Enzyme-Mimic Peptide Assembly To Achieve Amidolytic Activity

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

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Non-Fasting Factor VII Coagulant Activity (FVII:C) Increased by High-Fat Diet

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642518 ◽

1994 ◽

Vol 71 (06) ◽

pp. 755-758 ◽

Cited By ~ 46

Author(s):

E M Bladbjerg ◽

P Marckmann ◽

B Sandström ◽

J Jespersen

Keyword(s):

High Fat Diet ◽

Fat Content ◽

Factor Vii ◽

Amidolytic Activity ◽

High Fat ◽

Low Fat Diet ◽

Increased Risk ◽

Coagulant Activity ◽

High Fat Diets ◽

Fat Diets

SummaryPreliminary observations have suggested that non-fasting factor VII coagulant activity (FVII:C) may be related to the dietary fat content. To confirm this, we performed a randomised cross-over study. Seventeen young volunteers were served 2 controlled isoenergetic diets differing in fat content (20% or 50% of energy). The 2 diets were served on 2 consecutive days. Blood samples were collected at 8.00 h, 16.30 h and 19.30 h, and analysed for triglycerides, FVII coagulant activity using human (FVII:C) or bovine thromboplastin (FVII:Bt), and FVII amidolytic activity (FVIPAm). The ratio FVII:Bt/FVII:Am (a measure of FVII activation) increased from fasting levels on both diets, but most markedly on the high-fat diet. In contrast, FVII: Am (a measure of FVII protein) tended to decrease from fasting levels on both diets. FVII:C rose from fasting levels on the high-fat diet, but not on the low-fat diet. The findings suggest that high-fat diets increase non-fasting FVII:C, and consequently may be associated with increased risk of thrombosis.

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Enzyme-mimic activity study of superstable and ultrasmall graphene encapsuled CoRu nanocrystal

APL Materials ◽

10.1063/5.0048777 ◽

2021 ◽

Vol 9 (5) ◽

pp. 051110

Author(s):

Phouphien Keoingthong ◽

Shengkai Li ◽

Zhaotian Zhu ◽

Liang Zhang ◽

Jieqiong Xu ◽

...

Keyword(s):

Enzyme Mimic

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Kinetic study of the inhibition of the amidolytic activity of thrombin by benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581)

International Journal of Biochemistry ◽

10.1016/0020-711x(87)90313-2 ◽

1987 ◽

Vol 19 (11) ◽

pp. 1105-1112 ◽

Cited By ~ 2

Author(s):

C. Izquierdo ◽

F.J. Burguillo ◽

J.L. Usero ◽

A. Del Arco

Keyword(s):

Kinetic Study ◽

Amidolytic Activity

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Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia

Biological Chemistry ◽

10.1515/hsz-2014-0256 ◽

2015 ◽

Vol 396 (3) ◽

pp. 261-275 ◽

Cited By ~ 14

Author(s):

Miroslaw Ksiazek ◽

Abdulkarim Y. Karim ◽

Danuta Bryzek ◽

Jan J. Enghild ◽

Ida B. Thøgersen ◽

...

Keyword(s):

Serine Protease ◽

Calcium Ions ◽

Enzyme Stability ◽

Amidolytic Activity ◽

Tannerella Forsythia ◽

Calcium Dependent ◽

Genes Encoding ◽

Mature Enzyme ◽

Almost All ◽

Unique Mechanism

Abstract The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

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Potential of enzyme mimics in biomimetic sensors: a modified-cyclodextrin as a dehydrogenase enzyme mimic

Biosensors and Bioelectronics ◽

10.1016/s0956-5663(03)00077-0 ◽

2003 ◽

Vol 18 (11) ◽

pp. 1407-1417 ◽

Cited By ~ 33

Author(s):

Ritu Kataky ◽

Edward Morgan

Keyword(s):

Enzyme Mimic ◽

Enzyme Mimics ◽

Modified Cyclodextrin ◽

Dehydrogenase Enzyme

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Arginine Inhibits Serpins

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029606299047 ◽

2007 ◽

Vol 13 (2) ◽

pp. 213-218

Author(s):

Thomas W. Stief

Keyword(s):

Esterase Activity ◽

Serine Proteases ◽

Biological Fluid ◽

Microtiter Plate ◽

Human Albumin ◽

Amidolytic Activity ◽

Peptide Substrates ◽

Plate Reader ◽

High Concentrations ◽

Very High

Serine protease inactivators (serpins) are important regulators in biochemistry. Often it is necessary to block the serpin action, that is, to stabilize the sample. The guanidine group of arginine is the ligand for the active center pocket of many serine proteases. Arginine or guanidine inhibits serine proteases, and arginine belongs to the reactive P1-P1' center of many serpins. The plasmatic antithrombin, antiplasmin, or anti-C1-esterase activity was determined: A total of 20 µL of pooled normal plasma or 7% human albumin was added to 100 µL of 0—2.67 M arginine, pH 8.6, 10 µL of 26 mIU/mL thrombin in 7% human albumin, and 30 µL of 1.7 mM CHG-Ala-Arg-pNA (37°C). ΔA at 405 nm was determined, by using a microtiter plate reader. Thrombin was substituted by plasmin or C1-esterase, and the chromogenic peptide substrates <Glu-Phe-Lys-pNA or MeOC-Lys(eCBO)-Gly-Arg-pNA, respectively, were used. The IC50 of arginine against plasmatic antithrombin activity is 580 mM; the IC 25 is 440 mM. The IC25 of arginine against plasmatic α 2-antiplasmin or C1-inactivator is 1650 mM. The amidolytic activity of thrombin, plasmin, and C1-esterase is inhibited similarly by arginine: the IC50 for arginine against the amidolytic activity of these proteases is about 400 mM. Arginine at very high concentrations inhibits serpins. This is important, if stabilization of a biological fluid is a prerequisite for valid activities of serine proteases. In addition, these high concentrations of arginine might be a new gentle principle to inhibit pathogens that need serpins for their pathophysiology.

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