Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases

2014 ◽  
Vol 460 (2) ◽  
pp. 187-198 ◽  
Author(s):  
Todd A. Naumann ◽  
Donald T. Wicklow ◽  
Neil P. J. Price
Keyword(s):  
Protease Activity ◽  
Peptide Bonds ◽  
Linker Region ◽  
Plant Chitinases ◽  
Specific Peptide ◽  
Class Iv

Fungal polyglycine hydrolases cleave multiple specific peptide bonds in the linker domains of targeted plant chitinases. These proteases somehow recognize polyglycine linkers in targeted chitinases. Describing this new protease activity improves understanding of how proteases target their substrates.

Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

1980 ◽  
Vol 26 (1) ◽  
pp. 77-86 ◽  
Author(s):  
S. E. Jensen ◽  
L. Phillippe ◽  
J. Teng Tseng ◽  
G. W. Stemke ◽  
J. N. Campbell
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Protease Activity ◽  
Gel Filtration ◽  
Laboratory Strain ◽  
Exchange Chromatography ◽  
The Other ◽  
Protease Production ◽  
Peptide Bonds

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a molecular weight of 34 850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two proteases against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.

scholarly journals Spectrophotometric measurement of the liberation of the α-amino group of cysteine residues in polypeptides

1966 ◽  
Vol 100 (2) ◽  
pp. 309-314 ◽  
Author(s):  
S Streichman ◽  
Y Avi-Dor
Keyword(s):  
Spectral Characteristics ◽  
Cysteine Residues ◽  
Amino Group ◽  
Peptide Bonds ◽  
Spectral Changes ◽  
Sh Group ◽  
Induced Changes ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Specific Peptide

1. The reaction between beta-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R.S.CH(2).CO.CO(2)H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the alpha-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.

scholarly journals Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

2021 ◽  
Vol 22 (12) ◽  
pp. 6362
Author(s):  
Emmiliisa Vuorinen ◽  
Salla Valtonen ◽  
Nazia Hassan ◽  
Randa Mahran ◽  
Huda Habib ◽  
...  
Keyword(s):  
Protease Activity ◽  
Microtiter Plate ◽  
Probe Method ◽  
Label Free ◽  
Target Class ◽  
Signaling Mechanism ◽  
Peptide Bonds ◽  
Time Resolved ◽  
Drug Candidates ◽  
Catalytic Function

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.

scholarly journals Towards SERS-based multiplexed monitoring of protease activity using non-natural aromatic amino acids

2020 ◽  
Vol 238 ◽  
pp. 04001
Author(s):  
Nina Turk ◽  
Hans Demol ◽  
Andre Skirtach ◽  
Roel Baets ◽  
Kris Gevaert
Keyword(s):  
Amino Acids ◽  
Protease Activity ◽  
Aromatic Amino Acids ◽  
Region Of Interest ◽  
Surface Enhanced Raman Spectroscopy ◽  
Detection Sensitivity ◽  
Peptide Substrates ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Specific Peptide

Surface Enhanced Raman Spectroscopy (SERS) allows sensitive and selective detection of protease activity by monitoring the cleavage of specific peptide substrates. Furthermore, it offers the possibility for multiplexing, during which the activity of two (or more) proteases with different specificities is detected simultaneously. To distinguish between the contributions of different proteases, different aromatic amino acids with non-overlapping SERS peaks need to be used as Raman reporters. As the three natural aromatic amino acids only offer limited possibilities for multiplexing, we examined several non-natural aromatic amino acids with the aim of expanding multiplexing possibilities. We recorded their SERS spectra for the Raman shifts of 300-1700 cm–1 and identified their characteristic SERS peaks. Of the examined nonnatural aromatic amino acids, 3-nitro-tyrosine and two phenylalanines containing stable heavy isotopes seem particularly promising for multiplexing applications. Besides exhibiting characteristic SERS peaks in the spectral region of interest, these non-natural aromatics provide strong SERS peaks compared to natural aromatic amino acids, consequently improving detection sensitivity.

X-Ray Investigation of Fragment 1 of Bovine Prothrombin

1979 ◽  
Author(s):  
J.M. Burridge ◽  
R. Aschaffenburg ◽  
C.C.F. Blaxe ◽  
M.P. Esnouf
Keyword(s):  
Three Dimensional ◽  
Factor Xa ◽  
Factor V ◽  
Peptide Bonds ◽  
X Ray ◽  
Space Groups ◽  
Carboxyglutamic Acid ◽  
Cell Dimensions ◽  
Ph Range ◽  
Specific Peptide

The conversion of prothrombin to thrombin during the penultimate stage of the blood cloting system is achieved by the cleavage by factor Xa of two specific peptide bonds in the inactive prothrombin molecule; it is thought that prothrombin, factor Xa and factor V are organised into a specific prothrombin-converting complex on a phospholipid surface prothrombin and factor Xa binding via Ca2+ ions which bind to γ carboxyglutamic acid residues in both proteins. Fragment 1 is released by the action of thrombin, and is thought to inhibit excess prothrombin conversion. The fragment consists of the first 1 residues of prothrombin, molecular weight 22,000, it contains all ten γ-carboxyglutamic acid residues, and two of the three carbohydrate chains of the prothrombin molecule, together with one of the so-called “Kringle” units, repeated in fragment 2, and occurring five times in plasminogen. Fragment 1 crystallises from 2M phosphate, pH range 6.0 to 7.5, in one of the tetragonal space groups P43212 or its enantiomorph P41212; cell dimensions a = b = 78.87Å, c = 84.99Å, with one molecule per asymmetric unit. Isomorpho,. derivative survey work has led to the collection of low resolution (6Å) photographic oru jection data and three dimensional difftactometer data for crystals grown at pH 7.5 Experiments are in progress on crystals grown at pH 6.0, and on methods of modifyin protein before crystallisation; trial data collection at 3Å has suggested that st tural analysts at this intermediate resolution should be possible.

Hydrolysis of Dipeptide N-Heptyl Esters with Newlase F

2003 ◽  
Vol 2003 (1) ◽  
pp. 1-1 ◽  
Author(s):  
Zhong-Zhou Chen ◽  
Yan-Mei Li ◽  
Yu-Fen Zhao
Keyword(s):  
Lipase Activity ◽  
Protease Activity ◽  
Crude Enzyme ◽  
Protecting Groups ◽  
Peptide Conjugates ◽  
Peptide Bonds ◽  
Hydrolysis Conditions ◽  
Hydrolysis Of

Newlase F, a crude enzyme containing lipase and protease, can be used in the synthesis of peptide conjugates; the hydrolysis conditions were optimized to increase lipase activity and suppress protease activity and in addition, N-terminal protecting groups and peptide bonds were not affected.

scholarly journals Molecular identification of a new isolate of actinobacteria ATIS61 and characterization of the protease activities

2021 ◽  
Vol 22 (3) ◽  
Author(s):  
FADHLIAH AMFAR ◽  
Lenni Fitri ◽  
SUHARTONO SUHARTONO
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Identification ◽  
Protease Activity ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Peptide Bonds ◽  
Activity Test ◽  
The 16S Rrna Gene

Abstract. Amfar F, Fitri L, Suhartono. 2021. Molecular identification of a new isolate of actinobacteria ATIS61 and characterization of the protease activities. Biodiversitas 22: 1564-1569. Protease is an enzyme that catalyzes the hydrolysis of peptide bonds in protein. Actinobacteria are one of bacterial groups that is able to produce protease. Actinobacteria are Gram-positive bacteria and mostly aerobic. This study aimed to identify protease-producing actinobacteria ATIS61 isolate using 16S rRNA gene and to characterize the protease activity. This study was experimental research, consisted of amplification and sequencing of the 16S rRNA gene, and protease activity test. The 16S rRNA gene analysis showed that ATIS61 isolate was closely related to Nocardia sp. strain 335427 with a 99.88% similarity and the result of phylogenetic tree construction was related to Nocardia farcinica strain ARS8 with Bootstrap 94%. Protease activity test showed the highest activity was on the eighth day of incubation at 0.115 U/mL. Protease activity based on temperature showed the highest activity at 40°C of 0.156 U/mL and the stability of protease towards temperature was stable at 40°C and 50°C. Protease activity showed that the highest protease activity was at pH 8 of 0.096 U/mL and the highest protease stability was also at pH 8. The addition of HgCl2 showed that it could inhibit protease activity with a value of 0.059 U/mL.

751 Long-term benefit of a hospital based heart failure programme: downgrading the risk of class IV patients

2003 ◽  
Vol 2 (1) ◽  
pp. 161
Author(s):  
E RYAN ◽  
C OLOUGHLIN ◽  
M LEDWIDGE ◽  
B TRAVERS ◽  
M RYDER ◽  
...  
Keyword(s):  
Heart Failure ◽  
Term Benefit ◽  
Class Iv

Azocoll protease activity assay

2007 ◽  
Author(s):  
Naxin Jiang ◽  
Nguan Soon Tan ◽  
Bow Ho ◽  
Jeak Ling Ding
Keyword(s):  
Protease Activity ◽  
Activity Assay
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