scholarly journals Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

1979 ◽  
Vol 181 (3) ◽  
pp. 733-736 ◽  
Author(s):  
A L Fink ◽  
R Feldman ◽  
J Zehnder
Keyword(s):  
Methyl Ester ◽  
Wide Temperature Range ◽  
Methyl Esters ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Low Ph ◽  
Time Dependent ◽  
Acidic Ph ◽  
Spectral Changes ◽  
Tryptophan Methyl Ester

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347–352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213–217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.

scholarly journals Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

2019 ◽  
Author(s):  
Max Hebditch ◽  
Ryan Kean ◽  
Jim Warwicker
Keyword(s):  
Experimental Data ◽  
Design Principle ◽  
Ph Dependence ◽  
Low Ph ◽  
Viral Clearance ◽  
Acidic Ph ◽  
Antibody Therapeutics ◽  
Acid Environment ◽  
Pass Through ◽  
Acid Stable

AbstractEngineered proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral clearance. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of saltbridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable proteins.

The Effect Of Alpha-Tocopherol On Urokinase

1981 ◽  
Author(s):  
D Ogston
Keyword(s):  
Vitamin E ◽  
Methyl Ester ◽  
Serum Albumin ◽  
Potent Inhibitor ◽  
Sodium Succinate ◽  
Gel Filtration ◽  
Alpha Tocopherol ◽  
Time Dependent ◽  
Physiological Relevance ◽  
Single Protein

Alpha-tocopherol (vitamin E) in the form of its succinate markedly inhibited the activity of urokinase (5 CTA u/ml) on fibrin plates when tested at a concentration of 10-5m . The amidolytic (S-2444) and esterase (N-α-acetylglycyl-L-lysine methyl ester) activities of urokinase were also inhibited by α-tocopherol. Inhibition was concentration and time-dependent. Sodium succinate (10-4M) did not influence urokinase activity.The inhibition of urokinase by α-tocopherol was abolished in the presence of dilutions of whole plasma. All fractions of plasma separated by gel filtration on Sephadex G-200 interfered with the inhibition of urokinase by α-tocopherol indicating that the effect of plasma does not reside in a single protein. The inhibition of urokinase by α-tocopherol could be reversed by serum albumin.It is concluded that α-tocopherol is a potent inhibitor of urokinase in purified systems, but the effect of protein on the reaction suggests that this inhibition has little physiological relevance.

scholarly journals Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein

2009 ◽  
Vol 83 (9) ◽  
pp. 4670-4677 ◽  
Author(s):  
Zhao-ling Qin ◽  
Yan Zheng ◽  
Margaret Kielian
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Ph Dependence ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Acidic Ph ◽  
Virus Growth ◽  
Histidine Residues

ABSTRACT A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.

scholarly journals fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

1998 ◽  
Vol 72 (5) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sallie Glomb-Reinmund ◽  
Margaret Kielian
Keyword(s):  
Ammonium Chloride ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Ph Dependence ◽  
Low Ph ◽  
Spike Protein ◽  
Acidic Ph ◽  
Fusion Activity ◽  
Protein Subunit ◽  
Subunit Interactions

ABSTRACT Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity offus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 andfus-1 E2. Sequence analysis demonstrated that thefus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.

Enzymic characterization of epidermis-derived 12-lipoxygenase isoenzymes

2001 ◽  
Vol 355 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Malte SIEBERT ◽  
Peter KRIEG ◽  
Wolf D. LEHMANN ◽  
Friedrich MARKS ◽  
Gerhard FÜRSTENBERGER
Keyword(s):  
Arachidonic Acid ◽  
Catalytic Activity ◽  
Linoleic Acid ◽  
Methyl Ester ◽  
Docosahexaenoic Acid ◽  
Methyl Esters ◽  
Acid Methyl Ester ◽  
Dienoic Acid ◽  
Acidic Ph ◽  
Acid Methyl

Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an ω-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca2+ content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca2+ and with an acidic pH, Ca2+ had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate.

scholarly journals Acidic pH Mediates Changes in Antigenic and Oligomeric Conformation of Herpes Simplex Virus gB and Is a Determinant of Cell-Specific Entry

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Darin J. Weed ◽  
Stephen J. Dollery ◽  
Tri Komala Sari ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Conformational Changes ◽  
Viral Entry ◽  
Ph Dependence ◽  
Low Ph ◽  
Acidic Ph ◽  
Class Iii ◽  
Simplex Virus

ABSTRACTHerpes simplex virus (HSV) is an important human pathogen with a high worldwide seroprevalence. HSV enters epithelial cells, the primary site of infection, by a low-pH pathway. HSV glycoprotein B (gB) undergoes low pH-induced conformational changes, which are thought to drive membrane fusion. When neutralized back to physiological pH, these changes become reversible. Here, HSV-infected cells were subjected to short pulses of radiolabeling, followed by immunoprecipitation with a panel of gB monoclonal antibodies (MAbs), demonstrating that gB folds and oligomerizes rapidly and cotranslationally in the endoplasmic reticulum. Full-length gB from transfected cells underwent low-pH-triggered changes in oligomeric conformation in the absence of other viral proteins. MAbs to gB neutralized HSV entry into cells regardless of the pH dependence of the entry pathway, suggesting a conservation of gB function in distinct fusion mechanisms. The combination of heat and acidic pH triggered irreversible changes in the antigenic conformation of the gB fusion domain, while changes in the gB oligomer remained reversible. An elevated temperature alone was not sufficient to induce gB conformational change. Together, these results shed light on the conformation and function of the HSV-1 gB oligomer, which serves as part of the core fusion machinery during viral entry.IMPORTANCEHerpes simplex virus (HSV) causes infection of the mouth, skin, eyes, and genitals and establishes lifelong latency in humans. gB is conserved among all herpesviruses. HSV gB undergoes reversible conformational changes following exposure to acidic pH which are thought to mediate fusion and entry into epithelial cells. Here, we identified cotranslational folding and oligomerization of newly synthesized gB. A panel of antibodies to gB blocked both low-pH and pH-neutral entry of HSV, suggesting conserved conformational changes in gB regardless of cell entry route. Changes in HSV gB conformation were not triggered by increased temperature alone, in contrast to results with EBV gB. Acid pH-induced changes in the oligomeric conformation of gB are related but distinct from pH-triggered changes in gB antigenic conformation. These results highlight critical aspects of the class III fusion protein, gB, and inform strategies to block HSV infection at the level of fusion and entry.

Activity of Plasmin and Streptokinase-Activator on Substituted Arginine and Lysine Esters

1966 ◽  
Vol 16 (01/02) ◽  
pp. 018-031 ◽  
Author(s):  
S Sherry ◽  
Norma Alkjaersig ◽  
A. P Fletcher
Keyword(s):  
Molecular Structure ◽  
Methyl Ester ◽  
Comparative Studies ◽  
Esterase Activity ◽  
Methyl Esters ◽  
Hydrolytic Activity ◽  
The Other ◽  
Other Hand

SummaryComparative studies have been made of the esterase activity of plasmin and the streptokinase-activator of plasminogen on a variety of substituted arginine and lysine esters. Human plasmin preparations derived by different methods of activation (spontaneous in glycerol, trypsin, streptokinase (SK) and urokinase) are similar in their esterase activity; this suggests that the molecular structure required for such esterase activity is similar for all of these human plasmins. Bovine plasmin, on the other hand, differs from human plasmin in its activity on several of the substrates studied (e.g., the methyl esters of benzoyl arginine and tosyl, acetyl and carbobenzoxy lysine), a finding which supports the view that molecular differences exist between the two animal plasmins. The streptokinase-activator hydrolyzes both arginine and lysine esters but the ratios of hydrolytic activity are distinct from those of plasmin and of other activators of plasminogen. The use of benzoyl arginine methyl ester as a substrate for the measurement of the esterase activity of the streptokinase-activator is described.

scholarly journals Evaluation of 3-Methylbutanoic Acid Methyl Ester as a Factor Influencing Flavor Cleanness in Arabica Specialty Coffee

2021 ◽  
Vol 11 (12) ◽  
pp. 5413
Author(s):  
Keiko Iwasa ◽  
Harumichi Seta ◽  
Yoshihide Matsuo ◽  
Koichi Nakahara
Keyword(s):  
Methyl Ester ◽  
Methyl Esters ◽  
Acid Methyl Ester ◽  
Chemical Compounds ◽  
Gas Chromatography Mass Spectrometry ◽  
Arabica Coffee ◽  
Acid Methyl ◽  
Coffee Brew ◽  
Coffee Beans ◽  
Specialty Coffee

This paper reports on the chemical compounds in arabica coffee beans with a high Specialty Coffee Association (SCA) cupping score, especially those in specialty coffee beans. We investigated the relationship between the chemical compounds and cupping scores by considering 16 types of Coffea arabica (arabica coffee) beans from Guatemala (SCA cupping score of 76.5–89.0 points). Non-targeted gas chromatography-mass spectrometry-based chemometric profiling indicated that specialty beans with a high cupping score contained considerable amounts of methyl-esterified compounds (MECs), including 3-methylbutanoic acid methyl ester (3-MBM), and other fatty acid methyl esters. The effect of MECs on flavor quality was verified by spiking the coffee brew with 3-MBM, which was the top-ranked component, as obtained through a regression model associated with cupping scores. Notably, 3-MBM was responsible for the fresh-fruity aroma and cleanness of the coffee brew. Although cleanness is a significant factor for specialty beans, the identification of compounds that contribute to cleanness has not been reported in previous research. The chemometric profiling approach coupled with spiking test validation will improve the identification and characterization of 3-MBM commonly found in arabica specialty beans. Therefore, 3-MBM, either alone or together with MECs, can be used as a marker in coffee production.

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