scholarly journals The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation

1993 ◽  
Vol 290 (2) ◽  
pp. 583-590 ◽  
Author(s):  
P V Attwood ◽  
W Johannssen ◽  
A Chapman-Smith ◽  
J C Wallace
Keyword(s):  
Rate Constants ◽  
Pyruvate Carboxylase ◽  
Electron Microscopic Observation ◽  
Enzyme Concentration ◽  
Enzymic Activity ◽  
Chicken Liver ◽  
Electron Microscopic ◽  
Acetyl Coa ◽  
Degree Of Dissociation ◽  
Tetrameric Structure

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

scholarly journals Inactivation of chicken liver pyruvate carboxylase by 1,10-phenanthroline

1988 ◽  
Vol 252 (2) ◽  
pp. 501-507 ◽  
Author(s):  
J A Carver ◽  
G S Baldwin ◽  
D B Keech ◽  
R Bais ◽  
J C Wallace
Keyword(s):  
Pyruvate Carboxylase ◽  
Chelating Agent ◽  
Order Rate Constant ◽  
Enzyme Concentration ◽  
Chicken Liver ◽  
First Order ◽  
Tetrameric Structure ◽  
Inactivation Mechanism ◽  
Phenanthroline Complex ◽  
Exchange Activity

Inactivation of chicken liver pyruvate carboxylase by the chelating agent 1,10-phenanthroline follows pseudo-first-order kinetics. The hyperbolic dependence of the apparent first-order rate constant on 1,10-phenanthroline concentration is consistent with a two-step inactivation mechanism, in which 1,10-phenanthroline binds firstly to the enzyme, and secondly to the enzyme-bound Mn(II) ion. Binding of 1,10-phenanthroline to pyruvate carboxylase results in complete loss of ATP/Pi exchange activity, but only a 61% decrease in pyruvate/oxaloacetate exchange activity. The rate of inactivation is greater at low enzyme concentrations, implying that binding of 1,10-phenanthroline to monomers and dimers is preferred relative to that of tetramers. Furthermore, in the presence of acetyl-CoA, which stabilizes the tetrameric structure, no dependence of inactivation on enzyme concentration is observed. As monitored by gel-permeation liquid chromatography, formation of the enzyme-Mn(II)-phenanthroline complex results in loss of the tetrameric structure of the enzyme. From atomic-absorption measurements, inactivation by 1,10-phenanthroline also causes some loss of Mn(II) from the enzyme. It is concluded that the Mn(II) atom does not participate directly in the reaction mechanism, but may play a structural role essential to the integrity of the enzyme's tetrameric structure.

scholarly journals Distribution and degradation of biotin-containing carboxylases in human cell lines

1985 ◽  
Vol 232 (2) ◽  
pp. 385-393 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Cell Lines ◽  
Rate Constants ◽  
Pyruvate Carboxylase ◽  
Degradation Rate ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Degradation Rates

Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.

scholarly journals The carboxybiotin complex of chicken liver pyruvate carboxylase. A kinetic analysis of the effects of acetyl-CoA, Mg2+ ions and temperature on its stability and on its reaction with 2-oxobutyrate

1986 ◽  
Vol 235 (2) ◽  
pp. 359-364 ◽  
Author(s):  
P V Attwood ◽  
J C Wallace
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Protein Conformation ◽  
Pyruvate Carboxylase ◽  
Activation Parameters ◽  
Slow Phase ◽  
Acetyl Coa ◽  
First Order ◽  
Thermodynamic Activation Parameters ◽  
Pseudo First Order

The enzyme-[14C]carboxybiotin complex of chicken liver pyruvate carboxylase has been isolated and shown to be relatively stable, with a half-life at 0 degree C of 342 min. The kinetic properties of the decay of this complex, in both the presence and the absence of the substrate analogue, 2-oxobutyrate, have been examined. The data for the reaction with 2-oxobutyrate at 0 degree C fitted a biphasic exponential decay curve, enabling the calculation of rate constants for both the fast and slow phases of the reaction at this temperature. The effect of temperature on the observed pseudo-first-order rate constant for the slow phase of the reaction with 2-oxobutyrate, and that for the decay of the enzyme-[14C]carboxybiotin complex alone, have been examined. Arrhenius plots of these data revealed that the processes being studied in each type of experiment were single reactions represented by one rate constant in each case. For the decay of the enzyme-[14C]carboxybiotin complex in the absence of 2-oxobutyrate, the rate-determining process may be the movement of carboxybiotin from the site of the first partial reaction to the site of the second. The calculated thermodynamic activation parameters indicate that this reaction is accompanied by a large change in protein conformation. With 2-oxobutyrate present, the observed process in the slow phase of the reaction was probably the dissociation of the carboxybiotin from the first subsite. Here, the activation parameters suggest that a much smaller change in protein conformation accompanies this reaction. Both sets of experiments were also performed in the presence of acetyl-CoA, but this activator had little effect on the measured thermodynamic activation parameters. However, in both cases the observed pseudo-first-order rate constants in the presence of acetyl-CoA were about 75% of those in its absence. The effects of Mg2+ on the reaction kinetics of the enzyme-[14C]carboxybiotin complex with 2-oxobutyrate were similar to those observed with the sheep enzyme by Goodall, Baldwin, Wallace & Keech [(1981) Biochem. J. 199, 603-609].

scholarly journals Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

1979 ◽  
Vol 177 (2) ◽  
pp. 697-705 ◽  
Author(s):  
Karen S. McGurk ◽  
H. Olin Spivey
Keyword(s):  
Secondary Structure ◽  
Pyruvate Carboxylase ◽  
Conformational Transitions ◽  
Sulphonic Acid ◽  
Chicken Liver ◽  
Stopped Flow ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Fluorescence Measurements ◽  
Secondary Structure Composition

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO3 and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10–13%; ATP, 21–24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO3 had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the ε-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499–3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% β-pleated sheet and 66% other structural types.

Electron Microscopic Observation of the Longitudinal Organization of Poly (p-Phenylene Terephthalamide) Fibers

1982 ◽  
Vol 40 ◽  
pp. 680-681
Author(s):  
Li Li-Sheng ◽  
L.F. Allard ◽  
W.C. Bigelow
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Scanning Electron Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Aromatic Polyamides ◽  
Radial Arrangement ◽  
Scanning Electron ◽  
Better Than

The aromatic polyamides form a class of fibers having mechanical properties which are much better than those of aliphatic polyamides. Currently, the accepted morphology of these fibers as proposed by M.G. Dobb, et al. is a radial arrangement of pleated sheets, with the plane of the pleats parallel to the axis of the fiber. We have recently obtained evidence which supports a different morphology of this type of fiber, using ultramicrotomy and ion-thinning techniques to prepare specimens for transmission and scanning electron microscopy.

Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 608-609
Author(s):  
Neng-Bo He ◽  
S.W. Hui
Keyword(s):  
Lipid Bilayers ◽  
Erythrocyte Membrane ◽  
Lipid Membranes ◽  
Surface Film ◽  
Biological Membranes ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Black Lipid Membranes ◽  
Fine Mesh ◽  
Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

The Scanning Electron Microscopic Observation of the Vestibular Sensory Epithelia

1969 ◽  
Vol 27 ◽  
pp. 40-41
Author(s):  
D.J. Lim ◽  
W.C. Lane
Keyword(s):  
Semicircular Canals ◽  
Electron Microscopic Observation ◽  
Gravitational Acceleration ◽  
Electron Microscopic ◽  
Sensory Epithelia ◽  
Scanning Electron Microscopic ◽  
Vestibular Sensory Epithelia ◽  
And Function ◽  
Sand Piles ◽  
Active Investigation

The morphology and function of the vestibular sensory organs has been extensively studied during the last decade with the advent of electron microscopy and electrophysiology. The opening of the space age also accelerated active investigation in this area, since this organ is responsible for the sensation of balance and of linear, angular and gravitational acceleration.The vestibular sense organs are formed by the saccule, utricle and three ampullae of the semicircular canals. The maculae (sacculi and utriculi) have otolithic membranes on the top of the sensory epithelia. The otolithic membrane is formed by a layer of thick gelatin and sand-piles of calcium carbonate crystals (Fig.l).

Plastic deformation of θ ' in Al-4%Cu alloy

1978 ◽  
Vol 36 (1) ◽  
pp. 576-577
Author(s):  
Shrikant P. Bhat
Keyword(s):  
Deformation Behavior ◽  
Room Temperature ◽  
Electron Microscopic Observation ◽  
Bulk Alloy ◽  
Electron Microscopic ◽  
Cu Alloy ◽  
Cu Alloys ◽  
Orowan Mechanism ◽  
The Subject ◽  
Cyclic Straining

deformation behavior of Al-Cu alloys aged to contain θ ' has been the subject of many investigations (e.g., Ref. 1-5). Since θ ' is strong and hard, dislocations bypass θ ' plates (Orowan mechanism) at low strains. However, at high strains the partially coherent θ ' plates are probably sheared, although the mechanism is complex, depending on the form of deformation. Particularly, the cyclic straining of the bulk alloy is known to produce gross bends and twists of θ '. However, no detailed investigation of the deformation of θ ' has yet been reported; moreover, Calabrese and Laird interpreted the deformation of θ ' as largely being elastic.During an investigation of high temperature cyclic deformation, the detailed electron-microscopic observation revealed that, under reversed straining conditions, θ ' particles are severely distorted--bent and twisted depending on the local matrix constraint. A typical electronmicrograph, showing the twist is shown in Fig. 1. In order to establish whether the deformation is elastic or plastic, a sample from a specimen cycled at room temperature was heated inside the microscope and the results are presented in a series of micrographs (Fig. 2a-e).

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