Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

Oliveira, Vitor; Ferro, Emer S.; Gomes, Marcelo D.; Oshiro, Maria E.M.; Almeida, Paulo C.; Juliano, María A.; Juliano, Luiz

doi:10.1002/(sici)1097-4644(20000301)76:3<478::aid-jcb14>3.0.co;2-h

Cited by 11 publications

(2 citation statements)

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“…On the other hand, membrane associated form of TOP [10] and neurolysin have been identified [11,12]. The secretion of TOP has been reported in AtT20 [13,14] and MDCK cells [15] while neurolysin was showed to be secreted by astrocytes [16].…”

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confidence: 99%

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Oliveira

Gatti

Rioli

et al. 2002

European Journal of Biochemistry

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We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 M for NaCl, KCl, NH 4 Cl and NaI, and 0.025-0.1 M for Na 2 SO 4 . Concentration higher than 0.2 N NH 4 Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na 2 SO 4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na 2 SO 4 , while NH 4 Cl and NaI have very modest effect. High positive values of enthalpy (DH*) and entropy (DS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.Keywords: protease; peptide; metalloprotease; fluorescence; enthalpy of activation in proteolysis; entropy of activation in proteolysis.Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zinc-dependent peptidases, members of the metallopeptidase M3 family and contain in their primary sequence the HEXXH motif [1,2]. Rat neurolysin has been the first member of the M3 family of which the 3D structure has been determined [3]. As it has been shown that this enzyme and thermolysin have common ancestors, the M3 family was thus included in the clan MA [4]. It is interesting to note that in the structure of neurolysin, the catalytic center is located in a deep channel [3], which limits the access only to short peptides [5,6]. This selectivity toward hydrolysis of oligopeptides was also verified for TOP [5][6][7][8][9]. The high primary sequence identity found for these related peptidases, which is about 65% [2], allows hypothesizing that they may share a similar folding including the deep channel that constitutes the neurolysin active site. This feature seems to prevent the unspecific cleavage of other proteins and it is of particular relevance for TOP and neurolysin as they are not expressed as inactive precursors and they are...

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confidence: 99%

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Oliveira

Gatti

Rioli

et al. 2002

European Journal of Biochemistry

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“…The use of the THOP specific inhibitor JA2 was shown to potentiate BK-induced hypotension, without affecting the hypertensive effects of angiotensins I and II, strongly suggesting its role in blood pressure control [22]. THOP also significantly affects BK in the kidney, having a role in blood flow, glomerular filtration rate, urinary sodium and potassium excretion and urine volume [33][34][35][36]. Furthermore, THOP also converts angiotensin I in vitro to the biologically active peptide angiotensin 1-7 [37] synergizing with the effects of bradykinin in vascular smooth muscle cells culture [38,39] and in rat hindlimb perfusate ex vivo [40].…”

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Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles

et al. 2020

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The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been conducted to develop biotechnological tools to make possible the use of both proteases to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases.

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The effect of intracrystalline and surface-bound osteopontin on the degradation and dissolution of calcium oxalate dihydrate crystals in MDCKII cells

2011

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In vivo, urinary crystals are associated with proteins located within the mineral bulk as well as upon their surfaces. Proteins incarcerated within the mineral phase of retained crystals could act as a defence against urolithiasis by rendering them more vulnerable to destruction by intracellular and interstitial proteases. The aim of this study was to examine the effects of intracrystalline and surface-bound osteopontin (OPN) on the degradation and dissolution of urinary calcium oxalate dihydrate (COD) crystals in cultured Madin Darby canine kidney (MDCK) cells. [(14)C]-oxalate-labelled COD crystals with intracrystalline (IC), surface-bound (SB) and IC + SB OPN, were generated from ultrafiltered (UF) urine containing 0, 1 and 5 mg/L human milk OPN and incubated with MDCKII cells, using UF urine as the binding medium. Crystal size and degradation were assessed using field emission scanning electron microscopy (FESEM) and dissolution was quantified by the release of radioactivity into the culture medium. Crystal size decreased directly with OPN concentration. FESEM examination indicated that crystals covered with SB OPN were more resistant to cellular degradation than those containing IC OPN, whose degree of disruption appeared to be related to OPN concentration. Whether bound to the crystal surface or incarcerated within the mineral interior, OPN inhibited crystal dissolution in direct proportion to its concentration. Under physiological conditions OPN may routinely protect against stone formation by inhibiting the growth of COD crystals, which would encourage their excretion in urine and thereby perhaps partly explain why, compared with calcium oxalate monohydrate crystals, COD crystals are more prevalent in urine, but less common in kidney stones.

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Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

Cited by 11 publications

References 49 publications

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles

The effect of intracrystalline and surface-bound osteopontin on the degradation and dissolution of calcium oxalate dihydrate crystals in MDCKII cells

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