scholarly journals The Peptide Ligase Activity of Human Legumain Depends on Fold Stabilization and Balanced Substrate Affinities

ACS Catalysis ◽  
2021 ◽  
Vol 11 (19) ◽  
pp. 11885-11896
Author(s):  
Elfriede Dall ◽  
Vesna Stanojlovic ◽  
Fatih Demir ◽  
Peter Briza ◽  
Sven O. Dahms ◽  
...  
Keyword(s):  
Substrate Affinities

scholarly journals Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina

2021 ◽  
Vol 7 (2) ◽  
pp. 80
Author(s):  
Bo Pilgaard ◽  
Marlene Vuillemin ◽  
Jesper Holck ◽  
Casper Wilkens ◽  
Anne S. Meyer
Keyword(s):  
Alginate Lyase ◽  
Marine Fungus ◽  
Brown Macroalgae ◽  
Polysaccharide Lyases ◽  
Alginate Lyases ◽  
Encoding Genes ◽  
Substrate Affinities ◽  
Alginate Degradation ◽  
Insight Into

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Multiple cryptic species with divergent substrate affinities in the Serpula himantioides species complex

2011 ◽  
Vol 115 (1) ◽  
pp. 54-61 ◽  
Author(s):  
Tor Carlsen ◽  
Ingeborg Bjorvand Engh ◽  
Cony Decock ◽  
Mario Rajchenberg ◽  
Håvard Kauserud
Keyword(s):  
Cryptic Species ◽  
Species Complex ◽  
Substrate Affinities

Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine

1992 ◽  
Vol 262 (1) ◽  
pp. G178-G184 ◽  
Author(s):  
A. M. Reisenauer ◽  
E. A. Lee ◽  
R. O. Castillo
Keyword(s):  
Protein Digestion ◽  
Soluble Enzyme ◽  
Rat Intestine ◽  
Membrane Enzyme ◽  
Intestinal Membrane ◽  
Related Proteins ◽  
Relationship Of ◽  
Soluble Enzymes ◽  
Substrate Affinities ◽  
Antigenic Epitopes

Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme.

scholarly journals The effects of nitrogen-heme-iron coordination on substrate affinities for cytochrome P450 2E1

2011 ◽  
Vol 193 (1) ◽  
pp. 50-56 ◽  
Author(s):  
Jeffrey P. Jones ◽  
Carolyn A. Joswig-Jones ◽  
Michelle Hebner ◽  
Yuzhuo Chu ◽  
Dennis R. Koop
Keyword(s):  
Cytochrome P450 ◽  
Heme Iron ◽  
Cytochrome P450 2E1 ◽  
Iron Coordination ◽  
Substrate Affinities

Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes

2000 ◽  
Vol 203 (19) ◽  
pp. 2991-3001 ◽  
Author(s):  
T. Verri ◽  
M. Maffia ◽  
A. Danieli ◽  
M. Herget ◽  
U. Wenzel ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Single Carrier ◽  
Catalytic Function ◽  
Negative Membrane Potential ◽  
Cotransport System ◽  
Polymerase Chain ◽  
Substrate Affinities

H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(−1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane ‘low-affinity’-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.

Solubilization and partial purification of particulate catechol-O-methyltransferase from rat liver

1977 ◽  
Vol 55 (10) ◽  
pp. 1108-1113 ◽  
Author(s):  
J. H. Tong ◽  
A. D'Iorio
Keyword(s):  
Rat Liver ◽  
Partial Purification ◽  
Catecholamine Metabolism ◽  
Ph Profile ◽  
Significant Difference ◽  
Solubilized Enzyme ◽  
Relationship Of ◽  
The Relationship ◽  
Substrate Affinities

Particulate catechol-O-methyltransferase (COMT) from rat liver has been solubilized by acetone treatment and partially purified. Results from the present study demonstrate that the solubilized, partially purified enzyme is similar to the cytosol COMT with respect to molecular weight, pH profile, sensitivity toward inhibitors, Mg2+ requirement, and substrate affinities. However, a comparison of the crude particulate COMT and the solubilized enzyme shows that there is a significant difference in their affinity for catechol substrates. This finding suggests that membrane protein and (or) lipid components may play an important role in catecholamine metabolism. The relationship of particulate COMT to [3H]norepinephrine binding was investigated. No correlation between the COMT and [3H]norepinephrine binding activities was observed in vitro.

Robust Electrophysiological Assays using Solid Supported Membranes: the Organic Cation Transporter OCT2

2011 ◽  
Vol 64 (1) ◽  
pp. 31 ◽  
Author(s):  
Olga Gaiko ◽  
Ingo Janausch ◽  
Sven Geibel ◽  
Henning Vollert ◽  
Petra Arndt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Organic Cation ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Supported Membranes ◽  
Cho Cell ◽  
Drug Candidates ◽  
Substrate Affinities ◽  
Solid Supported Membranes

An electrophysiological assay platform based on solid supported membranes (SSM) for the organic cation transporter (OCT) is presented. Stable Chinese hamster ovary (CHO) cell lines overexpressing the human (hOCT2) and rat transporters (rOCT2) were generated and validated. Membrane preparations from the cell lines were investigated using SSM-based electrophysiology. Baculovirus transfected insect cells (HighFive and Mimic Sf9) were also tested with the same assay but yielded less than optimal results. The assays were validated by the determination of substrate affinities and inhibition by standard inhibitors. The study demonstrates the suitability of the SSM-based electrophysiological OCT assay for rapid and automatic screening of drug candidates.

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