scholarly journals Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.

1988 ◽  
Vol 107 (6) ◽  
pp. 2679-2688 ◽  
Author(s):  
W L Dentler
Keyword(s):  
Membrane Proteins ◽  
Atpase Activity ◽  
Tetrahymena Thermophila ◽  
High Molecular Mass ◽  
Integral Membrane Proteins ◽  
Matrix Proteins ◽  
Membrane Atpase ◽  
The Matrix ◽  
Triton X

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.

Comparative Isolation of Cilia and Flagella from the Lamellibranch Mollusc, Aequipecten irradians

1973 ◽  
Vol 12 (2) ◽  
pp. 345-367
Author(s):  
R. W. LINCK
Keyword(s):  
Atpase Activity ◽  
Cross Sections ◽  
Basal Body ◽  
Electron Dense Material ◽  
The Matrix ◽  
Fine Structures ◽  
Dynein Arms ◽  
Cilia And Flagella ◽  
Triton X ◽  
Ciliary Ultrastructure

Gill cilia and sperm flagella from the lamellibranch mollusc Aequipecten irradians were compared with respect to their ultrastructures and adenosinetriphosphatase activities. Cilia were isolated from excised gills using 3 different solutions: twice-concentrated seawater, 10 % ethanol-10 mM CaCl2 and 60% glycerol. In each case deciliation occurs by the severance of the cilium at the junction of the transition zone and the basal body, and in each case the ciliary ultrastructure is maintained. Sperm flagella were purified by mechanical decapitation. Cilia and sperm flagella have similar fine structures, except that the matrix of the cilia contains substantially more electron-dense material than that of flagella. The ATPase activity of purified cilia is approximately 0.09,µmol P1/min/mg protein; that of flagella is 0.13. Ciliary and flagellar axonemes were prepared by repeated extraction of the membranes with 1% Triton X-100. Ciliary axonemes maintain their 9 + 2 cylindrical orientation, whereas flagellar axonemes often appear as opened or fragmented arrays of the 9 + 2 structure, due to the partial breakdown of the flagellar nexin fibres. A-subfibre arms which were obvious in whole organelles are rarely seen in axoneme preparations. Again the ciliary matrix is considerably more amorphous than in flagellar axonemes. The ATPase activities of ciliary and flagellar axonemes are 0.13 and 0.12 µmol P1/min/mg protein respectively; however, activities of ciliary axonemes may vary by a factor of 2, depending on the method of isolation. The difficulty in observing A-subfibre arms in cross-sections of ciliary and flagellar axonemes is discussed in terms of random, non-reinforcing arrangements of the dynein arms.

Evidence for a second class of membrane glycoprotein involved in cell adhesion

1989 ◽  
Vol 93 (4) ◽  
pp. 631-640
Author(s):  
W.E. Norris
Keyword(s):  
Extracellular Matrix ◽  
Biological Activity ◽  
Cell Adhesion ◽  
Membrane Proteins ◽  
Soluble Fraction ◽  
Biological Activities ◽  
Integral Membrane Proteins ◽  
Membrane Glycoprotein ◽  
Transmembrane Receptors ◽  
Triton X

It is believed that transmembrane relationships exist between the cytoskeleton and the extracellular matrix through integral membrane proteins, almost certainly glycoproteins, which would act as transmembrane receptors. Such receptors would include those involved in cell adhesion. I have been able to isolate a detergent-soluble fraction from chick embryo fibroblasts that is enriched in these integral membrane proteins by making use of their amphipathic character to phase-separate them in the detergent Triton X-114. Antisera raised to this fraction had biological activities interfering with cell adhesion and motility. A 45 X 10(3) Mr glycoprotein unique to this fraction appears to be responsible for this biological activity and is a candidate for a transmembrane receptor involved in cell adhesion.

scholarly journals Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Dario Mizrachi ◽  
Yujie Chen ◽  
Jiayan Liu ◽  
Hwei-Ming Peng ◽  
Ailong Ke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Water Soluble ◽  
Integral Membrane Proteins ◽  
Protein Fusion ◽  
Fusion Strategy

scholarly journals Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

2021 ◽  
Vol 5 (1) ◽  
pp. e202101162
Author(s):  
Yuta Endo ◽  
Yuko Shimizu ◽  
Hanako Nishikawa ◽  
Katsuhiro Sawasato ◽  
Ken-ichi Nishiyama
Keyword(s):  
Membrane Proteins ◽  
Molecular Basis ◽  
Terminal Region ◽  
Integral Membrane Proteins ◽  
The Other ◽  
Other Hand ◽  
Model Protein

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

scholarly journals Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation.

1987 ◽  
Vol 105 (1) ◽  
pp. 215-227 ◽  
Author(s):  
L Yuan ◽  
J G Barriocanal ◽  
J S Bonifacino ◽  
I V Sandoval
Keyword(s):  
Monoclonal Antibodies ◽  
Sialic Acid ◽  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Chemical Characteristics ◽  
Dibutyryl Cyclic Amp ◽  
Complex Type ◽  
Golgi System ◽  
Different Parts

The localization and chemical characteristics of two Golgi integral membrane proteins (GIMPs) have been studied using monoclonal antibodies. The two proteins are segregated in different parts of the Golgi system and whereas GIMPc(130 kD) is located in the cis and medial cisternae, GIMPt (100 kD) is confined in the trans-most cisterna and trans-tubular network. Both GIMPs are glycoproteins that contain N- and O-linked carbohydrates. The N-linked carbohydrates were exclusively of the complex type. Although excluded from the trans-side of the Golgi system, where sialylation is believed to occur, GIMPc acquires sialic acid in both its N- and O-linked carbohydrates. Sialic acid was also detected in the N-linked carbohydrates of GIMPt. GIMPc is apparently phosphorylated in the luminal domain in vivo. Phosphorylation occurred exclusively on serine and was stimulated by dibutyryl cyclic AMP. GIMPc and GIMPt displayed half-lives of 20 and 9 h, respectively.

scholarly journals Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.

1986 ◽  
Vol 103 (6) ◽  
pp. 2389-2402 ◽  
Author(s):  
W A Muller ◽  
M A Gimbrone
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Umbilical Vein ◽  
Integral Membrane Proteins ◽  
Cellular Polarity ◽  
Differential Distribution ◽  
Endothelial Monolayers ◽  
Apical Side

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane-associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.

Proteins From Oyster Shell: Biomineralization Regulators and Commercial Polymer Analogs

1999 ◽  
Vol 599 ◽  
Author(s):  
A. P. Wheeler ◽  
C. S. Sikes
Keyword(s):  
Crystal Growth ◽  
Aspartic Acid ◽  
Large Scale ◽  
Matrix Protein ◽  
Gene Families ◽  
Eastern Oyster ◽  
Matrix Proteins ◽  
The Matrix

AbstractMolluscan shell is a composite made up of μm-sized CaCO3 crystals and an organic phase (matrix). This report outlines our studies on the structure and activities of matrix proteins isolated from the inner calcite layer of shell of the Eastern oyster, including their cellular origin and structure and their relationship to the crystalline mineral phase. In addition, we present results of the synthesis and commercialization of polypeptide polymers which are based on the structure and activities of the oyster proteins. Extracted shell proteins are polyanionic and range in size from relatively small soluble forms to those which are crosslinked and insoluble. The soluble forms are capable of adsorbing to calcite in vitro and in the process changing its growth habit and acting as threshold growth inhibitors. Their function in vivo is not understood, but they may serve to control shell crystal morphology. The insoluble protein forms gels readily and may serve to provide resiliency to the shell and, from in vitro and in situ observations, appears to serve as a site for nucleation of crystals. However, from studies in vitro, these gels do not lower the energy of activation for nucleation, as previously expected. Matrix protein aggregates are identifiable by AFM on the surface of crystals, but as such do not serve as nucleation sites for new crystal growth. If the aggregates are removed, then ectopic crystal growth proceeds readily revealing orientation of the underlying crystals. All the matrix proteins contain domains rich in aspartic acid, are heavily phosphorylated, crossreact in antibody studies and may belong to a limited number of gene families with individuals modified post-synthesis. The proteins are made by a specialized group of cells located primarily some distance from the growing edge of the shell and appear to be assembled into sheets soon after secretion. Soluble anti-scalants and crosslinked insoluble water absorbents have been developed based on the structure and activity of the matrix proteins. These are primarily poly(aspartates) which can be made in large scale via thermal polycondensation of aspartic acid. The soluble forms are commercially used as biodegradable water treatment chemicals among other applications.

scholarly journals Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?

2001 ◽  
Vol 114 (1) ◽  
pp. 9-19 ◽  
Author(s):  
C.J. Hutchison ◽  
M. Alvarez-Reyes ◽  
O.A. Vaughan
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Dna Sequences ◽  
Genetic Disorders ◽  
Integral Membrane Proteins ◽  
Partial Lipodystrophy ◽  
Nuclear Lamins ◽  
Disease Phenotypes

The nuclear lamina is a filamentous structure composed of lamins that supports the inner nuclear membrane. Several integral membrane proteins including emerin, LBR, LAP1 and LAP2 bind to nuclear lamins in vitro and can influence lamin function and dynamics in vivo. Results from various studies suggest that lamins function in DNA replication and nuclear envelope assembly and determine the size and shape of the nuclear envelope. In addition, lamins also bind chromatin and certain DNA sequences, and might influence chromosome position. Recent evidence has revealed that mutations in A-type lamins give rise to a range of rare, but dominant, genetic disorders, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction-system disease and Dunnigan-type familial partial lipodystrophy. An examination of how lamins A/C, emerin and other integral membrane proteins interact at the INM provides the basis for a novel model for how mutations that promote disease phenotypes are likely to influence these interactions and therefore cause cellular pathology through a combination of weakness of the lamina or altered gene expression.

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