Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors.

Abstract

We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay . We find that the reaction has at least two steps, requires 0.5 mM ATP and 1 .5 MM MgC12, and is inhibited by micromolar Ca" . The optimum pH is 7.0 and temperature, 25'-30'C . The rate ofthe reaction is increased by cold preincubation with both MgC12 and ATP . Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either oftwo purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca" . This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca" that regulate the gelation process .The soluble proteins extracted from a variety of cells in the cold form a solid gel when warmed to room temperature under physiological conditions (5,12,13,18,19,20,25,27,28,31) . This so-called gelation reaction has attracted considerable attention, because analysis of it promises to reveal how cells regulate the consistency of their cytoplasm. It is already clear that gelation requires polymerization of actin (12,20) and the cross-linking of the actin filaments by accessory proteins (2,3,15,25,30). These cross-linking proteins seem to differ from cell to cell.One goal of research in this area is to explain the gelation phenomenon at the molecular level . This will require the purification of all of the essential components of the gelation system and characterization of their interactions . To prove that all of the essential structural and regulatory components of the gel have been identified, one must show that the purified components account quantitatively for the gelation properties of the crude extract . To make this comparison, it is necessary to define in some detail the properties of the gelation reaction in the crude extract .In the present study we use a low-shear falling ball viscometer to describe in detail how environmental conditions influence the rate and extent of the gelation reaction in crude Acanthamoeba extracts . We confirm previous qualitative evidence (18, 19) that gelation of Acanthamoeba extract requires Mg`and ATP, is reversed by cold, and is inhibited by cytochalasin B . New findings include evidence for an Mg"-and ATP-requiring "potentiation reaction" that precedes gelation and for inhibition of gelation by micromolar Ca" . Furthermore, we confirm the finding of Maruta and Korn (15) that the extract can be fractionated into a number of low molecular weight compo-J . CELL BIOLOGY

The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin.

“…Low shear rate apparent viscosity was measured with a miniature falling ball device (Maclean-Fletcher and Pollard, 1980) at 25°C. Quantitative physical measurements were made with an R18 Weissenberg rheogoniometer (Sangamo Controls, Ltd., Bognor Regis, Sussex, England) in the small amplitude, forced oscillation mode as described by Sato et al .…”

Light‐ and nucleotide‐dependent increase in apparent viscosity in a suspension of retinal disks

“…For the performance of the dark experiments all procedures were performed with aid of infrared image converters. For a discussion of the falling ball method for measuring the viscosity of nonNewtonian fluids see [12].…”

“…Viscosity measurements were made with the falling ball method [12]. Either fully bleached, native bovine membranes or dark-adapted toad membranes reconstituted with bovine peripheral enzymes (G protein and PDE) were used.…”

Developmental Biology of Neoplastic Growth

“…Viscosity reflects polymer length but is affected by gelation, and orientation of the filaments, which decreases the sensitivity of measurement. Although having these faults, viscometry using Ostwald's viscometer or the falling ball apparatus is a good choice for just checking the effect of toxins on actin polymerization since these instruments are simple and easy to use (MacLean-Fletcher and Pollard 1980). Light scattering can be also used for estimating the size of aggregates, by measuring actin solution turbidity.…”