scholarly journals Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

1982 ◽  
Vol 95 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M S Mooseker ◽  
T D Pollard ◽  
K A Wharton
Keyword(s):  
Ionic Strength ◽  
Intestinal Epithelial ◽  
Capping Protein ◽  
The Core ◽  
Nucleated Polymerization ◽  
Myosin Subfragment ◽  
Brush Borders ◽  
Pointed End ◽  
Low Ionic Strength

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

scholarly journals Characterization and localization of myosin in the brush border of intestinal epithelial cells

1978 ◽  
Vol 79 (2) ◽  
pp. 444-453 ◽  
Author(s):  
MS Mooseker ◽  
TD Pollard
Keyword(s):  
Ionic Strength ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Gel Filtration ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders ◽  
Low Ionic Strength

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro.

1988 ◽  
Vol 107 (5) ◽  
pp. 1793-1797 ◽  
Author(s):  
W S Sale ◽  
L A Fox
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Glass Surface ◽  
Functional Assay ◽  
Atpase Subunits ◽  
Low Ionic Strength ◽  
Intermediate Chains ◽  
Sea Urchin Sperm

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.

scholarly journals Accessibility of epitopes on fibrin clots and fibrinogen gels

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1469-1475 ◽  
Author(s):  
R Procyk ◽  
B Kudryk ◽  
S Callender ◽  
B Blomback
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Trials ◽  
Ionic Strength ◽  
Factor Xiii ◽  
Imaging Agent ◽  
Low Ionic Strength ◽  
Clot Structure ◽  
Radiolabeled Antibodies ◽  
Fibrin Clots

Abstract Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

Contractile proteins from the tomato

1980 ◽  
Vol 58 (7) ◽  
pp. 797-801 ◽  
Author(s):  
Maryanne Vahey ◽  
Stylianos P. Scordilis
Keyword(s):  
Ionic Strength ◽  
Ethylenediaminetetraacetic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Sds Page ◽  
Myosin Subfragment 1 ◽  
Parenchymal Cells ◽  
Low Ionic Strength

Proteins exhibiting all of the basic structural and biochemical characteristics of actin and myosin have been isolated from the parenchymal cells of the fruit of the tomato, Lycopersicon esculentum. Crude cytoplasmic extracts of these cells contain filaments that can be decorated by rabbit skeletal muscle myosin subfragment-1 (S-1). Polymerized tomato actin activates the Mg2+–ATPase of both skeletal and tomato myosin at physiological ionic strength. Tomato actin comigrates with skeletal actin on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) indicating an apparent molecular weight of 45 000. High ionic strength extracts of tomato contain a myosin whose ATPase activity in 0.5 M KCl is maximal in the presence of K+-ethylenediaminetetraacetic acid (K+-EDTA) and is inhibited by Mg2+. Tomato myosin interacts with skeletal F-actin to form an actomyosin complex that can be dissociated by ATP. At low ionic strength the Mg2+–ATPase of the myosin can be activated by actin.

The assembly of ribbon-shaped structures in low ionic strength extracts obtained from vertebrate smooth muscle

1973 ◽  
Vol 265 (867) ◽  
pp. 203-212 ◽  
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Diffraction Analysis ◽  
Average Diameter ◽  
Smooth Muscle Myosin ◽  
Optical Diffraction ◽  
The Core ◽  
Regular Arrangement ◽  
Low Ionic Strength ◽  
Muscle Myosin

In actomyosin extracts from smooth muscle obtained at low ionic strength, an assembly of protein into long ribbon-shaped elements is observed to take place. These ribbons which range up to about 100 nm in width and up to many micrometres in length exhibit a strong repeat period of about 5.6 nm. Optical diffraction analysis shows that they possess a long repeat of 39.1 nm ± 0.4 nm. Tropomyosin purified from vertebrate smooth muscle can be induced to form the same ribbon-shaped elements. On removal of salt from solution the ribbons dissociate into fine filaments of average diameter about 8 nm which show subfilaments of about 2 to 3 nm diameter. In crude preparations the ribbons occur in solution together with myosin. If such preparations are left to stand for several days, ribbons may be found that show a visible 14 nm period which appears to arise from the presence of a regular arrangement of projections. Smooth muscle myosin alone assembles into cylindrical filaments which exhibit a regular arrangement of projections along their entire length, indicating an absence of polarity. These results indicate, as have those recently obtained from section material, that the myosin-containing component of vertebrate smooth muscle contains a protein that forms the core of the filament, which is responsible for its ribbon-like shape and which probably determines the polarity of the attached myosin molecules. It is proposed that this protein is tropomyosin.

scholarly journals Affinity chromatography of immobilized actin and myosin

1975 ◽  
Vol 149 (2) ◽  
pp. 365-379 ◽  
Author(s):  
R C Bottomley ◽  
I P Trayer
Keyword(s):  
Ionic Strength ◽  
Salt Concentration ◽  
Free Solution ◽  
Heavy Meromyosin ◽  
Chemical Modifications ◽  
Low Concentrations ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
One Step ◽  
Low Ionic Strength

Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.

scholarly journals STUDIES ON THE ORGANIZATION OF THE BRUSH BORDER IN INTESTINAL EPITHELIAL CELLS

1965 ◽  
Vol 26 (3) ◽  
pp. 693-706 ◽  
Author(s):  
Jane Overton ◽  
Alexander Eichholz ◽  
Robert K. Crane
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Preceding Paper ◽  
Enzymic Activity ◽  
Negative Staining ◽  
Morphological Identification ◽  
Intestinal Epithelial ◽  
The Core ◽  
Brush Borders

Two of the fractions obtained by density gradient centrifugation of Tris-disrupted brush borders from hamster intestinal mucosa have been identified as the microvillus cores and their surrounding membranous coats, respectively. This identification has the following morphological basis. In shadowed preparations one fraction (cores) appears as rounded, compact rods, and the other fraction (coats) appears as flattened sheets. Both rods and sheets have dimensions appropriate to the identities assigned to them. In addition, negative staining shows that the rods are composed of aligned particles of roughly 60 A, consistent with the appearance of the core in tissue section, where 60-A fibrils are characteristic. The sheets are covered by non-aligned particles of approximately the same size. Sectioned preparations show that the core fraction contains predominantly fibrous material with some membranous contamination and that the coat fraction is apparently composed exclusively of elongated sacs with a unit membrane structure. Some details of the structure of the core are evident in cases where the compact rod appears to be loosened, revealing a doubled strand. The strand is approximately 350 A wide; the compact rod is roughly twice this width. With negative staining the strand shows a dense central region. The morphological identification presented here is consistent with the distribution of enzymic activity among the density gradient fractions described in the preceding paper.

DIFFERENTIAL STABILITY OF IODINE POOR AND IODINE RICH RAT THYROGLOBULIN

1974 ◽  
Vol 75 (1) ◽  
pp. 33-49 ◽  
Author(s):  
Lubomir J. Valenta
Keyword(s):  
Ionic Strength ◽  
Succinic Anhydride ◽  
Iodine Concentration ◽  
Freezing And Thawing ◽  
Sedimentation Pattern ◽  
Differential Stability ◽  
Chaotropic Anions ◽  
Low Ionic Strength ◽  
Covalent Interactions

ABSTRACT In certain environments thyroglobulin is dissociated into 12S subunit or unfolded into 14–17S species. The unfolding and dissociating ability of various agents was examined on poorly iodinated (PIT) or iodine rich (IRT) rat thyroglobulins. According to the efficiency of provoking unfolding or dissociation these agents could be grouped in the following order: low ionic strength < chaotropic anions < chaotropic anions plus freezing and thawing < succinic anhydride and mild alkali < SDS < strong alkali. The degree of thyroglobulin changes was further dependent on the iodine concentration. No change of sedimentation pattern was obtained in the presence of 2-mercaptoethanol or after incomplete reduction and alkylation of the iodine rich protein. No stabilization of PIT was brought about by a number of oxidizing agents. Stabilization was only obtained by in vitro iodination. The main difference between the uneffected and unfolded or dissociated thyroglobulin species was in the amount of thyroxine, or, at lower levels of iodination, DIT. The results suggest that non-covalent, especially hydrophobic, rather than covalent interactions are involved in stabilization of conformation of the thyroglobulin molecule.

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