An Isozyme of Earthworm Serine Proteases Acts on Hydrolysis of Triacylglycerol

2005 ◽  
Vol 69 (10) ◽  
pp. 2009-2011 ◽  
Author(s):  
Nobuyoshi NAKAJIMA ◽  
Manabu SUGIMOTO ◽  
Sadao TSUBOI ◽  
Hideaki TSUJI ◽  
Kohji ISHIHARA
Keyword(s):  
Serine Proteases ◽  
Hydrolysis Of

scholarly journals Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis

2021 ◽  
Vol 22 (13) ◽  
pp. 6896
Author(s):  
Bianca op den Brouw ◽  
Parviz Ghezellou ◽  
Nicholas R. Casewell ◽  
Syed Abid Ali ◽  
Behzad Fathinia ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Serine Proteases ◽  
Human Melanoma ◽  
Snake Venoms ◽  
Lead Compounds ◽  
Bioactive Properties ◽  
Fibrinogenolytic Activity ◽  
Pharmacological Potential ◽  
Hydrolysis Of ◽  
Viper Venoms

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

Degradation of phthalic acid esters (PAEs) by an enzyme mimic and its application in the degradation of intracellular DEHP

2019 ◽  
Vol 55 (89) ◽  
pp. 13458-13461 ◽  
Author(s):  
Xia Li ◽  
Jianpeng Li ◽  
Junxiang Zhu ◽  
Sijia Hao ◽  
Guozhen Fang ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Phthalic Acid ◽  
Enzyme Mimic ◽  
Phthalic Acid Esters ◽  
Hydrolysis Of ◽  
Acid Esters

An enzyme mimic inspired by serine proteases was developed for the degradation of PAEs and applied in the hydrolysis of intracellular DEHP.

scholarly journals Peptidyl Activity-Based Probes for Imaging Serine Proteases

2021 ◽  
Vol 9 ◽  
Author(s):  
Paulina Kasperkiewicz
Keyword(s):  
Posttranslational Modifications ◽  
Serine Proteases ◽  
Visual Observation ◽  
Biological Processes ◽  
Basic Principles ◽  
Peptide Bonds ◽  
Endogenous Inhibitors ◽  
Hydrolysis Of ◽  
Enormous Number

Proteases catalyze the hydrolysis of peptide bonds. Products of this breakdown mediate signaling in an enormous number of biological processes. Serine proteases constitute the most numerous group of proteases, accounting for 40%, and they are prevalent in many physiological functions, both normal and disease-related functions, making them one of the most important enzymes in humans. The activity of proteases is controlled at the expression level by posttranslational modifications and/or endogenous inhibitors. The study of serine proteases requires specific reagents not only for detecting their activity but also for their imaging. Such tools include inhibitors or substrate-related chemical molecules that allow the detection of proteolysis and visual observation of active enzymes, thus facilitating the characterization of the activity of proteases in the complex proteome. Peptidyl activity-based probes (ABPs) have been extensively studied recently, and this review describes the basic principles in the design of peptide-based imaging agents for serine proteases, provides examples of activity-based probe applications and critically discusses their strengths, weaknesses, challenges and limitations.

Factor VII Activating Protease (FSAP): Functional Implications of the “Marburg-1” Polymorphism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2126-2126
Author(s):  
Fabian Stavenuiter ◽  
Alexander B Meijer ◽  
Erica Sellink ◽  
Koen Mertens
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Proteolytic Processing ◽  
Factor Vii ◽  
Maximal Effect ◽  
Single Chain ◽  
Surface Binding ◽  
Positive Effects ◽  
Charged Surfaces ◽  
Hydrolysis Of

Abstract Abstract 2126 Poster Board II-104 Introduction FSAP is a plasma serine protease first reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and Factor VII (FVII), suggesting a key role in hemostasis and thrombosis. Numerous additional functions have been proposed, including inhibition of smooth muscle cell proliferation and migration. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources due to autocatalytic activity which is stimulated through interaction with negatively charged surfaces. About 5-10% of healthy individuals carry a polymorphism (Marburg-1) at position c221 (G534Q) located in one of the 8 surface binding loops of the serine protease domain. This polymorphism has been proposed to be associated with impaired activation of scuPA in vitro, suggesting a putative defect in fibrinolysis. Epidemiological studies have remained inconclusive with regard to prothrombotic implications of this polymorphism. Residue c221 has been described as highly important in serine proteases. For prothrombin the D221Q mutation has been associated with a severe defect in fibrinogen clotting. Similarly, patients who are hemizygous for a c221 substitution in FIX (A221V) suffer from haemophilia B. In general, in Na+ -dependent serine proteases like FVII, FIX, and thrombin, residue c221 contributes to activity and substrate specificity. Objectives: Our aim was to investigate, using intact recombinant (r) FSAP, the effect of the M1-polymorphism on FSAP biological activity. Results Various stable cell lines (HEK293-, BHK-, LOVO-, and CHO cells) expressing normal rFSAP (wt) and its Marburg-1 (M1) variant were produced. Irrespective to the cell type used, rFSAP was found to be cleaved after expression due to autocatalytic cleavage. However, wtFSAP was found to be more sensitive to proteolytic processing than its M1-variant. Moreover, wtFSAP was found to be completely inactivated whereas the M1-variant could be purified in its two-chain form. To overcome the problem of autocatalytic degradation, for wtFSAP we constructed a FSAP-variant in which the natural activation site (R313-I314) was replaced by a cleavage site for the bacterial protease thermolysin. Thermolysin-activated rFSAP displayed the same affinity for chromogenic peptide substrates (S2288) as pdFSAP (Km 0.38 mM) and retained its capability to activate scuPA (Km 62 nM). Vmax for scuPA activation was increased through interaction with negative charged surfaces like polyphosphate and heparin (2- and 3-fold, respectively), whereas no effect on the hydrolysis of S2288 was found. In contrast, the M1-variant displayed severely reduced affinity for S2288 (6.5-fold) and hardly any scuPA activation. Interestingly, addition of heparin or polyphosphate showed positive effects on the hydrolysis of both substrates by the M1-variant. Compared to wtFSAP, however, both the Km and Vmax were still heavily affected. Surprisingly, wtFSAP proved incapable of cleaving purified FVII, even in the presence of calcium-ions and lipid vesicles of varying composition, including up to 40 mol% negative phospholipids such as phosphatidylserine and cardiolipin (CL). On membranes of 100% CL FVII cleavage did occur, but this resulted in transient activation and rapid degradation. The M1-variant, however, displayed no FVII cleavage under any of the conditions tested. Finally, we found that Na+, in absence of CaCl2, affects the maximal rate of S2288 hydrolysis by rFSAP, with a maximal effect at physiological relevant concentrations. The Na+ concentrations needed to reach maximal catalytic activity of the M1-variant were found 8 - 10 fold above physiologically relevant levels. Conclusions While rFSAP indeed activates scuPA, FVII appears surprisingly resistant to activation by rFSAP. The M1-variant does not activate FVII either, but does display reduced scuPA activation. The M1-polymorphism, being a Gly to Glu substitution at position c221, makes the protease less responsive to Na+. This is compatible with its location in the putative Na+-binding loops. Whether or not the reduced scuPA activation has any physiological impact remains unclear. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Studies on a Novel Circulating Anticoagulant in a Female with Bleeding Diathesis

1977 ◽  
Author(s):  
Harry L. Messmore ◽  
J. Fareed ◽  
Roberta L. Chang ◽  
Gerald M. Gawlik ◽  
Gerald F. Kozuh
Keyword(s):  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Surgical Trauma ◽  
Thrombin Time ◽  
At Iii ◽  
Life Threatening ◽  
History Of ◽  
Induced Aggregation ◽  
Hydrolysis Of

A 42 year old Caucasian female has had a history of bleeding from surgical trauma since the age of 3. She has bled massively from surgical trauma and has had life threatening postpartum hemorrhages. Laboratory tests showed marked prolongation of thrombin time, prothrombin time, partial thromboplastin time, low α1 antitrypsin and lipoprotein lipase activity. Fibrinogen level was normal. Mixing of patient’s plasma and serum with citrated normal plasma suggested the presence of a potent antithrombin (AT) substance. Heating of both the plasma and serum at 56°C for 2 hours failed to destroy the AT activity. The AT activity of the patient’s plasma was quantitated to be equivalent to 0.75–0.9 u/ml heparin. Platelets aggregated normally with ADP, collagen, epinephrine, but not with Ristocetin and thrombin (0.5–5.0 u/ml). Her plasma and serum blocked thrombin induced aggregation of normal platelets. Her plasma also inhibited the factor Xa induced hydrolysis of S-2222 (Bz-Ile-Glu-[γ-OR]-Gly-Arg-pNA) and the action of thrombin on S-2160 (Bz-Phe-val-Arg-pNA). The inhibitor was not adsorbed with barium sulfate or reduced in activity by protamine sulfate, toluidine blue or heparinase. It was chromatographed on Sephadex G-100 and G-50, and eluted in the void volume and a low molecular (10–20,000) fraction. Incubation of the patient’s plasma with chymotrypsin and trypsin destroyed the anticoagulant activity. Although anti-AT-III serum totally neutralized the anticoagulant activity of heparin-ized plasma, it failed to neutralize the patient’s plasma anticoagulant activity. Albumin partially neutralized the anticoagulant activity. These studies suggest that this AT substance is a potent inhibitor of serine proteases and behaves as activated AT-III.

Amino Acid And Peptide Thioeters: New Sensitive Substrates For Bovine Factors IXa , Xa , XIa , XIIa , Thrombin, Plasma Kallikrein And Trypsin

1981 ◽  
Author(s):  
James C Powers ◽  
Brian J McRae
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Turnover Rate ◽  
Hydrolysis Product ◽  
Amino Acid Residues ◽  
Sensitive Assay ◽  
High Turnover ◽  
Leaving Group ◽  
Thiol Reagent ◽  
Hydrolysis Of

A series of amino acid and peptide thioesters have been synthesized as sensitive assay substrates for the serine proteases involved in blood coagulation. Each substrate had a P1 Arg residue. The thiol leaving group (P1 ') and the P2 amino acid residues were varied. The thioesters have much higher Kcat/KM values than the corresponding amides such as 4-nitroanilides and 7-amino-4-methylcou- marin amides. In addition, the thiol released upon enzymatic hydrolysis of the substrates, can be measured chromogenically by use of a thiol reagent such as 4,4'- dithiodipyridine contained in the assay mixture. Thioesters are among the most sensitive assay substrates for coagulation serine proteases due to the combination of a high turnover rate with the ease of detection of the hydrolysis product. In addition they can be utilized with enzymes such as factor IXa for which no other suitable synthetic substrate is currently available.

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