Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry

Pollender, Jean-Marc; Gruda, Julian

doi:10.1139/o79-006

Cited by 8 publications

(4 citation statements)

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“…While the interaction of phalloidin and F-actin does produce an ultraviolet difference spectrum, it was not possible to detect a conformational change in F-actin-phalloidin mixtures by the method of susceptibility to proteolytic digestion because such mixtures do not show a measurable rate of digestion. This lack of digestbility of phalloidin-stabilized actin, compared with the measurable rate of digestion of F-actin in the absence of phalloidin (Figure 4; Rich & Estes, 1976), has been also reported by other investigators (deVries & using different enzymes (Pollender & Gruda, 1979). These findings could be evidence that a conformational change occurs in actin upon binding phalloidin.…”

Section: Discussionsupporting

confidence: 84%

Mechanism of action of phalloidin on the polymerization of muscle actin

Estes¹,

Selden²,

Gershman³

1981

Biochemistry

215

137

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Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.

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Section: Discussionsupporting

confidence: 84%

Mechanism of action of phalloidin on the polymerization of muscle actin

Estes¹,

Selden²,

Gershman³

1981

Biochemistry

215

137

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“…It is well known that proteins most susceptible to in vitro proteolysis have short in vivo half-lives [34]. Thus, the experiments of Pollender and Gruda [37] offer strong supportive evidence to ours that the major mechanism by which phalloidin induces increases in liver actin content in vivo is by inhibiting actin degradation.…”

Section: Discussionsupporting

confidence: 60%

“…Pollender and Gruda [37] have shown that phalloidin protects actin against trypsin-or pronase-induced proteolysis in vitro. It is well known that proteins most susceptible to in vitro proteolysis have short in vivo half-lives [34].…”

Section: Discussionmentioning

confidence: 99%

Effect of phalloidin on liver actin distribution, content, and turnover

Low

Chaponnier

et al. 1982

J of Cellular Biochemistry

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Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Planimetric analyses of SDS-polyacrylamide gels showed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 micrograms/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dualisotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.

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“…In the polymer of intact actin, these sites, all located in subdomain 2 of the monomer [2][3][4], are protected. Proteolysis by subtilisin and trypsin was, however, observed at high enzyme concentrations [31,32]. It seemed to be of particular interest to investigate the effects of removal of the C-terminal residues on the accessibility of the cleavage sites within the surface loop 39-51 in F-actin, since, according to the atomic model of the actin filament [1], this loop is directly involved in the inter-monomer interaction with the C-terminal phenylalanine.…”

Section: Polymerization Properties Of Intact and C-terminal Truncatedmentioning

confidence: 99%

Long-range conformational effects of proteolytic removal of the last three residues of actin

Strzelecka‐Gołaszewska

Mossakowska

Wozniak

et al. 1995

Biochemical Journal

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Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.

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Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry

Cited by 8 publications

References 0 publications

Mechanism of action of phalloidin on the polymerization of muscle actin

Mechanism of action of phalloidin on the polymerization of muscle actin

Effect of phalloidin on liver actin distribution, content, and turnover

Long-range conformational effects of proteolytic removal of the last three residues of actin

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