scholarly journals A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Jia Wei ◽  
Yixiao Zhang ◽  
Tai-Yuan Yu ◽  
Kianoush Sadre-Bazzaz ◽  
Michael J Rudolph ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Potent Inhibitor ◽  
Acetyl Coa Carboxylase ◽  
Single Chain ◽  
Electron Microscopic ◽  
Acetyl Coa ◽  
Microscopic Studies ◽  
And Function

Abstract Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3–AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

scholarly journals Coenzyme A is a potent inhibitor of acetyl-CoA carboxylase from rat epididymal fat-pads

1992 ◽  
Vol 283 (1) ◽  
pp. 35-38 ◽  
Author(s):  
S K Moule ◽  
N J Edgell ◽  
A C Borthwick ◽  
R M Denton
Keyword(s):  
Potent Inhibitor ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetyl Coa Carboxylase ◽  
Inhibitory Effects ◽  
Fat Pad ◽  
Maximal Inhibition ◽  
Acetyl Coa ◽  
Epididymal Fat ◽  
Fat Pads

Rat epididymal fat-pad extracts have previously been shown to contain an insulin-stimulated acetyl-CoA carboxylase kinase, which is co-eluted from Mono Q ion-exchange chromatography with a potent inhibitor of acetyl-CoA carboxylase [Borthwick, Edgell & Denton (1990) Biochem. J. 270, 795-801]. A variety of tests, including reactivity with thiol reagents, identify this inhibitor as CoA. Inhibition requires the presence of MgATP, but is independent of any phosphorylation of the enzyme. The effect is complete in about 5 min and is associated with depolymerization of acetyl-CoA carboxylase. Half-maximal inhibition is observed at about 40 nM-CoA. The inhibitory effects of CoA can be partially reversed by incubation with citrate and more fully overcome by treatment of the enzyme with the insulin-stimulated acetyl-CoA carboxylase kinase.

Electron microscopic analysis of the microtubular structure of HEp-2 cells

1990 ◽  
Vol 48 (3) ◽  
pp. 470-471
Author(s):  
Miklós S.Z. Kellermayer ◽  
Tamás Henics ◽  
György Szücs ◽  
Gerald H. Pollack
Keyword(s):  
Microscopic Analysis ◽  
Unknown Origin ◽  
Tubular Structure ◽  
Minimal Essential Medium ◽  
Electron Microscopic ◽  
Brij 58 ◽  
Cacodylate Buffer ◽  
Microscopic Studies ◽  
And Function ◽  
Hank’S Solution

The HEp-2 cell line was first established by Moore, et al. and today it serves as a widely used subject for a large variety of microbiological and cell-biological experiments. A tubular structure of unknown origin and function has been described first in virally infected, and later in uninfected HEp-2 cells. However, this tubular structure has not been further analyzed. We performed transmission (TEM) and whole cell mount electron microscopic studies of monolayer HEp-2 cells to morphologically describe the structure and relate it to other organelles of the cell.For embedding, HEp-2 cells were grown on glass coverslips in Eagle's minimal essential medium. 24 hours after passage, three types of procedures were carried out: (1) The cells were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate buffer. (2) The cells were treated for 10 min. with Hank's solution containing 0.2% Brij-58 and 2.5% glutaraldehyde. Fixation was completed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate. (3) Cells were treated with 0.2% Brij-58 in Hank’s solution for 10 min. Subsequently, they were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate.

scholarly journals Erratum: A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

2017 ◽  
Vol 3 (1) ◽  
Author(s):  
Jia Wei ◽  
Yixiao Zhang ◽  
Tai-Yuan Yu ◽  
Kianoush Sadre-Bazzaz ◽  
Michael J Rudolph ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

The Microenvironment of Neurons

1970 ◽  
Vol 28 ◽  
pp. 136-137
Author(s):  
William Bondareff
Keyword(s):  
Electron Micrographs ◽  
Extracellular Space ◽  
Brain Volume ◽  
Freeze Substitution ◽  
Electron Microscopic ◽  
Morphological Studies ◽  
Microscopic Studies ◽  
Distribution Composition ◽  
And Function ◽  
The Brain

Neurons in the central nervous system are separated by extracellular spaces, the distribution, composition and function of which are not unequivocally known. Earlier electron microscopic studies of chemically-fixed tissues demonstrated extracellular spaces composed of uniformly narrow, apparently empty channels constituting 3-5% of the brain volume. The results of more recent morphological and non-morphological studies support the existence of less uniform intercellular channels, varying in dimension from 100 Å to almost 1 μ, and constituting 20-25% of the brain volume. Although this space is not revealed in electron micrographs of chemicallyfixed nervous tissues, it is demonstrated readily in specimens fixed by freeze-drying or freeze-substitution (Fig. 1). The dynamic nature of those extracellular spaces, as visualized in electron micrographs of nervous tissue fixed by freeze-substitution, was demonstrated in studies of normalbrain maturation. An extracellular space of 40% became gradually smaller as development proceeded to reach the smaller extracellular space characteristic of mature animals.

scholarly journals Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action

1990 ◽  
Vol 270 (3) ◽  
pp. 795-801 ◽  
Author(s):  
A C Borthwick ◽  
N J Edgell ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Insulin Action ◽  
Kinase Activity ◽  
Potent Inhibitor ◽  
Fold Increase ◽  
Epididymal Adipose Tissue ◽  
Acetyl Coa Carboxylase ◽  
Serine Kinase ◽  
Acetyl Coa ◽  
Carboxylase Activity

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide (‘I’-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.

scholarly journals In meso crystallogenesis. Compatibility of the lipid cubic phase with the synthetic digitonin analogue, glyco-diosgenin

2020 ◽  
Vol 53 (2) ◽  
pp. 530-535 ◽  
Author(s):  
Leendert van Dalsen ◽  
Dietmar Weichert ◽  
Martin Caffrey
Keyword(s):  
Membrane Protein ◽  
Synthetic Surfactant ◽  
Phase Method ◽  
Cubic Phase ◽  
Electron Microscopic ◽  
Protein Preparation ◽  
Microscopic Studies ◽  
Pre Treatment ◽  
And Function ◽  
Integral Proteins

Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the in meso or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the in meso method for crystallogenesis.

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