scholarly journals Allosteric control of Zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate

1997 ◽  
Vol 326 (3) ◽  
pp. 731-735 ◽  
Author(s):  
Roberts K. SCOPES
Keyword(s):  
Ionic Strength ◽  
Zymomonas Mobilis ◽  
Phosphate Concentration ◽  
Hill Coefficient ◽  
Allosteric Control ◽  
Low Glucose ◽  
Low Ionic Strength ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
T State ◽  
Micromolar Range

The second enzyme of the Entner–Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis–Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod–Wyman–Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.

scholarly journals The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1982 ◽  
Vol 202 (1) ◽  
pp. 53-58 ◽  
Author(s):  
C. Peter Downes ◽  
Robert H. Michell
Keyword(s):  
Ionic Strength ◽  
Erythrocyte Membrane ◽  
Erythrocyte Membranes ◽  
Phosphodiesterase 4 ◽  
Maximal Activation ◽  
Human Erythrocyte Membranes ◽  
The Right ◽  
Phosphatidylinositol 4 ◽  
Low Ionic Strength ◽  
Micromolar Range

1. Both the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca2+ in the micromolar range. We have therefore compared the mechanisms and affinities for Ca2+ activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca2+-pump ATPase was activated by Ca2+ in a highly co-operative manner, with half-maximal activation occurring at about 0.3μm-Ca2+. At an optimal Ca2+concentration, calmodulin stimulated the Ca2+-sensitive ATPase activity about 10-fold. 3. Ca2+ activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca2+ requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca2+ activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg2+, half-maximal activation of the phosphodiesterase was at about 3μm-Ca2+. The presence of 1mm-Mg2+ shifted the Ca2+ activation curve to the right, as did elevation of the ionic strength. When the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg2+concentration, the ATPase was fully activated at 3μm-Ca2+, whereas no polyphosphoinositide phosphodiesterase activity was detected below 100μm-Ca2+. 5. The Ca2+-pump ATPase of the erythrocyte membrane normally maintains the Ca2+ concentration of healthy erythrocytes below approx. 0.1μm. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.

scholarly journals Regulation of glucose-6-phosphate dehydrogenase activity in sea urchin eggs by reversible association with cell structural elements.

1986 ◽  
Vol 103 (4) ◽  
pp. 1509-1515 ◽  
Author(s):  
R R Swezey ◽  
D Epel
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Time Course ◽  
Calcium Concentration ◽  
Metabolic Activation ◽  
Structural Elements ◽  
Ionic Detergent ◽  
Low Ionic Strength ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Substrate Concentrations

In unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, glucose-6-phosphate dehydrogenase (G6PDH) associates with the particulate elements remaining either after homogenization or extraction of eggs with non-ionic detergent in low ionic-strength media. At physiological ionic strength, the extent of G6PDH binding to these particulate elements is proportional to the total protein concentration in the extracts. In fertilized eggs this association is prevented by one or more low molecular weight solutes. The dissociation is reversible, and there are no permanent modifications of either G6PDH or its particulate binding site that affect binding. After fertilization, the time course of dissociation of G6PDH from particulate elements is too fast to be caused by a change in intracellular pH, but it could be triggered, but not maintained, by an increase in the intracellular calcium concentration. Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations. Therefore, release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.

scholarly journals Effect of cross-bridge kinetics on apparent Ca2+ sensitivity.

1982 ◽  
Vol 79 (6) ◽  
pp. 997-1016 ◽  
Author(s):  
P W Brandt ◽  
R N Cox ◽  
M Kawai ◽  
T Robinson
Keyword(s):  
Ionic Strength ◽  
Phosphate Concentration ◽  
Hill Coefficient ◽  
Cross Bridge ◽  
Left Shift ◽  
Hill Coefficients ◽  
Tension Curve ◽  
The Cross ◽  
Cross Bridges ◽  
The Hill

Three different ways of shifting the pCa/tension curve on the pCa axis have been studied and related to changes in the rate constants of the cross-bridge cycle. The curve midpoint shifts to higher pCa's when the substrate (Mg-ATP) is reduced from 5 to 0.25 mM, when the phosphate concentration is reduced from 7.5 mM to 0, and when the ionic strength is reduced from 0.200 to 0.120. The Hill coefficients of the pCa/tension curve in our standard saline (5 mM substrate, 5 mM free ATP, 7.5 mM phosphate, ionic strength 0.200, 15 degree C) are between 5.1 and 5.6 and fall to 3.0 with the left shift of the curve brought about by reducing both substrate and phosphate. Left shifts of the curve produced by reduction in the ionic strength do not result ina lower Hill coefficient. Reducing eigher substrate or phosphate is associated with a reduction in the optimal frequency for oscillatory work, but reduction in ionic strength is not so associated. Maximum tension increases with the left shift of the curve brought about by reducing phosphate concentration or ionic strength, but tension decreases with the left shift of the curve accompanying substrate concentration reduction in phosphate-free saline. We argue that one mechanism for the observed shift of the curve along the pCa axis is the relationship between the time a cross-bridge takes to complete a cycle and the time Ca2+ stays bound to troponin C (TnC). If the cycle rate is decreased, a smaller fraction to TnC sites must be occupied to keep a given fraction of cross-bridges active. To illustrate this concept, we present a simplified model of the cross-bridge cycle incorporating the kinetics of Ca binding to TnC.

scholarly journals Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin

1993 ◽  
Vol 296 (3) ◽  
pp. 785-796 ◽  
Author(s):  
L Agius ◽  
M Peak
Keyword(s):  
Ionic Strength ◽  
Ionic Composition ◽  
Bound State ◽  
Lactate Dehydrogenase Release ◽  
Intracellular Binding ◽  
Low Ionic Strength ◽  
Different Characteristics ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Small Inhibition ◽  
Substrate Concentrations

The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocytes have been preincubated. In hepatocytes pre-cultured with 5 mM glucose the release of glucokinase was much slower than that of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 microM-1 mM) markedly increased glucokinase release. This suggests that, in cells maintained in 5 mM glucose, glucokinase is present predominantly in a bound state and this binding is dependent on the presence of Mg2+. The enzyme can be released or translocated from its bound state by an increase in [glucose] (A50 15 mM) or by fructose (A50 50 microM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insulin and counteracted by glucagon. They were inhibited by cyanide, but not by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-like effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D-glucose and 2-deoxyglucose were ineffective. When hepatocytes were incubated with [2-3H, U-14C]glucose, the incorporation of 3H/14C label into glycogen correlated with the extent of glucokinase release. Since 2-3H is lost during conversion of glucose 6-phosphate into fructose 6-phosphate, substrate-induced translocation of glucokinase from a Mg(2+)-dependent binding site to an alternative site might favour the partitioning of glucose 6-phosphate towards glycogen, as opposed to phosphoglucoisomerase.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

An Evaluation of a Plasma Fractionation System

1960 ◽  
Vol 4 (01) ◽  
pp. 031-044
Author(s):  
George Y. Shinowara ◽  
E. Mary Ruth
Keyword(s):  
Biological Activity ◽  
Ionic Strength ◽  
Plasma Proteins ◽  
High Concentration ◽  
Significant Evidence ◽  
Native Proteins ◽  
Mobility Data ◽  
Fractionation Procedure ◽  
The Individual ◽  
Low Ionic Strength

SummaryFour primary fractions comprising at least 97 per cent of the plasma proteins have been critically appraised for evidence of denaturation arising from a low temperature—low ionic strength fractionation system. The results in addition to those referable to the recovery of mass and biological activity include the following: The high solubilities of these fractions at pH 7.3 and low ionic strengths; the compatibility of the electrophoretic and ultracentrifugal data of the individual fractions with those of the original plasma; and the recovery of hemoglobin, not hematin, in fraction III obtained from specimens contaminated with this pigment. However, the most significant evidence for minimum alterations of native proteins was that the S20, w and the electrophoretic mobility data on the physically recombined fractions were identical to those found on whole plasma.The fractionation procedure examined here quantitatively isolates fibrinogen, prothrombin and antithrombin in primary fractions. Results have been obtained demonstrating its significance in other biological systems. These include the following: The finding of 5 S20, w classes in the 4 primary fractions; the occurrence of more than 90 per cent of the plasma gamma globulins in fraction III; the 98 per cent pure albumin in fraction IV; and, finally, the high concentration of beta lipoproteins in fraction II.

scholarly journals A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

1981 ◽  
Vol 193 (1) ◽  
pp. 375-378 ◽  
Author(s):  
A R Ashton ◽  
L E Anderson
Keyword(s):  
Ionic Strength ◽  
Pisum Sativum ◽  
Deae Cellulose ◽  
High Concentrations ◽  
Rapid Purification ◽  
Low Ionic Strength

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

scholarly journals Crystallization of tuna ferricytochrome c at low ionic strength.

1990 ◽  
Vol 265 (8) ◽  
pp. 4177-4180
Author(s):  
M H Walter ◽  
E M Westbrook ◽  
S Tykodi ◽  
A M Uhm ◽  
E Margoliash
Keyword(s):  
Ionic Strength ◽  
Ferricytochrome C ◽  
Low Ionic Strength
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