Characterization of peptidases of adult Trichuris muris

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 623-630 ◽  
Author(s):  
L. J. Drake ◽  
A. E. Bianco ◽  
D. A. P. Bundy ◽  
F. Ashall
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Matrix Protein ◽  
Sodium Dodecyl ◽  
Time Dependent ◽  
Extracellular Matrix Protein ◽  
Peptidase Activity ◽  
Trichuris Muris ◽  
Hydrolysis Of

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, Mr 85 and 105 kDa, which degrade gelatin optimally at pH 6·0 in sodium dodecyl sulphate–polyacrylamide gels. The peptidases were inactivated diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

scholarly journals Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

2021 ◽  
Author(s):  
Aniel Moya-Torres ◽  
Monika Gupta ◽  
Fabian Heide ◽  
Natalie Krahn ◽  
Scott Legare ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hollow Fiber ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Continuous Production ◽  
Extracellular Matrix Protein ◽  
Close Monitoring ◽  
High Quality ◽  
Biolayer Interferometry ◽  
Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

scholarly journals Characterization of the human extracellular matrix protein 1 gene on chromosome 1q21

1997 ◽  
Vol 16 (5) ◽  
pp. 289-292 ◽  
Author(s):  
Maureen R. Johnson ◽  
Douglas J. Wilkin ◽  
Hans L. Vos ◽  
Rosa Isela Ortiz De Luna ◽  
Anindya M. Dehejia ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Extracellular Matrix Protein 1 ◽  
Chromosome 1Q21

scholarly journals Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Lifang Sun ◽  
Pu Chen ◽  
Yintao Su ◽  
Zhixiong Cai ◽  
Lingwei Ruan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sodium Dodecyl ◽  
Titration Calorimetry ◽  
Pseudomonas Sp ◽  
Sterol Carrier Protein ◽  
Dimerization Domain ◽  
Alkyl Chains ◽  
Hydrolysis Of

A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76 Å and reveals that SdsAP contains the characteristic metallo-β-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of ‘primary alkyl’ chains, confirming that SdsAP is a primary alkylsulfatase.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components

1978 ◽  
Vol 176 (1) ◽  
pp. 283-294 ◽  
Author(s):  
J G Heathcote ◽  
C H J Sear ◽  
M E Grant
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lens Capsule ◽  
Time Dependent ◽  
Lower Molecular Weight Species ◽  
Direct Incorporation ◽  
Isolated Rat

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.

scholarly journals Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity

1979 ◽  
Vol 177 (1) ◽  
pp. 107-114 ◽  
Author(s):  
T G Villa ◽  
V Notario ◽  
J R Villanueva
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Beta Glucanase ◽  
Hydrolysis Of

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

1991 ◽  
Vol 58 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Teresa Requena ◽  
Carmen Peláez ◽  
Michel J. Desmazeaud
Keyword(s):  
Leucine Aminopeptidase ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Titratable Acidity ◽  
Aminopeptidase Activity ◽  
Whole Cells ◽  
Alanine Aminopeptidase ◽  
Triton X ◽  
Hydrolysis Of

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

scholarly journals 3317 Localization and characterization of a novel extracellular matrix protein β-IG-H3

1995 ◽  
Vol 35 ◽  
pp. S136
Author(s):  
I. Rawe ◽  
Q. Zhan ◽  
Y. Komai-Hori ◽  
R. Burrows ◽  
C.L. Kublin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Extracellular Matrix Protein

scholarly journals A docked mutation phenocopies dumpy oblique alleles via altered vesicle trafficking

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12175
Author(s):  
Suresh Kandasamy ◽  
Kiley Couto ◽  
Justin Thackeray
Keyword(s):  
Matrix Protein ◽  
Vesicle Trafficking ◽  
Notch Pathway ◽  
Vesicle Transport ◽  
Snare Proteins ◽  
Extracellular Matrix Protein ◽  
Functional Connection ◽  
Core Proteins ◽  
Genes Encoding

The Drosophila extracellular matrix protein Dumpy (Dpy) is one of the largest proteins encoded by any animal. One class of dpy mutations produces a characteristic shortening of the wing blade known as oblique (dpyo), due to altered tension in the developing wing. We describe here the characterization of docked (doc), a gene originally named because of an allele producing a truncated wing. We show that doc corresponds to the gene model CG5484, which encodes a homolog of the yeast protein Yif1 and plays a key role in ER to Golgi vesicle transport. Genetic analysis is consistent with a similar role for Doc in vesicle trafficking: docked alleles interact not only with genes encoding the COPII core proteins sec23 and sec13, but also with the SNARE proteins synaptobrevin and syntaxin. Further, we demonstrate that the strong similarity between the doc1 and dpyo wing phenotypes reflects a functional connection between the two genes; we found that various dpy alleles are sensitive to changes in dosage of genes encoding other vesicle transport components such as sec13 and sar1. Doc’s effects on trafficking are not limited to Dpy; for example, reduced doc dosage disturbed Notch pathway signaling during wing blade and vein development. These results suggest a model in which the oblique wing phenotype in doc1 results from reduced transport of wild-type Dumpy protein; by extension, an additional implication is that the dpyo alleles can themselves be explained as hypomorphs.

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