The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba.
The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.
D000199:Actins / Q000302:isolation & purification / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000656:Amoeba / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D005782:Gels / Q000032:analysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010455:Peptides / Q000032:analysis; D011189:Potassium Chloride / Q000494:pharmacology; D012995:Solubility; D013053:Spectrophotometry
D000199:Actins; D005782:Gels; D010455:Peptides; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D008274:Magnesium; D002122:Calcium Chloride; D002118:Calcium