with aprotinin and its active center concentrations, was 692 nM. Hydrolysis of D-Val- Leu-Arg-Nan by rK1, at pH 9.0 and 37oC, was carried out in the absence and in thepresence of increasing concentrations of aprotinin. The results showed that the hydrolyses followed Michaelis-Menten kinetics on the wide range of substrate concentration (120-640 mM). Km and kcat values are 104.2 + 24.6 and 8641 + 572 20 min-1, respectively. The data indicated that inhibition of rK1 by aprotinin is not asimple competitive inhibition, but that it is a competitive inhibition of the parabolic type. Parabolic competitive inhibition is a rare type of enzyme inhibition, in wich two aprotinin molecules binds to one rK1 molecule. The calculated values of the constants Ki and Kii were, 26.4 + 12.0 and 16.9 + 8.8 nM, respectively. Parabolic competitive inhibition was also reported for the hK1 inhibition by aprotinin with DVal-Leu-Arg-Nan as substrate.