scholarly journals The purification and regulatory properties of α-oxoglutarate dehydrogenase from Acinetobacter lwoffi

1973 ◽  
Vol 135 (1) ◽  
pp. 215-223 ◽  
Author(s):  
Malcolm G. Parker ◽  
P. David J. Weitzman
Keyword(s):  
Protein Concentration ◽  
Analytical Ultracentrifugation ◽  
Energy Charge ◽  
Kinetic Studies ◽  
Dilute Solution ◽  
Site Of Action ◽  
Oxoglutarate Dehydrogenase ◽  
Sites Of Action ◽  
High Degree ◽  
Regulatory Properties

The α-oxoglutarate dehydrogenase multienzyme complex was purified from Acinetobacter lwoffi to a high degree of homogeneity as shown by gel electrophoresis and analytical ultracentrifugation. Sedimentation-velocity analyses gave s20,w values which increased with increasing protein concentration, suggesting dissociation of the complex in dilute solution. The maximum s20,w value thereby obtained and the value determined by active enzyme centrifugation were both in the range 28–29S. Electron micrographs of the complex indicated a molecular diameter of 20–22nm (200–220Å). The overall activity of the complex was inhibited by NADH, and kinetic studies indicated sites of action on the first and third enzyme components. AMP and ADP relieved this inhibition and also stimulated enzyme activity. Assays specific for the first enzyme component showed this to be the site of action of the adenylates. The activity of the complex varied with energy charge in a manner consistent with its role in energy metabolism.

scholarly journals Some regulatory properties of the 2-oxoglutarate dehydrogenase complex from European bison heart.

1995 ◽  
Vol 42 (3) ◽  
pp. 339-346 ◽  
Author(s):  
J Markiewicz ◽  
S A Strumiło
Keyword(s):  
Reaction Rate ◽  
Kinetic Studies ◽  
European Bison ◽  
Bison Bonasus ◽  
Maximum Reaction Rate ◽  
Phosphate Ions ◽  
Maximum Reaction ◽  
Oxoglutarate Dehydrogenase ◽  
Regulatory Properties ◽  
Dehydrogenase Complex

Basic regulatory properties of the 2-oxoglutarate dehydrogenase complex (OGDC) isolated and purified from the heart muscle of European bison (Bison bonasus) were studied. Kinetic studies have shown that in the absence of phosphate ions OGDC exhibits kinetic attributes of negative cooperativity with respect to 2-oxoglutarate. ADP and phosphate lower S0.5 value of OGDC for 2-oxoglutarate without changing the maximum reaction rate. NADH inhibits OGDC versus both 2-oxoglutarate and NAD+. Moreover, bison heart OGDC shows negative kinetic cooperativity for NAD+ and positive kinetic cooperativity for CoA at low CoA concentrations. The latter property has not been observed in earlier studies on OGDC from bovine and pig heart and other tissues of these animals.

Localization of diuretic action in microperfused rat distal tubules: Ca and Na transport

1985 ◽  
Vol 248 (4) ◽  
pp. F527-F535 ◽  
Author(s):  
L. S. Costanzo
Keyword(s):  
Distal Tubule ◽  
Maximal Concentration ◽  
Na Transport ◽  
Tubule Cell ◽  
Ca Transport ◽  
Site Of Action ◽  
Distal Tubules ◽  
Sites Of Action ◽  
Distal Site

Experiments were performed in rats to examine the distal site of action of thiazide diuretics and the additive hypocalciuric properties of thiazides and amiloride. In clearance experiments, the maximal natriuretic and hypocalciuric dose of chlorothiazide was established. When amiloride was added, there was further augmentation of Ca reabsorption (P less than 0.025) but no additional natriuresis. Amiloride blunted thiazide-induced kaliuresis (P less than 0.001). Localization of the thiazide effect was studied in early and late distal tubules microperfused in vivo with control and thiazide-containing solutions. The maximally effective luminal drug concentration, 5 X 10(-4) M, inhibited Na transport (P less than 0.001) and enhanced Ca transport (P less than 0.01) in the early distal segments; late segments were on the average unaffected. It is suggested that thiazides interact with the distal convoluted tubule cell, whose predominant location is the early distal tubule. In two long distal tubules, with early and late segments, a maximal concentration of chlorothiazide increased Ca transport and decreased Na transport. Addition of 10(-5) M amiloride caused an additional increment in Ca reabsorption. As amiloride's action is located in the late distal tubule, it is suggested from these experiments that a basis for additive hypocalciuric actions of thiazides and amiloride is separate sites of action in the distal tubule.

Impact of Labeling Herbicides by Site of Action: A University View

1999 ◽  
Vol 13 (3) ◽  
pp. 662-662 ◽  
Author(s):  
Carol Mallory-Smith
Keyword(s):  
Herbicide Resistant ◽  
Site Of Action ◽  
Private And Public ◽  
Public Sectors ◽  
Sites Of Action

One of the most consistent recommendations, from both the private and public sectors, for the prevention and management of herbicide-resistant weeds is to rotate herbicides with different sites of action. Therefore, it is imperative that those who make herbicide recommendations and those who apply herbicides know which herbicides have the same or different sites of action. An herbicide's site of action is not obvious from its trade, common, or chemical name, and it is not possible to determine from the label if it has the same site of action as another herbicide.

Simazine-Induced Nitrate Absorption Related to Plant Protein Content

Weed Science ◽  
1972 ◽  
Vol 20 (6) ◽  
pp. 569-572 ◽  
Author(s):  
S. K. Ries ◽  
V. Wert
Keyword(s):  
Hordeum Vulgare ◽  
Plant Growth ◽  
Avena Sativa ◽  
Protein Concentration ◽  
Nitrate Uptake ◽  
Kinetic Studies ◽  
Regression Analyses ◽  
Plant Weight ◽  
Companion Plants ◽  
Nutrient Solutions

Barley(Hordeum vulgareL. ‘Coho’), rye(Secale cerealeL. ‘MSU Exp.’) and oat(Avena sativaL. ‘Gary’) seedlings were grown in nutrient solutions containing 0 and 10−9to 10−7M concentrations of 2-chloro-4,6-bis(ethylamino)-s-triazine (simazine). In kinetic studies with rye and barley seedlings, simazine increased water and nitrate uptake after 3 days' exposure to 10−9M simazine compared to controls; there was no effect from 10−7M simazine. The effect was manifested in an increase in both plant weight and total protein. After 12 days, companion plants treated with 10−7M simazine were the same size as controls but contained a higher protein concentration especially in the shoots. Regression analyses showed that the simazine-induced increases in total nitrogen content of the three grain species were closely related to the nitrate and water uptake, whether manifested by greater plant growth or more protein per gram of plant.

scholarly journals Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

2011 ◽  
Vol 286 (27) ◽  
pp. 24417-24425 ◽  
Author(s):  
Chi-Yuan Chou ◽  
Liang Tong
Keyword(s):  
Binding Sites ◽  
Analytical Ultracentrifugation ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
The Other ◽  
Biotin Carboxylase ◽  
Binding Modes ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

Nitrate poisoning in cattle. 6. Tungsten (wolfram) as a prophylactic against nitrate-nitrite intoxication in ruminants.

1981 ◽  
Vol 29 (1) ◽  
pp. 37-47
Author(s):  
A. Korzeniowski ◽  
J.H. Geurink ◽  
A. Kemp
Keyword(s):  
Body Weight ◽  
Sodium Tungstate ◽  
Kinetic Studies ◽  
Potassium Nitrate ◽  
Triangular Diagram ◽  
Degree Of Protection ◽  
Nitrate Intake ◽  
High Degree ◽  
The Relationship ◽  
Nitrite Intoxication

The amount of nitrite formed in the rumen after supply of potassium nitrate was decreased considerably by tungsten, which was administered as sodium tungstate to cows orally and by rumen fistula in daily doses of up to 6.6 mg per kg body weight. This effect of tungsten depends on the molybdenum content of the fodder and can be overcome in the case of high molybdenum levels. The relationship between the nitrate intake of the animal, the dose of tungsten administered to the animal daily and the highest concentration of nitrite reached in the rumen is given as a mathematical equation as well as in the form of a triangular diagram. By means of this three-element relationship and as a result of kinetic studies on the action of tungsten, a dosage pattern of tungsten was elaborated. The effectiveness of tungsten as a protective against nitrate intoxication was proved on cows dosed repeatedly as well as with single doses of nitrate up to 500 mg NO3 per kg body weight. It is concluded finally that tungsten offers a high degree of protection against nitrate toxicity to ruminants. (Abstract retrieved from CAB Abstracts by CABI’s permission)

scholarly journals Adenosine 5′-pyrophosphate sulphurylase in baker's yeast

1972 ◽  
Vol 128 (3) ◽  
pp. 647-654 ◽  
Author(s):  
C. A. Adams ◽  
D. J. D. Nicholas
Keyword(s):  
Protein Concentration ◽  
Kinetic Studies ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
High Affinity ◽  
Heat Labile ◽  
Linear Kinetics ◽  
Inorganic Sulphate

ADP sulphurylase from baker's yeast was purified and its properties were studied. The enzyme is very heat-labile and its activity shows linear kinetics over narrow ranges of time and protein concentration. It is not activated by metals and is inhibited by thiol-reactive compounds. The enzyme, which replaces inorganic sulphate in adenosine 5′-sulphatophosphate with Pi to yield ADP, also catalyses an exchange of Pi into ADP. Kinetic studies show that the enzyme has a high affinity for adenosine 5′-sulphatophosphate, although concentrations in excess of 1.0mm are inhibitory. However, the kinetics for Pi are more complex and the enzyme is not inhibited by Pi up to 20.0mm.

scholarly journals Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1972 ◽  
Vol 126 (5) ◽  
pp. 1081-1090 ◽  
Author(s):  
S. E. Halford ◽  
M. J. Schlesinger ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Steady State ◽  
Analytical Ultracentrifugation ◽  
Kinetic Studies ◽  
Product Formation ◽  
E Coli ◽  
Enzyme Subunits ◽  
Self Association ◽  
The Stability

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

Dynamics of single polyelectrolyte chains in salt-free dilute solutions investigated by analytical ultracentrifugation

2015 ◽  
Vol 17 (24) ◽  
pp. 15896-15902 ◽  
Author(s):  
Zhonglin Cao ◽  
Sha Wu ◽  
Guangzhao Zhang
Keyword(s):  
Analytical Ultracentrifugation ◽  
Complex Dynamics ◽  
Electrostatic Repulsion ◽  
Dilute Solution ◽  
Dilute Solutions

Two concentration regimes are distinguished in polyelectrolyte salt-free dilute solution. The complex dynamics of polyelectrolytes arises due to the interchain electrostatic repulsion.

scholarly journals Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver

1989 ◽  
Vol 259 (3) ◽  
pp. 659-663 ◽  
Author(s):  
F Vanstapel ◽  
L Hammaker ◽  
K Pua ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Permeability Barrier ◽  
Membrane Bound ◽  
Marker Studies ◽  
High Degree ◽  
Regulatory Properties

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

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