scholarly journals Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain

1984 ◽  
Vol 219 (3) ◽  
pp. 727-733 ◽  
Author(s):  
P Schack ◽  
N C Kaarsholm
Keyword(s):  
Ionic Strength ◽  
Thiol Group ◽  
Peptide Inhibitor ◽  
Affinity Column ◽  
Sh Group ◽  
The One ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester ◽  
Thiol Proteinase ◽  
Immobilized Peptide

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

scholarly journals A kinetic and fluorimetric investigation of papain modified at tryptophan -69 and -177 by N–bromosuccinimide

1974 ◽  
Vol 141 (2) ◽  
pp. 503-515 ◽  
Author(s):  
Gordon Lowe ◽  
Alan S. Whitworth
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescence Spectra ◽  
Close Contact ◽  
Ph Dependence ◽  
Thiol Group ◽  
Structural Features ◽  
Enzymic Activity ◽  
Ph Profile ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan -69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pKa of the acidic limb of the pH profile by more than 1 pH unit for both kcat. and kcat./Km. The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan -69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan -177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pKa of 4.2. Histidine -159, which is in close contact with tryptophan -177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan -177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pKa determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N–benzyloxycarbonylglycylglycine amide and N–benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan -177 were shown to be structural features of the S2 and S1′ subsites respectively.

Fine Structural Localization of Acyltransferases Involved in Lipid Synthesis in Intestinal Mucosa.

1970 ◽  
Vol 28 ◽  
pp. 54-55
Author(s):  
R. J. Barrnett ◽  
J. A. Higgins
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Mucosal Cell ◽  
Structural Localization ◽  
Morphological Studies ◽  
Mucosal Cells ◽  
Initial Appearance ◽  
Sh Group ◽  
Hydrolysis Of ◽  
Intestinal Mucosal Cells

The main products of intestinal hydrolysis of dietary triglycerides are free fatty acids and monoglycerides. These form micelles from which the lipids are absorbed across the mucosal cell brush border. Biochemical studies have indicated that intestinal mucosal cells possess a triglyceride synthesising system, which uses monoglyceride directly as an acylacceptor as well as the system found in other tissues in which alphaglycerophosphate is the acylacceptor. The former pathway is used preferentially for the resynthesis of triglyceride from absorbed lipid, while the latter is used mainly for phospholipid synthesis. Both lipids are incorporated into chylomicrons. Morphological studies have shown that during fat absorption there is an initial appearance of fat droplets within the cisternae of the smooth endoplasmic reticulum and that these subsequently accumulate in the golgi elements from which they are released at the lateral borders of the cell as chylomicrons.We have recently developed several methods for the fine structural localization of acyltransferases dependent on the precipitation, in an electron dense form, of CoA released during the transfer of the acyl group to an acceptor, and have now applied these methods to a study of the fine structural localization of the enzymes involved in chylomicron lipid biosynthesis. These methods are based on the reduction of ferricyanide ions by the free SH group of CoA.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

scholarly journals One-Pot Ionic Liquid-Mediated Bioprocess for Pretreatment and Enzymatic Hydrolysis of Rice Straw with Ionic Liquid-Tolerance Bacterial Cellulase

2022 ◽  
Vol 9 (1) ◽  
pp. 17
Author(s):  
Malinee Sriariyanun ◽  
Nichaphat Kitiborwornkul ◽  
Prapakorn Tantayotai ◽  
Kittipong Rattanaporn ◽  
Pau-Loke Show
Keyword(s):  
Ionic Liquid ◽  
Saline Soil ◽  
Enzymatic Saccharification ◽  
Value Added ◽  
Specific Enzyme ◽  
One Pot ◽  
The One ◽  
Hydrolysis Of ◽  
Bacterial Cellulase ◽  
Value Added Products

Ionic liquid (IL) pretreatment of lignocellulose is an efficient method for the enhancement of enzymatic saccharification. However, the remaining residues of ILs deactivate cellulase, therefore making intensive biomass washing after pretreatment necessary. This study aimed to develop the one-pot process combining IL pretreatment and enzymatic saccharification by using low-toxic choline acetate ([Ch][OAc]) and IL-tolerant bacterial cellulases. Crude cellulases produced from saline soil inhabited Bacillus sp. CBD2 and Brevibacillus sp. CBD3 were tested under the influence of 0.5–2.0 M [Ch][OAc], which showed that their activities retained at more than 95%. However, [Ch][OAc] had toxicity to CBD2 and CBD3 cultures, in which only 32.85% and 12.88% were alive at 0.5 M [Ch][OAc]. Based on the specific enzyme activities, the sugar amounts produced from one-pot processes using 1 mg of CBD2 and CBD3 were higher than that of Celluclast 1.5 L by 2.0 and 4.5 times, respectively, suggesting their potential for further application in the biorefining process of value-added products.

Effect of Chloride Ions on the Hydrolysis of Trivalent Lanthanum, Praseodymium and Lutetium in Aqueous Solutions of 2 M Ionic Strength

2005 ◽  
Vol 34 (4) ◽  
pp. 427-441 ◽  
Author(s):  
H. López-González ◽  
M. Solache-Ríos ◽  
M. Jiménez-Reyes ◽  
J. J. Ramírez-García ◽  
A. Rojas-Hernández
Keyword(s):  
Ionic Strength ◽  
Aqueous Solutions ◽  
Chloride Ions ◽  
Hydrolysis Of

scholarly journals Anomalous Salt Dependence Reveals an Interplay of Attractive and Repulsive Electrostatic Interactions in α-synuclein Fibril Formation

QRB Discovery ◽  
2020 ◽  
Vol 1 ◽  
Author(s):  
Ricardo Gaspar ◽  
Mikael Lund ◽  
Emma Sparr ◽  
Sara Linse
Keyword(s):  
Ionic Strength ◽  
Electrostatic Interactions ◽  
Intrinsically Disordered Protein ◽  
Lipid Vesicles ◽  
Intrinsically Disordered ◽  
Catalytic Surfaces ◽  
Disordered Protein ◽  
Salt Dependence ◽  
Fluorescence Measurements ◽  
The One

Abstractα-Synuclein (α-syn) is an intrinsically disordered protein with a highly asymmetric charge distribution, whose aggregation is linked to Parkinson’s disease. The effect of ionic strength was investigated at mildly acidic pH (5.5) in the presence of catalytic surfaces in the form of α-syn seeds or anionic lipid vesicles using thioflavin T fluorescence measurements. Similar trends were observed with both surfaces: increasing ionic strength reduced the rate of α-syn aggregation although the surfaces as well as α-syn have a net negative charge at pH 5.5. This anomalous salt dependence implies that short-range attractive electrostatic interactions are critical for secondary nucleation as well as heterogeneous primary nucleation. Such interactions were confirmed in Monte Carlo simulations of α-syn monomers interacting with surface-grafted C-terminal tails, and found to be weakened in the presence of salt. Thus, nucleation of α-syn aggregation depends critically on an attractive electrostatic component that is screened by salt to the extent that it outweighs the screening of the long-range repulsion between negatively charged monomers and negative surfaces. Interactions between the positively charged N-termini of α-syn monomers on the one hand, and the negatively C-termini of α-syn on fibrils or vesicles surfaces on the other hand, are thus critical for nucleation.

ÉTUDE DES EFFETS ÉLECTROSTATIQUES SUR LE MÉCANISME D'ACTION CATALYTIQUE DE LA PHOSPHATASE ALCALINE INTESTINALE DE VEAU

1965 ◽  
Vol 43 (8) ◽  
pp. 2222-2235 ◽  
Author(s):  
Michel Lazdunski ◽  
Jacques Brouillard ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
High Substrate Concentration ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Activated Complex

The influence of dioxane and ethanol on the rate of hydrolysis of p-nitrophenyl phosphate in the presence of an intestinal alcaline phosphatase can be interpreted as a dielectric constant effect, at high substrate concentration. The dielectric constant effect is a function of the pH of the medium and is maximum around pH 9.4 at 25 °C and pH 9.0 at 15 °C. An interpretation suggesting that the change in diameter of the enzyme molecule becoming an activated complex is minimum at a pH of maximum activity is proposed. The same model can take into account the influence of the ionic strength on the same reaction.

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