In vitro investigation of the geometric contraction behavior of chemo-mechanical P-protein aggregates (forisomes).
We investigated the contracting behavior of forisomes from Vicia faba by carrying out precise measurements of their changing geometric parameters in vitro in the absence and in the presence of dissolved oxygen. Furthermore, we investigated the fine structure of forisomes by scanning electron microscopy. For the first time, single forisomes were titrated with Ca(2+), protons, and hydroxide ions recording the complete progression of their contractions. An apparent Ca(2+)-binding constant of (22+/-3) muM was calculated from two complete titration curves. The forisomes also contracted in the presence of Ba(2+) and Sr(2+) ions, but the amplitudes of contraction were smaller under the same measuring conditions. The time taken to change from the longitudinally expanded into the longitudinally contracted state was up to 2 s shorter in 10 mM Ca(2+) in comparison to 0.2mM Ca(2+). However, the contraction time was prolonged by decreasing the Ca(2+) concentration. In the absence of dissolved oxygen, the transition between the two final states of the forisomes was almost reversible and the amplitude of contraction remained almost constant during the first 25 contraction cycles. In the presence of dissolved oxygen the forisomes denaturated after a few cycles and lost their ability to contract, just after only a few cycles with 10 min in the contracted state. Denaturation of the forisomes occurred appreciably in the contracted state. We propose a cycle process to explain the thermodynamic basis of the Ca(2+)-induced contraction and its reversal by EDTA. Reducing the pH-value from 7.3 to 4.0 caused the forisomes to shorten by approximately 15%, while increasing the pH to 11.0 caused them to shorten by 28 to 30%. In both cases, the increases of the forisomes volume were greater than during the Ca(2+) induced contraction. The pH values of 4.7+/-0.3, and 10.2+/-0.2 marked the inflection points of the acid base titration of different forisomes.
Schwan
S
S
Fraunhofer Institute of Mechanics of the Materials, Heideallee 19, D-06120 Halle, Germany.
Fritzsche
M
M
Cismak
A
A
Heilmann
A
A
Spohn
U
U
D001696:Biomechanical Phenomena; D002118:Calcium / Q000494:pharmacology; D003285:Contractile Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008855:Microscopy, Electron, Scanning; D046912:Multiprotein Complexes; D010100:Oxygen; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D031307:Vicia faba
D003285:Contractile Proteins; D046912:Multiprotein Complexes; D010940:Plant Proteins; D010100:Oxygen; D002118:Calcium