Regulation of pyruvate kinases from Fusarium oxysporum

1977 ◽  
Vol 23 (10) ◽  
pp. 1346-1359 ◽  
Author(s):  
M. W. Zink
Keyword(s):  
Citric Acid ◽  
Fusarium Oxysporum ◽  
Adenosine Diphosphate ◽  
Major Type ◽  
Potassium Ions ◽  
Saturation Curve ◽  
Pyruvate Kinase Activity ◽  
Substrate Saturation ◽  
Pyruvate Kinases ◽  
Variable Substrate

Two types of pyruvate kinases were found in Fusarium oxysporum. One type (inducible) was present mainly during the early stages of growth on glucose or sucrose and displayed Michaelis–Menten kinetics with respect to phosphoenolpyruvate and adenosine diphosphate. The major type (constitutive) was present under all conditions of growth and displayed in the absence of potassium ions, a sigmoidal substrate saturation curve when phosphoenolpyruvate was used as the variable substrate. In the presence of potassium ions the saturation curve for phosphoenolpyruvate exhibits a plateau at half-maximal velocity.The effects of various metabolites on the activity of the inducible and constitutive kinases were also studied. Fructose-1,6-diphosphate, cyclic AMP, acetyl Co A, tryptophan, and phenylalanine had no effect on the activity of the enzymes. Citrate was a potent inhibitor of the constitutive pyruvate kinase activity and increased the sigmoidicity of the saturation curve for phosphoenolpyruvic acid. In the presence of K+, the bimodal plot observed in the absence of citrate gradually changed to a hyperbolic shape as the concentration of citric acid was increased. In the presence of K+ and ADP as the variable substrate citric acid converted the hyperbolic plot to a sigmoidal one. Citrate had no effect on the inducible enzyme.

scholarly journals Glucose 6-phosphate activation of pyruvate kinase from Mycobacterium smegmatis

1981 ◽  
Vol 193 (2) ◽  
pp. 435-440 ◽  
Author(s):  
R Kapoor ◽  
T A Venkitasubramanian
Keyword(s):  
Pyruvate Kinase ◽  
Mycobacterium Smegmatis ◽  
Kinase Activity ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Pyruvate Kinase Activity ◽  
Physiological Conditions ◽  
Sigmoidal Kinetics ◽  
High Degree ◽  
Variable Substrate

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.

scholarly journals The Increased Effectiveness of Platelet Concentrates Prepared in Acidified Plasma

Blood ◽  
1966 ◽  
Vol 27 (4) ◽  
pp. 449-459 ◽  
Author(s):  
FREDERICK A. FLATOW ◽  
EMIL J. FREIREICH
Keyword(s):  
Citric Acid ◽  
Acid Medium ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Red Cells ◽  
Platelet Concentrates ◽  
Standard Methods ◽  
Aggregation Of Platelets ◽  
Concentrate Preparation

Abstract Platelet concentrates prepared in acidified plasma (pH 6.5-6.7) are superior to concentrates prepared by standard methods, and are 80-90 per cent as effective as platelet rich plasma (PRP). The use of excess citric acid to acidify plasma promotes resuspension of the concentrate by eliminating clumping, which is a major factor in the decreased effectiveness of standard concentrates. Analysis of posttransfusion recovery and survival of platelets reveals no evidence of platelet injury in an acid medium. Acidification of PRP inhibits the aggregation of platelets by adenosine diphosphate (ADP). The presence of endogenous ADP may be an important factor in clumping during standard concentrate preparation. A method of acidification of PRP using citric acid is described which allows preparation of an effective concentrate from fresh whole blood without subjecting the red cells to acid pH. Reconstitution of the acidified platelet poor plasma and its native red cells increases the citrate molarity by less than 6 per cent and results in minimal decrease in pH of the whole blood.

scholarly journals Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

1969 ◽  
Vol 54 (1) ◽  
pp. 306-326 ◽  
Author(s):  
R. L. Post ◽  
S. Kume ◽  
T. Tobin ◽  
B. Orcutt ◽  
A. K. Sen
Keyword(s):  
Active Center ◽  
Adenosine Triphosphate ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Ion Concentration ◽  
Potassium Ions ◽  
Sodium Ions ◽  
Intact Cells ◽  
Electrophoretic Patterns

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.

scholarly journals Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1972 ◽  
Vol 129 (3) ◽  
pp. 571-581 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Aspartate Transcarbamoylase ◽  
Partial Purification ◽  
Saturation Curve ◽  
Phaseolus Aureus ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Sequential Reaction ◽  
Substrate Saturation

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required Pi for maximum stability, had an apparent molecular weight of 83000±5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5–10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the Km for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of Pi. Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.

scholarly journals Effects of the Tomato Pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the Biocontrol Bacterium Pseudomonas fluorescens WCS365 on the Composition of Organic Acids and Sugars in Tomato Root Exudate

2006 ◽  
Vol 19 (10) ◽  
pp. 1121-1126 ◽  
Author(s):  
Faina Kamilova ◽  
Lev V. Kravchenko ◽  
Alexander I. Shaposhnikov ◽  
Nataliya Makarova ◽  
Ben Lugtenberg
Keyword(s):  
Citric Acid ◽  
Organic Acids ◽  
Succinic Acid ◽  
Fusarium Oxysporum ◽  
Pseudomonas Fluorescens ◽  
Root Exudate ◽  
Soil Analysis ◽  
Strong Increase ◽  
Tomato Root ◽  
Biocontrol Strain

The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate revealed that the presence of F. oxysporum f. sp. radicis-lycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased, mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.

Pyruvate Kinase Activity as an Indicator of Temperature Attained During Cooking of Cured Pork

1988 ◽  
Vol 51 (10) ◽  
pp. 773-777 ◽  
Author(s):  
C. E. DAVIS ◽  
G. K. SEARCY ◽  
L. C. BLANKENSHIP ◽  
W. E. TOWNSEND
Keyword(s):  
Pyruvate Kinase ◽  
Water Extract ◽  
Adenosine Diphosphate ◽  
Model System ◽  
Pyruvate Kinase Activity ◽  
Meat Juice ◽  
Muscle Extract ◽  
Fluorescence Reaction ◽  
Pork Product ◽  
Muscle Pyruvate Kinase

Muscle pyruvate kinase (PYK) activity was established as a biochemical indicator of temperature attained during cooking in both a model system and a commercial-type pullman-style canned cured pork product. Water extract (model system) or pressed-out meat juice (commercial-type pullman-style canned cured pork) was incubated (37°C up to 40 min) with reagent containing adenosine diphosphate, phosphoenolpyruvic acid and NADH. Lactate dehydrogenase (LDH) necessary for the reaction is provided by the muscle extract or meat juice. When muscle PYK activity is present, NADH is oxidized resulting in a loss of fluorescence (reaction mixture spots on filter paper viewed under long-wave ultraviolet light). Model system results showed high PYK activity (no fluorescence) remained in samples heated to 67.7°C. PYK activity progressively declined in samples heated to 68.3°C and 68.9°C. No PYK activity was detected in samples heated to 69.5°C or 70°C. Canned product attaining a core temperature of 62.9°C had high PYK activity. PYK activity progressively declined in product heated to 65.6°C and 68.6°C. Essentially no PYK activity was detected from product processed to 69.9°C.

scholarly journals Modulation of guanine deaminase

1973 ◽  
Vol 131 (4) ◽  
pp. 683-687 ◽  
Author(s):  
K. Sree Kumar ◽  
A. Sitaramayya ◽  
P. S. Krishnan
Keyword(s):  
Rat Brain ◽  
Substrate Concentration ◽  
Mouse Liver ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Saturation Curve ◽  
Guanine Deaminase ◽  
Sigmoidal Kinetics ◽  
Substrate Saturation

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis–Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg2+ activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.

scholarly journals Application of the [3H]Leucine Incorporation Technique for Quantification of Bacterial Secondary Production Associated with Decaying Wetland Plant Litter

2006 ◽  
Vol 72 (9) ◽  
pp. 5948-5956 ◽  
Author(s):  
Jane E. Gillies ◽  
Kevin A. Kuehn ◽  
Steven N. Francoeur ◽  
Robert K. Neely
Keyword(s):  
Plant Species ◽  
Bacterial Production ◽  
Original Description ◽  
Plant Litter ◽  
Leucine Incorporation ◽  
Saturation Curve ◽  
Wetland Plant ◽  
Wide Range ◽  
Bacterial Uptake ◽  
Substrate Saturation

ABSTRACT The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 μM) required to achieve saturation-level uptake. We assessed the application of the [3H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 μM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [3H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [3H]leucine into protein exhibited a biphasic saturation curve, with lower apparent Km values ranging from 400 nM to 4.2 μM depending on the plant species studied. Upper apparent Km values ranged from 1.3 to 59 μM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent Km values possibly representing bacterial uptake and higher apparent Km values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

scholarly journals Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes

1984 ◽  
Vol 224 (1) ◽  
pp. 277-284 ◽  
Author(s):  
R Cacan ◽  
R Cecchelli ◽  
B Hoflack ◽  
A Verbert
Keyword(s):  
Plasma Membrane ◽  
Isotopic Dilution ◽  
Saturation Curve ◽  
Sugar Nucleotides ◽  
Acetylneuraminic Acid ◽  
Substrate Analogues ◽  
Carrier Mediated Transport ◽  
Carrier Molecule ◽  
Substrate Saturation

Treatment with NH4Cl of mouse thymocytes renders their plasma membrane permeable to sugar nucleotides both inwards and outwards. Using this model, we studied the entry and utilization of CMP-NeuAc, GDP-Fuc and UDP-Gal into intracellular vesicles in situ. It is shown that CMP-NeuAc and GDP-Fuc enter the vesicles in a manner indicating a carrier-mediated transport (substrate saturation curve, inhibition by substrate analogues, temperature dependence) and are entrapped in their uncleaved form. This leads to the formation of an intralumenal pool of these precursors which can be further utilized by the sialyltransferases and fucosyltransferases. The occurrence of an endogenous pool of CMP-NeuAc and GDP-Fuc is demonstrated by the fact that, when the vesicles are disrupted by detergent, the release of the endogenous sugar nucleotides causes an isotopic dilution of the labelled precursors added to measure the glycosyltransferase activities. In contrast, no accumulation of UDP-Gal has been detected, suggesting that transport and transfer reaction are simultaneous events. However, experiments with UDP 2′,3′-dialdehyde indicate that UDP-Gal is not transported through the membrane by galactosyltransferase action but by a distinct carrier molecule.

scholarly journals An automatic apparatus for the study of enzyme kinetics

1969 ◽  
Vol 115 (3) ◽  
pp. 511-515 ◽  
Author(s):  
J. A. Illingworth ◽  
K F Tipton
Keyword(s):  
Reaction Times ◽  
Flow Cell ◽  
Reaction Coil ◽  
Automatic Apparatus ◽  
Precise Control ◽  
Fixed Proportion ◽  
Substrate Saturation ◽  
Flow Apparatus ◽  
Variable Substrate ◽  
Linear Concentration

A continuous-flow apparatus is described for automatically plotting substrate saturation curves, and is suitable for use with a variety of enzymes. A linear concentration gradient of the variable substrate is combined with a fixed proportion of the other substrates and the enzyme, and after passing through a reaction coil the product concentrations are measured spectrophotometrically. Use of a 4cm. flow cell and modified spectrophotometer permits accurate measurement of NADH concentration in the region of 0·1μm. Precise control over reaction times and substrate concentration is achieved by using power-driven syringes with an integral mixer. Specimen results are given for yeast alcohol dehydrogenase.

Sign in / Sign up

Export Citation Format

Share Document