An actin depolymerizing protein from pig plasma

Harris, Harriet E.; Gooch, John

doi:10.1016/0014-5793(81)80017-8

Cited by 61 publications

(41 citation statements)

References 7 publications

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“…The presence of multiple gelsolin bands in electrophoresis under native conditions is not unexpected, because gelsolin is resolved into 3 components by isoelectric focussing in urea . The markedly different mobility of the mutant gelsolin at pH 8.6 cannot be explained by the loss of a single negative charge in a protein with an isoelectric point about 6.2 [19,20,22], containing 98 acidic and 99 basic residues. The mobility difference most likely reflects the electrophoretic behaviour of the proteolytically cleaved mutant gelsolin.…”

Section: Discussionmentioning

confidence: 92%

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

et al. 1993

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Familial amyloidosis, Finnish type is caused by a single base mutation in gelsolin, an actin filament severing and capping protein that is present in most tissues and in blood plasma. The mutation replaces aspartic acid with asparagine at residue 187 of the plasma sequence. This renders the gelsolin susceptible to proteolysis as a consequence of which amyloid protein is formed. Here it is shown that the mutant protein in plasma from a patient homozygous for this mutation lacks both actin severing and nucleating activities. Evidence is presented that the cleaved mutant gelsolin has dissociated under non‐denaturing conditions and that the resultant 65,000 and 55,000 M r C‐terminal fragments aggregate.

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Section: Discussionmentioning

confidence: 92%

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

et al. 1993

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“…However, it was not separated from immunoglobulins. Gelsolin binds to DEAE-cellulose at pH 8-8.5, but the extent of binding varied considerably [7]. Further experiments were carried out with Whatman DE-52 at p H 8 to try to understand this variability.…”

Section: Purijica T Ionmentioning

confidence: 99%

Preparation and characterization of pig plasma and platelet gelsolins

Weeds

Gooch

Pope

et al. 1986

European Journal of Biochemistry

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Pig plasma gelsolin has been prepared by a revised method involving poly(ethy1ene glycol) precipitation, chromatography on CM-cellulose and affinity chromatography on actin-Sepharose. Pig platelet gelsolin has been prepared by chromatography on DEAE-cellulose and actin-Sepharose. Partial chemical and proteolytic cleavage shows that the two proteins are closely related in their fragmentation patterns. The amino acid sequences are identical at the N-terminus of the platelet protein, but the plasma protein has an additional nine residues on the N-terminal side of the common sequence. Calcium binding studies show that the plasma protein has similar calcium binding properties to both macrophage and platelet gelsolins.Gelsolin was first isolated from cytoplasmic extracts of rabbit macrophages [l] and shown to be a calcium-dependent regulator of actin filament length [2]. An 'actin depolymerizing factor' was identified in blood plasma at about the same time [3 -51 and it is now clear that related proteins are present in plasma and in a variety of tissues [6]. Because blood plasma contains concentrations of gelsolin similar to those in cells, relatively large amounts of the protein can be obtained most readily from this source [5].In our earlier reports, gelsolin (formerly called 'actin depolymerizing factor') was prepared from pig plasma or serum by ammonium sulphate fractionation and ion-exchange chromatography [5, 71. It did not bind to DEAE-cellulose at pH 6, under conditions in which albumin was retained, but bound strongly to CM-cellulose at this pH and was largely separated from immunoglobulins G on elution with a salt gradient. Hydroxyapatite was used in the final stages of purification to remove residual immunoglobulins and other minor contaminants.A major problem with this lengthy procedure was proteolysis of the gelsolin even though inhibitors including diisopropyl fluorophosphate were present in the buffers. T h s proteolysis was most marked in the final stages of purification. A revised purification was described by Harris and Weeds [8] in which an initial precipitation by poly(ethy1ene glycol) was followed by two carboxymethyl-cellulose columns. Protein was applied to the first of these, a short column, in 50 mM NaC1. Although only a small amount of the protein bound, this included all the gelsolin, which was eluted with 0.35 M NaCl and, after dialysis, applied to a longer column for further fractionation using a salt gradient at a lower flow rate. The revised procedure resulted in improved yields and somewhat reduced proteolysis. Here we describe further modifications to the purification to improve yield, purity and speed by replacing hydroxyapatite with actin affinity columns. The preparation is greater than 95% pure and shows negligible proteolysis. Pig plasma gelsolin has been characterized in comparison to the related intracellular protein prepared from pig platelets. The chemical similarity between these two proteins is confirmed by partial enzyme cleavage and amino acid sequence analysis of their N...

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“…Furthermore, the shortening of F-actin by ADF was reported to be a Ca2+-dependent reaction [I]. A recent report by Harris and Gooch [13] described ADF as a 92000-daltons polypeptide with . In contrast to earlier findings, they supposed that the depolymerization of actin filaments by ADF was a Ca2+-independent reaction involving formation of stoichiometric 1 : 1 complexes with G-actin.…”

Section: Proteins Capable Of Dividing Actin Filaments Have Been Demonmentioning

confidence: 99%

“…The protein concentration was determined according to Lowry et al [22] with bovine serum albumin as a standard. In some experiments ADF was purified from human serum as described by Harris and Gooch [13].…”

Section: Pur$ication Of Adfmentioning

confidence: 99%

Further Characterization of the Ca²⁺‐Dependent F‐Actin‐Depolymerizing Protein of Human Serum

Thorstensson¹,

Utter²,

Norberg³

1982

European Journal of Biochemistry

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The F-actin-depolymerizing factor (ADF) of human serum was purified and identified as a 93 000-dakons protein. The concentration of ADF was calculated to 120-150 pg/ml in four normal sera and about half as much in three sera from leukemia patients treated with cytostatic drugs. In the presence of CaZC ADF shortened actin filaments into fragments, the size of which was correlated to the actin: ADF molar ratio, as judged by electron microscopy. The severing of F-actin was not necessarily followed by an increase in the quantities of monomeric actin, as determined by a DNase I inhibition assay and a sedimentation assay. The findings indicated that ADF shortens filamentous actin by breaking bonds between adjacent actin molecules thereby forming stable ADF-actin complexes, without a monomeric net release. The effect of ADF on F-actin was rapid and was reversed upon chelation of Ca2+. ADF cross-reacted immunologically and exhibited similarity in reaction mechanism with gelsolin, the Ca2+-dependent F-actin-severing protein from macrophages. This implies that the proteins are both structurally and functionally related. The physiological role of ADF may be to handle actin released at cell destruction, probably by forming ADF-G-actin 1 : 1 complexes thereby preventing formation of actin filaments.

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An actin depolymerizing protein from pig plasma

Cited by 61 publications

References 7 publications

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

Preparation and characterization of pig plasma and platelet gelsolins

Further Characterization of the Ca²⁺‐Dependent F‐Actin‐Depolymerizing Protein of Human Serum

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An actin depolymerizing protein from pig plasma

Cited by 61 publications

References 7 publications

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

Preparation and characterization of pig plasma and platelet gelsolins

Further Characterization of the Ca2+‐Dependent F‐Actin‐Depolymerizing Protein of Human Serum

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Further Characterization of the Ca²⁺‐Dependent F‐Actin‐Depolymerizing Protein of Human Serum