Studies on isocitrate lyase from the facultative autotroph Thiobacillus novellus

1973 ◽  
Vol 19 (4) ◽  
pp. 513-519 ◽  
Author(s):  
J. T. McCarthy ◽  
A. Michael Charles
Keyword(s):  
Isocitrate Lyase ◽  
Adenine Nucleotides ◽  
Potassium Phosphate ◽  
Forward Direction ◽  
Disc Electrophoresis ◽  
Reverse Direction ◽  
K Value ◽  
Absolute Requirement ◽  
Sigmoidal Kinetics ◽  
Hill Plots

Isocitrate lyase from Thiobacillus novellus was purified about eightfold from crude extracts of acetate-grown cells. Polyacrylamide gel "disc" electrophoresis revealed that the partly purified material contained one major and two minor peaks. The optimum pH for activity in the forward direction with glycylglycine buffer and the reverse direction with either glycylglycine or potassium phosphate was around pH 8.0. Plots of v vs. s gave Michaelis–Menten kinetics with an apparent Km of 4.3 × 10−5 M for isocitrate and 2.3 × 10−4 M for succinate. However, for glyoxylate, v vs. s plots were sigmoidal. Hill plots gave an n value of 2.6 and a k value of about 7.76 × 10−5 M. Since biosynthetic enzymes usually have sigmoidal kinetics, it follows that isocitrate lyase may be important in the synthesis of isocitrate. Also of interest was the finding that the enzyme did not show an absolute requirement for a divalent cation, but in the presence of 0.3 mM Mg2+ activity was stimulated about threefold over that observed in the absence of metal. Km for Mg2+ was 7.0 × 10−4 M. Complete loss of activity found on addition of 0.03 mM HgCl2 or CuSO4 to the enzyme in the absence of Mg2+, as well as the restoration of activity lost during storage by the addition of cysteine or glutathione supports the idea that sulfhydryl groups are required for activity. The effect of adenine nucleotides on activity revealed in decreasing order of inhibition that ATP > ADP > AMP. Adenosine or pyrophosphate were not inhibitory at any concentration.

Studies on the kinetics and regulation of glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 439-450 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Forward Direction ◽  
Product Inhibition ◽  
Reverse Direction ◽  
High Concentrations ◽  
Glutamate Synthesis ◽  
Inhibition Studies

A 190-fold purified preparation of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium was used for the determination of kinetic parameters of the substrates, NADPH, NH4+, and α-ketoglutarate, in the direction of glutamate synthesis, and NADP+ and glutamate in the reverse direction. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme. Based on an analysis of the data derived from initial velocity and product inhibition studies, the reaction mechanism was postulated to be ordered Ter Bi with NADPH as the first substrate to bind in the forward direction, and NADP+ binding first in the reverse direction.Of the several metabolites tested for a possible function in the regulation of GDH activity, only L-malate and L-glutamine appeared to exert an appreciable influence on the enzyme. ATP and AMP at a concentration of 0.8 mM were found to enhance GDH activity by 68% and 6%, respectively, but at high concentrations, both the adenine nucleotides proved to be inhibitory.

scholarly journals Brain-wide mapping of water flow perception in zebrafish

2020 ◽  
Author(s):  
Gilles Vanwalleghem ◽  
Kevin Schuster ◽  
Michael A. Taylor ◽  
Itia A. Favre-Bulle ◽  
Ethan K. Scott
Keyword(s):  
Water Flow ◽  
Lateral Line ◽  
Reverse Flow ◽  
Forward Direction ◽  
Brain Regions ◽  
Reverse Direction ◽  
Departure Point ◽  
Forward Flow ◽  
Larval Zebrafish ◽  
The Brain

AbstractInformation about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain specific aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, all neurons involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to forward flow, reverse flow, or both. Early in this pathway, in the lateral line ganglia, these are almost exclusively neurons responding to the simple presence of forward or reverse flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated volume of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to forward flow detection, and this is reinforced by our finding that details like flow velocity and the total volume of accumulated flow are only encoded for the simulated forward direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.Significance statementIn aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find some neurons that respond to the simple presence of water flow, and others that are attuned to the flow’s direction, speed, duration, or the accumulated volume of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating forward motion but rudimentary in processing flow in the reverse direction.

Tyrosine Biosynthesis in Sorghum bicolor: Characteristics of Prephenate Aminotransferase

1986 ◽  
Vol 41 (1-2) ◽  
pp. 79-86 ◽  
Author(s):  
Daniel L. Siehl ◽  
James A. Connelly ◽  
Eric E. Conn
Keyword(s):  
Reaction Rate ◽  
Deae Cellulose ◽  
Forward Direction ◽  
Reverse Direction ◽  
Aminotransferase Activity ◽  
Forward Reaction ◽  
High Affinity ◽  
Sorghum Plant ◽  
Arogenate Dehydrogenase ◽  
Tyrosine Biosynthesis

Abstract A stable activity which transfers the amino group from glutamate to prephenate was extracted from 4-day old etiolated shoots of sorghum. The activity was retained on DEAE cellulose and eluted as a single peak. Prephenate aminotransferase co-eluted with a very abundant α-ketoglutarate: aspartate aminotransferase, but heating at 70 °C resulted in loss of α-ketoglutarate: aspar­tate activity with nearly full retention of prephenate: glutamate aminotransferase activity. The heated enzyme displayed high affinity and specificity for prephenate. Among 7 donors tested, only glutamate, and aspartate at less than 20% the rate with glutamate, supported prephenate aminotransferase activity. In the reverse direction, a reaction rate comparable to that in the forward direction was unchanged as the concentration of α-ketoglutarate was reduced from 1.0 to 0.09 mᴍ. The apparent Km for arogenate was 0.8 mᴍ. The forward reaction was unaffected by the inclusion of tyrosine, phenylalanine or tryptophan. Together with the discovery of arogenate dehydrogenase in sorghum [3], these data indicate that, in the sorghum plant, tyrosine derives from prephenate by transamination and aromatization. rather than the reverse sequence.

RADIATED ACOUSTIC PRESSURE FROM A MOVING, NONUNIFORMLY VIBRATING CYLINDER WITH TWO SPHERICAL ENDCAPS

1994 ◽  
Vol 02 (01) ◽  
pp. 71-82 ◽  
Author(s):  
ZHAOXI WANG ◽  
SEAN F. WU
Keyword(s):  
Near Field ◽  
Translational Motion ◽  
Normal Component ◽  
Forward Direction ◽  
Numerical Results ◽  
Surface Velocity ◽  
Reverse Direction ◽  
Far Field ◽  
Field Source ◽  
Sound Diffraction

This paper presents numerical results of radiated acoustic pressures from a moving, nonuniformly vibrating cylinder with two spherical endcaps, based on an extended Kirchhoff integral formulation. Specifically, we consider cases in which the normal component of the surface velocity is nonzero on a portion of the surface, and zero elsewhere. Numerical results demonstrate that the radiation patterns depend critically on the frequency and source dimensions. For a noncompact source, the strongest radiation may not necessarily stem from a vibrating surface, but rather from a nonvibrating surface due to the effect of sound diffraction. The more noncompact the source is, the larger the number of side lobes in the near field and the more concentrated these side lobes will be. In the far field, however, the side lobes become smeared and less distinguishable. In other words, the effect of sound diffraction is greatly reduced in the far field. Source translational motion induces sound radiation in the perpendicular direction and enhances the radiated acoustic field in general. Enhancement in the forward direction is much greater than in the reverse direction.

AN EXPERIMENTAL STUDY ON THE TRANSIENT TEMPERATURE TEST OF THE PLANETARY GEAR REDUCER OF THE EPBM

2011 ◽  
Vol 10 (01) ◽  
pp. 37-43 ◽  
Author(s):  
LIFENG CHEN ◽  
XIAOLING WU ◽  
DATONG QIN ◽  
ZEJUN WEN
Keyword(s):  
Temperature Rise ◽  
Planetary Gear ◽  
Forward Direction ◽  
Test System ◽  
Gear Train ◽  
Reverse Direction ◽  
Transient Temperature ◽  
Pressure Balance ◽  
Planetary Gear Train ◽  
Temperature Test

A temperature test apparatus has been designed to measure the temperature rise of the planetary gear train. The experimental platform integrated the temperature test system, siemens operating system and industrial personal computer (IPC). The temperatures of various locations have been investigated under rated condition and the oil temperature-rise time under different speed was estimated. The experimental results indicated that the temperatures of the super-speed planetary gear train (PGT) of the earth pressure balance shield machine (EPBM) rise quickly than the low-speed PGT and the speed has significant influence on the temperature of the PGT. Through comparison the oil temperatures under reverse direction and forward direction, the direction of the motor has little influence on the temperature of the PGT.

scholarly journals pH-induced bistable dynamic behaviour in the reaction catalysed by glucose-6-phosphate dehydrogenase and conformational hysteresis of the enzyme

1989 ◽  
Vol 262 (3) ◽  
pp. 795-800 ◽  
Author(s):  
M A Aon ◽  
S Cortassa ◽  
J F Hervagault ◽  
D Thomas
Keyword(s):  
Dynamic Behaviour ◽  
Forward Direction ◽  
Memory Device ◽  
Reverse Direction ◽  
Protein Fluorescence ◽  
Ph Change ◽  
Before And After ◽  
Continuously Stirred Tank Reactor ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Intrinsic Protein Fluorescence

1. Bistable (multiple stationary states) dynamic behaviour in the activity of glucose-6-phosphate dehydrogenase that was subjected to successive pH change was demonstrated in an open continuously stirred tank reactor. Although the enzyme under study did not exhibit an autocatalytic effect and was homogeneously distributed, bistability was shown to occur. 2. The successive pH changes of the enzyme solution corresponded to a pH transition (8.3 in equilibrium 2), i.e. an acidification (forward direction) and an alkalinization (reverse direction). By use of intrinsic protein fluorescence methods, a glucose-6-phosphate dehydrogenase conformational hysteresis was shown to exist concomitant with the pH transition before and after enzyme injection into the reactor. 3. The results obtained suggest that the enzyme behaves, conformationally, as a memory device that stores information about its pH history (i.e. the enzyme records information in its structure about the environment to which it was previously exposed) and transduces it in a non-linear dynamic fashion, producing the bistable behaviour observed in the open reactor.

Performance of S-Cambered Profiles With Cut-Off Trailing Edges

1994 ◽  
Vol 116 (3) ◽  
pp. 522-527 ◽  
Author(s):  
Baby Chacko ◽  
V. Balabaskaran ◽  
E. G. Tulapurkara ◽  
H. C. Radha Krishna
Keyword(s):  
Lift Coefficient ◽  
Forward Direction ◽  
Aerodynamic Characteristics ◽  
Reverse Direction ◽  
Flow Conditions ◽  
Reversed Flow ◽  
Trailing Edge ◽  
Reverse Mode ◽  
Trailing Edges

The aerodynamic characteristics of an S-cambered profile are studied under forward and reversed flow conditions. The profile chord is cut by 3, 6, and 9 percent of the chord at the sharp trailing edge end and the performances of these profiles are compared. It is found that with increase in length of cutting the lift coefficient increases in forward direction and decreases in reverse direction of flow. Cutting off the sharp trailing edge improves the lift-drag characteristics in forward mode and deteriorates in the reverse mode.

Four- and five-step kinetic models of lactate dehydrogenase

1976 ◽  
Vol 54 (10) ◽  
pp. 915-918 ◽  
Author(s):  
Uwe Borgmann ◽  
Keith J. Laidler ◽  
Thomas W. Moon
Keyword(s):  
Lactate Dehydrogenase ◽  
Forward Direction ◽  
Reverse Direction ◽  
Similar Data ◽  
State Data ◽  
Incomplete Model ◽  
Step Model ◽  
The Relationship ◽  
Kinetics Of ◽  
Different Sources

A five-step model for the reaction catalyzed by beef heart lactate dehydrogenase (EC 1.1.1.27) reconciles differences observed in the four-step model if pre-steady-state data in the forward direction are compared with similar data in the reverse direction. The relationship between the four- and five-step models indicates what problems can develop when an incomplete model is proposed. Nevertheless, there are advantages to using the less complicated four-step model when comparing the molecular kinetics of enzymes catalyzing the same reaction but obtained from different sources.

scholarly journals Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase

1978 ◽  
Vol 175 (3) ◽  
pp. 987-998 ◽  
Author(s):  
A Lodola ◽  
J D Shore ◽  
D M Parker ◽  
J Holbrook
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Forward Direction ◽  
Divergent Evolution ◽  
Reverse Direction ◽  
Enzyme Form ◽  
Active Substrate ◽  
Rate Limiting ◽  
Transient Measurements

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows ‘all-of-the-sites’ behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.

Studies of the structure–function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran – Sepharose and Cibacron blue 3G-A

1980 ◽  
Vol 26 (5) ◽  
pp. 613-621 ◽  
Author(s):  
M. Kapoor ◽  
M. D. O'Brien
Keyword(s):  
Neurospora Crassa ◽  
Pyruvate Kinase ◽  
Adenine Nucleotides ◽  
Potassium Phosphate ◽  
Difference Spectrum ◽  
Cibacron Blue ◽  
Blue Dextran ◽  
Allosteric Effector ◽  
Noncompetitive Inhibitor ◽  
Incremental Addition

Blue dextran – Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G-A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kinase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental addition of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvate kinase – Cibacron blue complex.

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