scholarly journals Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger

1983 ◽  
Vol 209 (3) ◽  
pp. 669-676 ◽  
Author(s):  
A Habison ◽  
C P Kubicek ◽  
M Röhr
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Distinct Species ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Maximal Velocity ◽  
High Concentrations

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran-Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis-Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.

scholarly journals Sesquiterpene α-Farnesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Development in Apples

2000 ◽  
Vol 125 (1) ◽  
pp. 111-119 ◽  
Author(s):  
H.P. Vasantha Rupasinghe ◽  
Gopinadhan Paliyath ◽  
Dennis P. Murr
Keyword(s):  
Metal Ion ◽  
Maximal Activity ◽  
Exchange Chromatography ◽  
Cytosolic Fraction ◽  
Skin Tissue ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Farnesyl Pyrophosphate ◽  
Superficial Scald

To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg2+ or Mn2+ for activity. It exhibited allosteric kinetics, S(0.5) for farnesyl pyrophosphate being 84±18 μmol·L-1, and a Hill coefficient (nH) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.

Taurine transport in skate hepatocytes. I. Uptake and efflux

1992 ◽  
Vol 262 (3) ◽  
pp. G445-G450 ◽  
Author(s):  
N. Ballatori ◽  
J. L. Boyer
Keyword(s):  
Amino Acids ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Disulfonic Acid ◽  
Maximal Velocity ◽  
Small Contribution ◽  
Uptake System ◽  
High Concentrations ◽  
Efflux Pathway ◽  
Taurine Uptake

Taurine is an amino sulfonic acid maintained at relatively high concentrations in skate (Raja erinacea) hepatocytes (65 mmol/l intracellular water). To identify the transport mechanisms involved in intracellular taurine accumulation, uptake and efflux of [14C]taurine was measured in freshly isolated skate hepatocytes. Uptake of 50 microM taurine at 15 degrees C was essentially linear over 3 h when measured in normal elasmobranch Ringer, was diminished by replacement of extracellular Cl- with NO3-, and was almost completely abolished by replacement of Na+ with choline+ or K+. Kinetic studies suggested the presence of two saturable Na(+)-dependent taurine uptake systems [apparent taurine Michaelis constant = 0.089 +/- 0.028 and 4.47 +/- 0.49 mM and maximal velocity (Vmax) = 0.19 +/- 0.07 and 1.65 +/- 0.42 (SE) nmol.microliter-1.15 min-1 for high- and low-affinity components, respectively; n = 4], as well as a small contribution from an Na(+)-independent uptake system. Uptake was inhibited by other beta-amino acids but not by alpha-amino acids, taurocholate, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.5 mM). In contrast to uptake, efflux of [14C]taurine was relatively slow (approximately 10% in 2 h) and was unaffected by isosmotic replacement of extracellular Na+ with choline+ or K+ or by replacing Cl- with NO3-. These findings suggest distinct mechanisms for taurine uptake and efflux in skate hepatocytes; uptake is largely Na+ dependent and requires Cl- for maximal activity, whereas the efflux pathway is independent of transmembrane Na+, K+, or Cl- gradients. Maintenance of high intracellular taurine levels in skate hepatocytes is achieved by active Na(+)-dependent uptake processes and a relatively slow efflux.

scholarly journals The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex

1988 ◽  
Vol 253 (3) ◽  
pp. 819-825 ◽  
Author(s):  
T Pawelczyk ◽  
R A Easom ◽  
M S Olson
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
Kidney Cortex ◽  
Complex Activity ◽  
Pig Kidney ◽  
Constant Strength ◽  
Dehydrogenase Complex

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.

Partial purification of galactinol synthase from leaves of Cucurbita pepo

1982 ◽  
Vol 60 (7) ◽  
pp. 1054-1059 ◽  
Author(s):  
John A. Webb
Keyword(s):  
Cucurbita Pepo ◽  
Partial Purification ◽  
Galactinol Synthase ◽  
Inositol 1 ◽  
Mature Leaves ◽  
Galactosyl Transferase ◽  
Naturally Occurring ◽  
High Concentrations ◽  
Uridine Nucleotides

An enzyme synthesizing galactinol, UDP-D-galactose:myo-inositol-1-α-D-galactosyl transferase (galactinol synthase), has been isolated and partially purified from mature leaves of Cucurbita pepo. The enzyme showed optimal activity between pH 7.5 and 8.0 and required Mn2+ and the presence throughout isolation, storage, and assay of a sulfhydryl protectant (β-mercaptoethanol). EDTA was completely inhibitory. From a range of metal ions only Mg2+ partially replaced Mn2+, while Co2+, Zn2+, Cu2+, and Ni2+ were inhibitory. The uridine nucleotides and UDP-glucose were from 40 to 80% inhibitory and probably constitute part of the in vivo control system. High concentrations of galactose, melibiose, and xylose were partially inhibitory. The enzyme appeared highly specific for myo-inositol and showed no ability for galactosyl transfer to any other naturally occurring sugar or sugar alcohol. Some reactivity was obtained with the isomeric scyllo-inositol but the product was not identified. A range of other sugar nucleotides were unreactive.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics

2018 ◽  
Vol 475 (11) ◽  
pp. 1979-1993 ◽  
Author(s):  
Andreas Sukmana ◽  
Zhaomin Yang
Keyword(s):  
Atpase Activity ◽  
Enrichment Culture ◽  
Physiological State ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Enzyme Analysis ◽  
Type Iv Pilus ◽  
Type Iv ◽  
High Concentrations

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

scholarly journals Enhanced Biosorption of Pb(II) Ions from Aqueous Solutions onto Citric Acid Treated Aspergillus niger Biomass: Equilibrium and Kinetic Studies

2020 ◽  
Vol 32 (3) ◽  
pp. 508-514
Author(s):  
Vinay Kumar Chintalapudi ◽  
Ramya Krishna S.L. Kanamarlapudi ◽  
Useni Reddy Mallu ◽  
Sudhamani Muddada
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Kinetic Studies ◽  
Order Kinetic ◽  
Ftir Analysis ◽  
Structural Variations ◽  
Order Kinetic Model ◽  
Pseudo Second Order ◽  
Optimized Conditions ◽  
Biosorption Kinetics

In the present study, initially Aspergillus niger was tested for biosorption of Pb(II) ions and then studied the effect of pretreatment for enhanced biosorption. It was found that the maximum biosorption potential was achieved with citric acid treatment (70.56 %) in comparison with the biomass without treatment (65.46 %) at a biosorbent dose of 20 mg/L, pH 4, 100 rpm, 37 ºC for 8 h. The optimized conditions for treated Aspergillus niger were determined by optimizing the biosorption parameters such as pH, temperature, biomass dose, incubation time and agitation speed. This study indicates that the citric acid treated Aspergillus niger is an effective biosorbent for removal of lead (II) at optimized conditions with the maximum biosorption potential of 83.6 % as compared to previous reported work. SEM-EDX and FTIR analysis showed the structural variations and the functional groups involved in lead biosorption, respectively. Biosorption kinetics showed that pseudo second order kinetic model as the better fit.

scholarly journals The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

1970 ◽  
Vol 120 (1) ◽  
pp. 145-150 ◽  
Author(s):  
A. P. F. Flint
Keyword(s):  
Luteinizing Hormone ◽  
Total Activity ◽  
Partial Purification ◽  
Kinetic Properties ◽  
Mevalonate Kinase ◽  
Farnesyl Pyrophosphate ◽  
Rat Ovary ◽  
High Concentrations ◽  
Geranyl Pyrophosphate

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6±0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The Km of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6±0.5μm; its Km for MgATP2− was 120±7.7μm. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3′-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP2−. The Ki for inhibition by geranyl pyrophosphate was 1.3±0.2μm; the Ki for inhibition by farnesyl pyrophosphate was 1.0±0.3μm. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.

scholarly journals Partial Purification and Characterization of Catalase from Banana Peels

2016 ◽  
Vol 13 (2) ◽  
pp. 392-398
Author(s):  
Baghdad Science Journal
Keyword(s):  
Gel Filtration ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Km Value ◽  
Almost All ◽  
Kinetics Of

Catalase (EC 1.11.1.6) is a well known enzyme which exists in almost all living creatures exposing to oxygen (such as plants, bacteria, and animals). It is a very necessary enzyme to protect the cell from oxidative detriment by reactive oxygen species (ROS). The aim of this study is the partial purification and characterization of Catalase enzyme from Banana peels. In this study, fresh banana peels are treated with 70 % ethanol ,further separated with chloroform ,water and ethyl acetate respectively .The supernatant of the enzymatic sample which is treated with chloroform is loaded into gel filtration column with Sephadex G-100 (1.0 x 90 cm) equilibrated with pH7 buffer media (phosphate buffer 0.1 M). Kinetic studies of the purified enzyme activity are measured and characterized .The maximal activity (26.04 units/mg) of catalase is observed with chloroform buffer extraction. The kinetics of catalase; Michalis constant Km and maximum velocity Vmax is determined using Linweaver- Burk plot, The Km value for catalase (434.7mM), Vmax (100 m mole min -1). Characterization results demonstrate that the optimal pH for activity is (7.6). And the optimal temperature for activity is 30?C .The present study indicates that Banana peels is a good source of catalase enzyme.

scholarly journals Purification and characterization of actinidin from Actinidia deliciosa and its utilization in inactivation of α-amylase

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Vivek Kumar Dhiman ◽  
Vivek Chauhan ◽  
Shamsher Singh Kanwar ◽  
Devendra Singh ◽  
Himanshu Pandey
Keyword(s):  
Digestive Enzyme ◽  
Fold Increase ◽  
Maximal Activity ◽  
In Vivo Studies ◽  
Maximal Velocity ◽  
Starch Digestion ◽  
Amylolytic Enzyme ◽  
Ph Range

Abstract Background Actinidin is an anionic thiol-proteinase predominant and unique to Chinese gooseberry or kiwifruit, whose strong digestibility enables proteins or enzymes vulnerable to digestion. The arrangement of active cysteine–thiol residues (Cys22-Cys65, Cys56-Cys98, and Cys156-Cys206) stabilizes the catalytic unit, thus allowing an effective Inhibition of α-amylase protein on exposure to the highest concentrations of actinidin under optimum conditions. When starch-rich foods are consumed with kiwifruit, starch digestion may be slowed by the inactivation of α-amylase (digestive enzyme), specifically reducing the blood sugar levels by hindering starch digestion that is helpful in diabetes mellitus. Thus, the study aimed at actinidin purification, optimization for maximal activity, and its demonstration as a potential to degrade α-amylase. Results Protease showed a molecular mass of 27 kDa on SDS-PAGE analysis. One factor at a time method was applied for process optimization, increasing the actinidin yield up to 176.03 U/mg. The enzyme was stable at a wide pH range; however, it was most active and stable at pH 7.5. The enzyme possessed half‐life at 35 °C of 5.5 h, at 40 °C of 4.5 h, at 45 °C of 2.5 h, and at 50 °C of 1 h. Lineweaver–Burk plot showed Michaelis–Menten constant (Km: 3.14 mg/ml) and maximal velocity (Vmax: 1.428 mmol/ml/min) using casein. The actinidin activity was enhanced with Ca2+ while it was inhibited by Cd2+ and Hg2+ ions. The α-amylase protein was successfully inactivated upon incubation with actinidin for 30 min; around ~ 85% of the α-amylase activity diminished. IC50 for inhibition of α-amylase was 2.54 mg/ml for crude actinidin and 1.86 mg/ml for purified actinidin. Conclusions Purified Actinidin showed a 1.28-fold increase in proteolytic activity. The proteinase showed an active pH range of 3.5–8.5 under varied buffer conditions and thermostability up to 50 °C. The results revealed a significant potential utility of actinidin to retard amylase as it effectively degraded the amylolytic enzyme under in vitro conditions and could be beneficial for lowering glycemic response to ingested starch. However, further in vitro as well as in vivo studies need to be conducted under gastrointestinal conditions to establish the hypothesis.

Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle

2001 ◽  
Vol 281 (5) ◽  
pp. R1681-R1688 ◽  
Author(s):  
R. Tupling ◽  
H. Green ◽  
G. Senisterra ◽  
J. Lepock ◽  
N. McKee
Keyword(s):  
Atpase Activity ◽  
Binding Capacity ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
In Vivo Model ◽  
Sprague Dawley ◽  
Atp Binding Site ◽  
Structural Modifications ◽  
The Hill

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 ± 6.7 g (mean ± SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (μmol · g protein−1 · min−1) of C (15,802 ± 1,246) and I (11,609 ± 1,029) was 90 and 73% ( P < 0.05) of CC (17,562 ± 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+ at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl- N′-(dimethylamino-α-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+ binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction ( P < 0.05) in FITC binding capacity (absolute units) in I (0.22 ± 0.01) compared with CC (0.29 ± 0.02) and C (0.29 ± 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.

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