Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay

2015 ◽  
Vol 10 (9) ◽  
pp. 2166-2174 ◽  
Author(s):  
Swe-Htet Naing ◽  
Krishna M. Vukoti ◽  
Jason E. Drury ◽  
Jennifer L. Johnson ◽  
Sibel Kalyoncu ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Aspartyl Protease ◽  
Peptide Cleavage ◽  
Cleavage Assay ◽  
Protease Substrate ◽  
Substrate Hydrolysis

P5449Neurohumoral regulation of the low-, medium- and high-renin HFrEF phenotypes

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Prausmueller ◽  
H Arfsten ◽  
G Spinka ◽  
J F Novak ◽  
A Cho ◽  
...  
Keyword(s):  
Heart Failure ◽  
Univariate Analysis ◽  
Renin Angiotensin System ◽  
Plasma Renin ◽  
Peptide Cleavage ◽  
Reduced Ejection Fraction ◽  
Ras Activation ◽  
Cleavage Assay ◽  
All Cause Mortality ◽  
Vasoactive Peptide

Abstract Background Previous investigations of plasma Renin-Angiotensin-System (RAS) fingerprints of patients with heart failure with reduced ejection fraction (HFrEF) revealed the existence of low, medium and high renin phenotypes independently of disease severity. Plasma renin serves as an excellent surrogate for angiotensin levels. The different renin phenotypes could not only hypothetically respond differently to RAS blockade but associated alterations of other vasoactive peptide systems could elucidate disease mechanisms and novel targets for heart failure therapy. The study aimed to investigate the relation between RAS regulation and pathophysiologically relevant vasoactive peptide systems based on different renin phenotypes. Methods We prospectively enrolled 369 patients with stable HFrEF. Laboratory markers including NT-proBNP and active renin concentration (ARC) were assessed. Plasma NEP levels (sNEP), bioactive adrenomedullin (bio-ADM) and big-endothelin1 (bigET-1) were measured by ELISA (R&D systems, UK; Sphingotec GmbH, Germany and Eagle Biosciences, Austria). NEP activity was determined by a fluorimetric peptide cleavage assay. The correlation between biomarkers and association with all-cause mortality was assessed. sNEP, bio-ADM and bigET-1 levels as well as NEP activity between the different renin phenotypes (i.e. <15. percentile, 15.-85. percentile and >85. percentile of ARC) was compared. Results Median age was 65 (IQR 53–73) years, 75% of patients were male. Median NT-proBNP levels were 1936 (IQR 855–4126) pg/mL. Median ARC was 155 (29–569) μIE/mL, the low, medium and high renin HFrEF phenotypes showed median ARC levels of 4.2μIE/mL (IQR 2.0–7.8), 155.1μIE/mL (IQR 43.3–353.5) and 2360μIE/mL (IQR 1483–3250) μIE/mL. Median bigET-1 was 0.62pmol/L (IQR 0.42–1.10), bio-ADM 26.0pg/mL (IQR 16.1–46.7), sNEP 413pg/mL (IQR 0–4111) and NEP activity 2.36nmol/mL/min (IQR 1.16–4.59). There was no correlation between sNEP and NEP activity [r=0.09, p=0.088]. ARC did not show a meaningful correlation with any of the four biomarkers [p=ns for sNEP, NEP activity and bigET-1; r=0.13, p=0.018 for bio-ADM]. In the univariate analysis ARC, bigET-1, bio-ADM but not sNEP and NEP activity, were associated with outcome. This association remained significant after adjustment for age, gender and kidney function for all three markers and for ARC after adding NT-proBNP [adj. HR per 1-IQR increase of ARC 1.27 (95% CI 1.04–1.22), p=0.003]. There were no differences in bigET-1, bio-ADM and sNEP or NEP activity stratified by the different renin phenotypes (Figure1). Figure 1 Conclusions ARC is a risk factor for mortality in HFrEF patients, independently of NT-proBNP. Plasma NEP levels and activity neither correlated with each other nor were associated with outcome. Bio-ADM and bigET-1 were strong risk factors for all-cause mortality. Interestingly, neither NEP nor bio-ADM or bigET-1 were related to RAS-activation, suggesting that there is no direct relationship with RAS regulation.

Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay

2008 ◽  
Vol 126 (1-2) ◽  
pp. 135-139 ◽  
Author(s):  
R RASOOLY ◽  
L STANKER ◽  
J CARTER ◽  
P DO ◽  
L CHENG ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Botulinum Neurotoxin A ◽  
Peptide Cleavage ◽  
Cleavage Assay

scholarly journals A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

10.3791/58892 ◽  
2019 ◽  
Author(s):  
Javier A. Jaimes ◽  
Jean K. Millet ◽  
Monty E. Goldstein ◽  
Gary R. Whittaker ◽  
Marco R. Straus
Keyword(s):  
Proteolytic Activity ◽  
Spike Protein ◽  
Peptide Cleavage ◽  
Protein Activation ◽  
Cleavage Assay ◽  
Fluorogenic Peptide

scholarly journals Enzymatic Activities of Human Cytomegalovirus Maturational Protease Assemblin and Its Precursor (pPR, pUL80a): Maximal Activity of pPR Requires Self-Interaction through Its Scaffolding Domain

2007 ◽  
Vol 81 (8) ◽  
pp. 4091-4103 ◽  
Author(s):  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Catalytic Domain ◽  
Covalent Binding ◽  
Point Mutations ◽  
Maximal Activity ◽  
Enzymatic Activities ◽  
Unexpected Finding ◽  
Peptide Cleavage ◽  
Metal Affinity ◽  
Cleavage Assay ◽  
Catalytic Sites

ABSTRACT Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered “tail” (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.

Microstructure and reactivity of small metal particles: The role of Ce additives

1992 ◽  
Vol 50 (1) ◽  
pp. 318-319
Author(s):  
L.D. Schmidt ◽  
K. R. Krause ◽  
J. M. Schwartz ◽  
X. Chu
Keyword(s):  
Atmospheric Pressure ◽  
Noble Metals ◽  
Mixed Oxides ◽  
Catalytic Properties ◽  
Chemical Shifts ◽  
Catalytic Converter ◽  
Structural Evolution ◽  
Single Particles ◽  
Small Metal Particles

The evolution of microstructures of 10- to 100-Å diameter particles of Rh and Pt on SiO2 and Al2O3 following treatment in reducing, oxidizing, and reacting conditions have been characterized by TEM. We are able to transfer particles repeatedly between microscope and a reactor furnace so that the structural evolution of single particles can be examined following treatments in gases at atmospheric pressure. We are especially interested in the role of Ce additives on noble metals such as Pt and Rh. These systems are crucial in the automotive catalytic converter, and rare earths can significantly modify catalytic properties in many reactions. In particular, we are concerned with the oxidation state of Ce and its role in formation of mixed oxides with metals or with the support. For this we employ EELS in TEM, a technique uniquely suited to detect chemical shifts with ∼30Å resolution.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Electron distribution change of small nickel particles encaged in X zeolite in presence of Ce3+ ions, related to their catalytic properties

1986 ◽  
Vol 83 ◽  
pp. 619-621 ◽  
Author(s):  
F. Vergand ◽  
B. Iraqi ◽  
C. Bonnelle ◽  
E. Ramaroson ◽  
M.F. Guilleux ◽  
...  
Keyword(s):  
Electron Distribution ◽  
Catalytic Properties ◽  
Distribution Change ◽  
Nickel Particles ◽  
X Zeolite

Countercation effects on surface and catalytic properties of 12-tungstophosphates

1992 ◽  
Vol 89 ◽  
pp. 681-693
Author(s):  
A Anwar ◽  
AA Abdel-Ghaffar
Keyword(s):  
Catalytic Properties
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