The activation of actin:DNase I complex with rat liver plasma membranes

Rohr, Gerhard; Mannherz, Hans Georg

doi:10.1016/0014-5793(79)80990-4

Cited by 28 publications

(4 citation statements)

References 18 publications

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“…in G-buffer (1) with actin and membrane protein concentra-In the previous paper (11, we have shown, in agreement with others, that liver plasm-membraneenriched fractions inactivate G-actin in respect to its ability to polymerize (2) and to inhibit DNase (2,3). This inactivation could not be attributed to actin phosphorylation by membranes as postulated by Grazi and Magi (2), since no phosphate incorporation into actin could be detected using either a radioactive or a colorimetric technique to measure protein-bound inorganic phosphate.…”

Section: Introductionsupporting

confidence: 89%

Effect of liver plasma membranes on G-actin. II. Fate of actin-bound nucleotide

Thérien¹,

Gruda²

1983

Can. J. Biochem. Cell Biol.

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G-actin incubated in presence of a liver fraction enriched in plasma membranes rapidly denatures, as evidenced by the biphasic loss of polymerizability and DNase inhibition. The inactivation is shown to result from the loss of actin-bound nucleotide induced by the rapid destruction of free nucleotides by membrane NPases. This is further supported by the observation that addition of either ATP or ADP to actin that has been exposed to membranes completely stops the denaturation process and partly restores polymerizing capacity. The biphasic aspect of inactivation is explained by the protective action of AMP and (or) adenosine formed in the course of the incubation.

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Section: Introductionsupporting

confidence: 89%

Effect of liver plasma membranes on G-actin. II. Fate of actin-bound nucleotide

Thérien¹,

Gruda²

1983

Can. J. Biochem. Cell Biol.

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“…Because buffer S provides ionic conditions that stabilize supramolecular forms of actin, we would expect that interactions between actin and accessory proteins should be preserved. Likewise, it also seems reasonable that nonsedimentable actin could be complexed with accessory proteins that could sterically inhibit the interaction between actin and DNase (32). It seems unlikely that the actin in the unbound fraction could be denatured actin, because actin treated with 0 .75 M guanidine retains its DNase-binding activity.…”

Section: Figure 10mentioning

confidence: 99%

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

Strauch¹,

Luna²,

LaFountain³

1980

The Journal of Cell Biology

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A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis, and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises^-1% of the total protein in homogenates of spermatocytes and spermatids . SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin . Under conditions that preserve native supramolecular states of actin, -80% of the spermatocyte actin is in a sedimentable form whereas only -30% of the spermatic! actin is sedimentable . These differences could be meaningful with regard to structural changes that occur during spermiogenesis . A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins . The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin .Actin has been found in a variety of nonmuscle cells (31) and, on that basis, many investigators have suggested that it may be a ubiquitous structural protein involved in several processes including cell locomotion (7,39), the maintenance of cell shape (6,35), and cell cleavage (33) . Included in the list of actincontaining cells are crane-fly gonial cells (spermatocytes, spermatids, and sperm of Nephrotoma suturalis) which have been reported by Forer and Behnke (11,12) to contain decorated microfilaments after treatment with glycerol and heavy meromyosin (19,20) . Those results raised the possibility that actin may be involved in the morphogenetic events of spermiogenesis . In addition, Forer and Behnke observed microfilaments in spindles of dividing spermatocytes (11) . That finding has been cited frequently as evidence in support of the idea that actin may play a role in the mechanism of chromosome transport (l0) .In an attempt to corroborate and extend the findings of Forer and Behnke (11, l2), we have conducted a biochemical analysis of the amount and supramolecular organization of actin in crane-fly gonial cells. Homogenates first were prepared in buffers that either stabilized actin pools in their native state or converted all cell actin to the monomeric DNase-binding THE JOURNAL OF CELL BIOLOGY " VOLUME 86 JULY 1980 315-325 © The Rockefeller University Press " 0021-9525/80/07/0315/11 $1 .00 form (3) and then were analyzed by a combination of DNase affinity chromatography and SDS polyacrylamide gel electrophoresis (PAGE) . This approach takes advantage of the specific interaction between actin and DNase I (27) and is especially applicable to cell types that contain limited amounts of actin . 43,000-dalton polypeptides that were isolated with DNase were characterized further by two-dimensional peptide mapping techniques .We have confirmed...

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“…Deoxyribonuclease I was measured by the hyperchromicity assay (Blickstad et al, 1978). The effect of 5'-nucleotidase on actin was determined by a modification of the method of Rohr & Mannherz (1979). Actin (50 #g/ml) was incubated with or without 5'-nucleotidase, in 100 1l of buffer (50 mm-Tris/HCl buffer, pH 7.5, containing 1 mM-CaCl2, 1 mM-MgSO4 and 0.0500 sulphobetaine 14).…”

Section: Protein Labelling In Primary Cultured Hepatocytesmentioning

confidence: 99%

The membrane topography of ecto-5′-nucleotidase in rat hepatocytes

Baron¹,

Pope²,

Luzio³

1986

Biochemical Journal

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The transmembrane topography of the rat hepatocyte ectoenzyme 5'-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5'-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5'-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5'-nucleotidase and actin, although the ability of preparations of 5'-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface.

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The activation of actin:DNase I complex with rat liver plasma membranes

Cited by 28 publications

References 18 publications

Effect of liver plasma membranes on G-actin. II. Fate of actin-bound nucleotide

Effect of liver plasma membranes on G-actin. II. Fate of actin-bound nucleotide

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

The membrane topography of ecto-5′-nucleotidase in rat hepatocytes

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