Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Vleurick, Lieve; Kühn, Eduard; Decuypere, Eddy; Veldhoven, Paul P. Van

doi:10.1002/(sici)1097-4644(19990301)72:3<349::aid-jcb4>3.0.co;2-h

Cited by 8 publications

(3 citation statements)

References 25 publications

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“…Plasma membranes were prepared from a low speed pellet (400 ϫ g) by flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient as described previously (51). This plasma membrane fraction has been positively identified by the presence of marker enzymatic activity (alkaline phosphodiesterase, EC 3.1.4.1) detected at pH 9 with sodium thymidine 5-monophoshate pnitrophenyl ester substrate (51). It also negatively tested for lysosomal enzyme ␤-hexosaminidase A activity (52).…”

Section: Methodsmentioning

confidence: 99%

Lysosomal Sialidase (Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex That Facilitates Elastic Fiber Assembly

Hinek

Pshezhetsky

Itzstein

et al. 2006

Journal of Biological Chemistry

149

140

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We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of ␤-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surfacetargeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.Mature elastic fibers and laminae present in the extracellular matrix of connective tissues of different organs and blood vessel walls are complex structures made of polymeric (insoluble) elastin in which polypeptide chains of tropoelastin are covalently cross-linked and assembled on a scaffold of 12-nm microfibrils consisting of several glycoproteins, e.g. fibrillins and microfibril-associated glycoproteins (1-11). Molecules of monomeric 70-kDa tropoelastin are synthesized by fibroblasts, chondrocytes, and smooth muscle cells and are secreted and properly aligned with one another for the subsequent lysyl oxidase-mediated cross-linking (12). The mechanisms governing tropoelastin secretion and assembly are not fully elucidated. The current data indicate that highly hydrophobic and non-glycosylated tropoelastin associates with several intracellular proteins that chaperone it through the intracellular compartments. Davis et al. (13) documented that, in the ergastoplasmic reticulum, tropoelastin binds to BiP and the peptidylprolyl cis,transisomerase FKPB65. Our immunohistochemical and biochemical studies have indicated that tropoelastin present in endosomal and Golgi compartments is escorted by the 67-kDa elastin-binding protein (EBP) 2 (14, 15), which protects tropoelastin from premature intracellular selfaggregation and an association with serine proteinases (16,17). We have established that, after delivering tropoelastin to...

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Section: Methodsmentioning

confidence: 99%

Lysosomal Sialidase (Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex That Facilitates Elastic Fiber Assembly

Hinek

Pshezhetsky

Itzstein

et al. 2006

Journal of Biological Chemistry

149

140

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“…Microsomal and Golgi fractions of chicken (White Leghorn) tissues were prepared as described previously (Bergeron et al 1973;Vleurick et al 1999) with some modifications. Briefly, 1 g of tissue was homogenized in 9 ml of homogenizing buffer (0.25 M sucrose and 5 mM Tris-HCl, pH 7.5).…”

Section: Preparation Of Microsomal and Golgi Fractionsmentioning

confidence: 99%

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

et al. 2011

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As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

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“…Γ s is the cell membrane, or the fluid-solid interface. Since the density of cell membrane is 1.18 g/ml [71], the density of fluid is assumed to be 1 g/ml, we take ρ r = 1.18 in our simulation.…”

Section: Dimensionless Governing Equations For Newtonian Fluid and Cementioning

confidence: 99%

A fictitious domain method with a hybrid cell model for simulating motion of cells in fluid flow

Hao

et al. 2015

Journal of Computational Physics

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In this study, we develop a hybrid model to represent membranes of biological cells and use the distributed-Lagrange-multiplier/fictitious-domain (DLM/FD) formulation for simulating the fluid/cell interactions. The hybrid model representing the cellular structure consists of a continuum representation of the lipid bilayer, from which the bending force is calculated through energetic variational approach, a discrete cytoskeleton model utilizing the worm-like chain to represent network filament, and area/volume constraints. For our computational scheme, a formally second-order accurate fractional step scheme is employed to decouple the entire system into three sub-systems: a fluid problem, a solid problem and a Lagrange multiplier problem. The flow problem is solved by the projection method; the solid problem based on the cell model is solved by a combination of level set method, ENO reconstruction, and the Newton method; and the Lagrange multiplier problem is solved by immerse boundary interpolation. The incompressibility of the material is implemented with the penalty function method. Numerical results compare favorably with previously reported numerical and experimental results, and show that our method is suited to the simulation of the cell motion in flow.

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Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Cited by 8 publications

References 25 publications

Lysosomal Sialidase (Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex That Facilitates Elastic Fiber Assembly

Lysosomal Sialidase (Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex That Facilitates Elastic Fiber Assembly

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

A fictitious domain method with a hybrid cell model for simulating motion of cells in fluid flow

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