scholarly journals Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase

1980 ◽  
Vol 187 (3) ◽  
pp. 727-732 ◽  
Author(s):  
T G Conley ◽  
D G Priest
Keyword(s):  
Metal Content ◽  
Protein Concentration ◽  
Thermal Inactivation ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Enthalpy Change ◽  
Prosthetic Group ◽  
Subunit Dissociation ◽  
Substrate Analogues ◽  
Thermal Stability Of

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was −30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and −30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was −15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and −18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.

scholarly journals Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation

1989 ◽  
Vol 261 (2) ◽  
pp. 415-421 ◽  
Author(s):  
O Geiger ◽  
H Görisch
Keyword(s):  
Acetic Acid ◽  
Active Site ◽  
Protein Concentration ◽  
Thermal Inactivation ◽  
Glucose Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Pyrroloquinoline Quinone ◽  
Soluble Form ◽  
Activation Process ◽  
Prosthetic Group

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

1997 ◽  
Vol 78 (05) ◽  
pp. 1372-1380 ◽  
Author(s):  
André L Fuly ◽  
Olga L T Machado ◽  
Elias W Alves ◽  
Célia R Carlinis
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Thermal Inactivation ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Crude Venom ◽  
Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

scholarly journals Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress

2006 ◽  
Vol 188 (23) ◽  
pp. 8070-8078 ◽  
Author(s):  
Shinya Sugimoto ◽  
Hiroyuki Yoshida ◽  
Yoshimitsu Mizunoe ◽  
Keigo Tsuruno ◽  
Jiro Nakayama ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Molecular Chaperone ◽  
Thermal Inactivation ◽  
Gel Filtration ◽  
Ring Structure ◽  
Stress Conditions ◽  
Gel Filtration Chromatography ◽  
Gram Positive ◽  
Tetragenococcus Halophilus

ABSTRACT In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB Tha ) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB Tha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB Tha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE Tha ) and ATP. Interestingly, the mixture of dimer and monomer ClpB Tha , which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB Tha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE Tha and ATP under poststress conditions.

Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Ismat Bibi ◽  
Haq Nawaz Bhatti
Keyword(s):  
Lignin Peroxidase ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Gel Filtration ◽  
Veratryl Alcohol ◽  
Exchange Chromatography ◽  
Different Temperatures ◽  
Scale Experiment ◽  
Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

scholarly journals Direct zinc binding to purified rhodopsin and disc membranes

1992 ◽  
Vol 282 (1) ◽  
pp. 123-128 ◽  
Author(s):  
T A Shuster ◽  
A K Nagy ◽  
D C Conly ◽  
D B Farber
Keyword(s):  
Retinal Degeneration ◽  
Concanavalin A ◽  
Visual Pigment ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Zinc Binding ◽  
Direct Binding ◽  
Filtration Step

Using the radionuclide 65Zn, we have demonstrated the direct binding of zinc to purified rhodopsin. 65Zn is eluted with detergent-solubilized rhodopsin from concanavalin A columns and remains bound to the visual pigment through a subsequent gel-filtration step. Zinc binding to purified disc membranes is highly specific and, of the ions tested, copper is the best competitor. Equilibrium-dialysis experiments indicate that zinc binding to detergent-solubilized forms of rhodopsin may increase on bleaching the photopigment. These results may have important implications for studies that indicate that zinc plays a role in retinal degeneration and normal photoreceptor physiology.

Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

The Protein Binding of Acetylsalicylic Acid and Salicylic Acid

1969 ◽  
Vol 15 (11) ◽  
pp. 1027-1038 ◽  
Author(s):  
M Amir Ali ◽  
Joseph I Routh
Keyword(s):  
Salicylic Acid ◽  
Human Serum Albumin ◽  
Acetylsalicylic Acid ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Progressive Increase ◽  
Lower Percentage ◽  
Therapeutic Implications ◽  
Dialysis Cell

Abstract Quantitative experiments to study the binding of acetylsalicylic acid (ASA) and salicylic acid (SA) by human serum albumin (HSA) were carried out using 14C-labeled ASA or SA and gel filtration to separate the free from the bound forms. Two binding procedures were employed: the ASA or SA was incubated with the HSA, or the mixture was placed in an equilibrium dialysis cell. By withdrawing samples at intervals, the extent of binding or the attainment of equilibrium could be assessed. Evidence that filtration by the gel caused unbinding of the bound SA was obtained, with resultant lower percentage binding of SA by HSA than that obtained by equilibrium dialysis without gel filtration. In either case, binding equilibrium was reached in 4-8 hr. The binding of ASA by HSA was markedly different from that of SA. The experiments both with or without gel filtration demonstrated a progressive increase in binding of ASA in the 20- to 53-hr periods studied. In addition, ASA apparently displaces SA from its binding sites on albumin, an observation that may have therapeutic implications.

scholarly journals Purification and characterization of the cell-wall-associated and extracellular α-glucosidases from Saccharomycopsis fibuligera

1995 ◽  
Vol 308 (3) ◽  
pp. 753-760 ◽  
Author(s):  
V Reiser ◽  
J Gašperík
Keyword(s):  
Cell Wall ◽  
Thermal Inactivation ◽  
Gel Filtration ◽  
Sole Source ◽  
Amino Acid Sequences ◽  
Optimum Activity ◽  
Saccharomycopsis Fibuligera ◽  
Peptide Pattern ◽  
Extracellular Form ◽  
Ph Range

Cell-wall-associated and extracellular alpha-glucosidases were purified to homogeneity from Saccharomycopsis fibuligera KZ growing on a medium containing cellobiose as the sole source of carbon; this substrate has the greatest inducing effect on the production of both forms of the enzyme. Depending on the source of carbon, 75-90% of the enzyme is associated with cell wall, from which it can be completely released by 1% Triton X-100 at 25 degrees C in 2 h. Both enzymes are glycoproteins in monomeric form with an apparent molecular mass of 132 kDa estimated by SDS/PAGE and 135 kDa estimated by gel filtration. N-linked carbohydrate accounts for 12% of the total mass. Both forms exhibited optimum activity at pH 5.5 and seem to be stable in the pH range 4.0-8.0 on incubation at 4 degrees C for 24 h. The cell-wall-associated form had an optimum activity at 42.5 degrees C and was stable in the absence of substrate up to 30 degrees C, while the extracellular form had optimal activity at 52.5 degrees C and was stable up to 40 degrees C. Both forms are unable to renature after thermal inactivation. The cell-wall-associated and extracellular alpha-glucosidases cleaved the same kind of substrates, from maltose to maltoheptaose, isomaltase and panose, although showing different rates of hydrolysis, and had little or no activity with polysaccharides. The extracellular form cross-reacts with antibody raised against the cell-wall-associated form, and both forms show the same peptide pattern after cleavage with chymotrypsin. The amino acid sequences of six peptides from both forms show marked similarity to those of Schwanniomyces occidentalis glucoamylase.

scholarly journals Further evidence for an allosteric model for ribonuclease

1976 ◽  
Vol 153 (2) ◽  
pp. 329-337 ◽  
Author(s):  
E J Walker ◽  
G B Ralston ◽  
I G Darvey
Keyword(s):  
Conformational Change ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Experimental Systems ◽  
Substrate Analogues ◽  
Multiple Binding ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Cyclic Cmp ◽  
Allosteric Model

Evidence is presented from three experimental systems to support the allosteric model of Walker et al. (1975) (Biochem. J. 147, 425-433) which explains the substrate-concentration-dependent transition observed in the RNAase (ribonuclease)-catalysed hydrolysis of 2‘:3’-cyclic CMP (cytidine 2‘:3’-cyclic monophosphate). 1. Kinetic studies of the initial rate of hydrolysis of 2‘:3’-cyclic CMP show that the midpoint of the transition shifts to lower concentrations of 2‘:3’-cyclic CMP in the presence of the substrate analogues 3′-CMP, 5′-CMP, 3′-AMP, 3′-UMP and Pi; 2′-CMP and 2′-UMP do not cause such a shift. 2. Trypsin-digestion studies show that a conformational change in RNAase to a form less susceptible to tryptic inactivation is induced in the presence of the substrate analogues 3′-CMP, 5′-CMP, 3′-AMP, and 3′-UMP. 2′-CMP, 2′-AMP and 2′-UMP do not induce this conformational change. 3. Equilibrium-dialysis experiments demonstrate the multiple binding of molecules of 3′-CMP, 3′-AMP and 5′-AMP to a molecule of RNAase. 2′-CMP binds the ratio 1:1 over the analogue concentration range studied.

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