Actin polymerization accompanies thy‐1‐capping on mouse thymocytes

Laub, Felix; Kaplan, Miriam R.; Gitler, Carlos

doi:10.1016/0014-5793(81)80048-8

Cited by 39 publications

(23 citation statements)

References 19 publications

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“…The possible involvement of ealmodulin, through, e.g., myosin light chain kinase activation, cannot be assessed in these experiments, since several aspects of cytoskeletal function (e.g., monomerpolymer steady-states [17,18])may be controlled by nucleotide levels.…”

Section: Resultsmentioning

confidence: 99%

Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

1982

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Section: Resultsmentioning

confidence: 99%

Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

1982

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“…Standard actin inhibition curves were generated utilizing actin (Sigma Chemical Co., St. Louis, MO) that was depolymerized in the dissociation buffer just before its use. Only values falling between 25 and 75% inhibition of the DNase I activity were used for quantitation (16)(17)(18).…”

Section: Methodsmentioning

confidence: 99%

Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.

1991

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Establishment and maintenance of a polar distribution of Na+,K+-ATPase is essential for efficient Na+ reabsorption by proximal tubule cells and is dependent upon the formation of a metabolically stable, detergent-insoluble complex of Na+,K+-ATPase with the actin membrane cytoskeleton. The present studies show that cellular ATP depletion results in a rapid duration-dependent dissociation of Na+,K+-ATPase from the actin cytoskeleton and redistribution of Na+,K+-ATPase to the apical membrane. During ATP depletion, total cellular Na+,K+-ATPase activity was unaltered, but the Triton-X-100-insoluble fraction (cytoskeleton associated) of Na+,K+-ATPase activity decreased (P < 0.01), with a corresponding increase in the detergent-soluble fraction of Na+,K+-ATPase (P < 0.01). Indirect immunofluorescent studies of cells with depleted ATP revealed a redistribution of Na+,K+-ATPase from the basolateral membrane into the apical membrane and throughout the cytoplasm. ATP depletion also resulted in the redistribution of F-actin from a primarily cortical concentration to a perinuclear location. There was also a rapid, duration-dependent conversion of monomeric G-actin to F-actin starting during the first 5 min of ATP depletion. Taken together, these data suggest that ATP depletion causes profound alterations in cell polarity by inducing major changes in the actin cytoskeletal architecture. (J. Clin. Invest. 1991.

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“…To optimize this procedure, we found two assays, a DNase I inhibition assay (14) and a gel scanning assay (3), that could measure relative yields of actin in crude root homogenates and in DNase I-agarose eluates, respectively. These allowed us to identify factors that affect the yield of actin isolated from pea roots by DNase I affinity chromatography.…”

mentioning

confidence: 99%

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography

Andersland¹,

Jagendorf²,

Parthasarathy³

1992

Plant Physiol.

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Native actin can be isolated from pea (Pisum sativum L.) roots by DNase I affinity chromatography, but the resulting yields and quality of actin are variable. By use of two assays for actin, a DNase I inhibition assay and a gel scanning assay, we identified several factors that increased actin yield. ATP is required for the actin in crude pea root extracts to bind to immobilized DNase I. We attempted to isolate actin from pea (Pisum sativum L.) roots with DNase I affinity chromatography, but our yields were variable and the purified actin was not always able to polymerize. To optimize this procedure, we found two assays, a DNase I inhibition assay (14) and a gel scanning assay (3), that could measure relative yields of actin in crude root homogenates and in DNase I-agarose eluates, respectively. These allowed us to identify factors that affect the yield of actin isolated from pea roots by DNase I affinity chromatography. MATERIALS AND METHODSPea plants (Pisum sativum L. cv Progress #9, Agway, Syracuse, NY) were grown in vermiculite in growth chambers (240C day, 180C night, 12-h day). Roots were cut from 5-to 8-d-old seedlings (secondary roots less than 2 cm long), washed free of vermiculite, blotted dry on paper towels, and weighed. All other procedures involving pea extracts or proteins were performed at 40C or on ice, unless otherwise specified.Stock solutions of Grade II Na2ATP (Sigma Chemical Co.), EDTA, and EGTA were adjusted to pH 7 to 7.5 with NaOH. Na2ATP concentrations were adjusted using a millimolar extinction coefficient of E259 = 15.4 mm-1 cm-'. A vanadate stock solution, prepared from V205 as described by Gallagher and Leonard (6), was a gift of Margherita De Biasi (Cornell University). Pyrophosphate stock solutions were prepared from the tetrasodium salt and adjusted to pH 8.0 with HCl. Formamide (non-ACS grade), soluble PVP (mol wt 40,000), polymerized calf thymus DNA (Type I), and prestained SDS mol wt standards (catalog #SDS-7B) were obtained from Sigma. DNase I, grade II, was from Boehringer (Indianapolis, IN). CNBr-activated Sepharose Cl-4B (Pharmacia, Piscataway, NJ) was prepared by the method of Kohn and Wilchek (9) and coupled to DNase I following the procedure of Lazarides and Lindberg (15). The DNase I-agarose was washed before each use with 40% (v/v) formamide in 25 mm Tris HCl, pH 7.5, and 5 mm CaCl2 for 20 min to remove traces of pancreatic or pea actin previously bound to the DNase I. The DNase I-agarose bound between 300 and 500 ,gg of muscle actin/mL, depending upon the batch and its age. DNase I-agarose was stored in 5 mm Tris HCl, pH 7.5, 0.5 mm CaCl2, and 0.02% NaN3.Muscle actin for controls and standards was isolated from rabbit back muscle by the method of Pardee and Spudich (25). The concentration of muscle G-actin and DNase I solutions were determined from their absorbances using extiuc-1716 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

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Actin polymerization accompanies thy‐1‐capping on mouse thymocytes

Cited by 39 publications

References 19 publications

Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography

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