scholarly journals Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea

1997 ◽  
Vol 326 (3) ◽  
pp. 649-655 ◽  
Author(s):  
Suren AGHAJANIAN ◽  
Paul C. ENGEL
Keyword(s):  
Glutamate Dehydrogenase ◽  
Time Course ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Potassium Phosphate ◽  
Enzyme Concentration ◽  
Size Exclusion ◽  
Native Enzyme ◽  
Opposite Pattern ◽  
Clostridium Symbiosum

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311, 905–910]. The extent of re-activation was not increased by raising the incubation temperature to 37 °C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time < 3–5 s) formation of ‘structured’ intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these ‘structured’ monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.

scholarly journals A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum

1990 ◽  
Vol 271 (2) ◽  
pp. 351-355 ◽  
Author(s):  
S E H Syed ◽  
P C Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Time Course ◽  
Enzyme Concentration ◽  
Complete Inactivation ◽  
Activation Rate ◽  
Clostridium Symbiosum ◽  
Activity Form ◽  
Ph Dependent ◽  
Time Courses ◽  
Low Ionic Strength

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.

scholarly journals Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme

1993 ◽  
Vol 292 (3) ◽  
pp. 797-803 ◽  
Author(s):  
E Mertens ◽  
J De Jonckheere ◽  
E Van Schaftingen
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Potassium Thiocyanate ◽  
Enzyme Concentration ◽  
Size Exclusion ◽  
Maximal Effect ◽  
Naegleria Fowleri ◽  
Exclusion Chromatography ◽  
Nægleria Fowleri ◽  
Fructose 1,6 Bisphosphate

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.

A Novel Chimaeric Derivative of Saruplase, rscu-PA-40kDA/Hir, Binds to Thrombin and Exerts Thrombus-specific Fibrinolysis in Arterial and Venous Thrombosis in Dogs

1997 ◽  
Vol 77 (03) ◽  
pp. 535-539 ◽  
Author(s):  
J Schneider ◽  
R Hauser ◽  
H-H Hennies ◽  
J Korioth ◽  
G Steffens ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Plasma Levels ◽  
Time Course ◽  
Bolus Injection ◽  
Specific Activity ◽  
Intravenous Bolus ◽  
Protease Domain ◽  
Parent Molecule ◽  
Significant Prolongation ◽  
Inhibitory Domain

SummaryThe chimaeric molecule rscu-PA-40kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino acid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. 125I-radiolabelled M23 (0.03 µM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 |xM) and hirudin (0.001-10 µµM) concentra-tion-dependently displaced 125I-M23 from its binding to thrombin. Saruplase (up to 10 (iM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 ± 4 vs 25 ± 11 min) and maximal height of reperfusion blood flow (98 ± 21 vs 108 ± 15 % of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated 125I-fibrin radioactivity in thrombosed saphenous veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of 125I-fibrin in the venous thrombosis model was 91 ± 1 % in M23-and 88 ± 5 % in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and a2-antiplasmin levels were slightly reduced (-27 ± 3 %) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 ± 19 %) and a2-antiplasmin (-75 ±11%) plasma levels. Template bleeding times virtually did not differ before (2.8 ± 0.3 min) and 60 min after bolus injection of M23 (3.1 ± 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 ± 0.2 min to 28 ± 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity

2001 ◽  
Vol 360 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Arun GOYAL ◽  
Xing-Guo WANG ◽  
Paul C. ENGEL
Keyword(s):  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Cysteine Residue ◽  
The Other ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Subunit Stoichiometry ◽  
Clostridium Symbiosum ◽  
Mutant Forms ◽  
Fold Activation

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

scholarly journals Enzyme optimization to reduce the viscosity of pitanga (Eugenia uniflora L.) juice

2016 ◽  
Vol 19 (0) ◽  
Author(s):  
Ricardo Schmitz Ongaratto ◽  
Luiz Antonio Viotto
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Incubation Time ◽  
Regression Models ◽  
Incubation Temperature ◽  
Correlation Coefficients ◽  
Enzyme Concentration ◽  
Optimum Conditions ◽  
Activity Time ◽  
Independent Variables

Summary The aim of this work was to separately evaluate the effects of pectinase and cellulase on the viscosity of pitanga juice, and determine the optimum conditions for their use employing response surface methodology. The independent variables were pectinase concentration (0-2.0 mg.g–1) and cellulase concentration (0-1.0 mg.g–1), activity time (10-110 min) and incubation temperature (23.2-56.8 °C). The use of pectinase and cellulase reduced the viscosity by about 15% and 25%, respectively. The results showed that enzyme concentration was the most important factor followed by activity time, and for the application of cellulase the incubation temperature had a significant effect too. The regression models showed correlation coefficients (R2) near to 0.90. The pectinase application conditions that led to the lowest viscosity were: concentration of 1.7 mg.g–1, incubation temperature of 37.6 °C and incubation time of 80 minutes, while for cellulase the values were: concentration of 1.0 mg.g-1, temperature range of 25 °C to 35 °C and incubation time of 110 minutes.

Mechanism of secretion of plasma insulin-like growth factor-I into milk of lactating goats

1991 ◽  
Vol 131 (3) ◽  
pp. 459-466 ◽  
Author(s):  
C. G. Prosser ◽  
I. R. Fleet ◽  
A. J. Davis ◽  
R. B. Heap
Keyword(s):  
Growth Factor ◽  
Time Course ◽  
Specific Activity ◽  
Gel Filtration ◽  
Free Form ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Lactating Goats ◽  
Igf I ◽  
Growth Factor I

ABSTRACT 125I-Labelled insulin-like growth factor-I (IGF-I) was infused as the free form directly into the pudic artery supplying one gland of lactating goats (n = 6). The infusion was for 60 min and 0·4±0·09% (s.e.m.) of the infusate was secreted into milk from the infused gland during its first passage through that gland. A large proportion of the 125I-labelled IGF-I escaped into the systematic circulation and was secreted into milk of both glands. A total of 5·2±0·4% of infused radioactivity was recovered in milk from both glands from 0 to 720 min. Radioactivity consisted of trichloroacetic acid (TCA)-precipitable and -soluble counts which were shown by gel filtration to be authentic IGF-I and degraded products of the peptide. The amount and time course of TCA-soluble radioactivity in milk from both glands was similar, suggesting degradation of 125I-labelled IGF-I at extramammary sites. Maximum specific activity for 125I-labelled IGF-I in milk from the infused gland was reached 80–120 min after the start of infusion and was 2·5-fold greater than milk from the non-infused gland. The time course of appearance of 125I-labelled IGF-I in milk suggests that transfer was via the transcellular pathway and this was further supported by comparing the pattern of transfer of [14C]sucrose and [14C]amino acids. When excess unlabelled IGF-I was included in the infusate, specific activity in milk from the infused gland was reduced to that of the non-infused gland, indicating a competitive and saturable mechanism of secretion for 125I-labelled IGF-I. Comparison of uptake and secretion of 125I-labelled IGF-I into milk from the non-infused gland with that of endogenous immunoreactive IGF-I suggests that vectorial transport of IGF-I across the mammary gland may be a significant contributor of IGF-I levels in milk. Journal of Endocrinology (1991) 131, 459–466

Estimation of the rate of appearance in the non-steady state with a two-compartment model

1992 ◽  
Vol 263 (2) ◽  
pp. E400-E415 ◽  
Author(s):  
A. Mari
Keyword(s):  
Steady State ◽  
Time Course ◽  
Specific Activity ◽  
Glucose Production ◽  
Compartment Model ◽  
New Method ◽  
Two Compartment Model ◽  
Glucose Clamp Technique ◽  
Glucose Output ◽  
The Relationship

A simple tracer-based method for calculating the rate of appearance of endogenous substances in the non-steady state, free from the inconsistencies of Steele's equation, is still lacking. This paper presents a method based on a two-compartment model by which the rate of appearance can be calculated with only a modest increase in complexity over Steele's approach. An equation is developed where the rate of appearance is expressed as a sum of three terms: a steady-state term, a term for the first compartment, and a term for the second compartment. The formula employs three parameters and makes the relationship between rate of appearance and specific activity changes explicit. An equation is also provided for estimating the error of the method in each individual run. The algorithm can be implemented with a spreadsheet on a personal computer. Simulated and experimental data obtained by the hyperinsulinemic euglycemic glucose clamp technique were used as a test. The accuracy with which the time course of glucose production could be reconstructed was clearly better than that using Steele's equation. Marked negative values for endogenous glucose output were calculated with Steele's equation but not with the new method. The characteristics of generality, simplicity, and accuracy and the availability of an error estimate make this new method suitable for routine application to non-steady-state tracer analysis.

scholarly journals Experimental Testing of a Size-Exclusion Chromatography Method Used for Evaluation of Molecular Parameters of Equine Anti-Ebola Immunoglobulin

2019 ◽  
Vol 19 (4) ◽  
pp. 261-267
Author(s):  
Е. Yu. Mishalova ◽  
E. V. Gordeev ◽  
V. N. Lebedev ◽  
S. A. Melnikov ◽  
S. A. Nimirskaya ◽  
...  
Keyword(s):  
Specific Activity ◽  
Experimental Testing ◽  
Horse Serum ◽  
Test Procedure ◽  
Size Exclusion ◽  
Array Detector ◽  
Test Procedures ◽  
Molecular Parameters ◽  
Hplc System

Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aim of the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».

scholarly journals EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Β-1,4-D-XYLANASE PRODUCTION

2017 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Anak Agung Istri Ratnadewi ◽  
Moch. Yoris Alidion ◽  
Agung Budi Santoso ◽  
Ika Oktavianawatia
Keyword(s):  
Large Scale ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Optimal Growth ◽  
Good Alternative ◽  
Growth Media ◽  
Gene Encoding ◽  
Xylanase Production ◽  
E Coli

<p>Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at  37 <sup>o</sup>C for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton</p>

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