AN EXPERIMENT ON PARTICIPATION OF Ca IONS IN MAINTAINING MEMBRANE STRUCTURE AND FUNCTION BY MEANS OF TRYPSINIZATION

Abstract

1) Washout experiments using ⁴⁵Ca and ²²Na ions in the sartorius muscles proved that ⁴⁵Ca ion dissociation from the surface membrane was suppressed by trypsinization. This phenomenon may be occurred by an attractive binding of mobile Ca⁺⁺ ions to newly developed or exposed anionic sites following conformational change of membrane protein. The conformational change, probably from helical to coil structure, did not tell us the detailed mechanism to produce the change in ion content of the muscle fibres or in membrane potential level by trypsin with an exception of a term of permeability increase. In addition, this change facilitated exchange of monovalent cations. Presumably, modification of the effect of trypsin on ⁴⁵Ca ion movement and secondarily on ²²Na efflux when combined with iodoacetate was a result of summation of Ca⁺⁺ ion association with anionic sites produced by trypsin and with those produced by protein disorganization by iodoacetate, not by glycolysis disturbance. The Ca⁺⁺ ions were already existing or were made free during rearrangement of protein structure. 2) ²²Na efflux was increased by trypsin. Correlation of ⁴⁵Ca ion association to ²²Na efflux increase is paradoxical, however, the results are reasonable if interpreted by independence principle. This type of increase in ²²Na efflux was conditionally different from that by Ca free Ringer. It was also passive in nature because of the appearance even in K free Ringer. However, when active ²²Na transport was completely inhibited, there remained only a small part of ²²Na efflux which was trypsin-sensitive.3) In this experiment, special attention was not paied to concrete geometrical organization of chemical components. The results of spontaneous twitching in sucrose suggested that the change after trypsin application was favorable for leaking out of internally ionized Ca⁺⁺ ions. Trypsin produced stabilization of Ca⁺⁺ ions on the membrane surface but caused labilization or ionization of calcium from the intracellular tubular systems. The stabilization is probably due to an attraction of mobile Ca⁺⁺ ions to newly developed anionic sites on carboxyl groups following trypsinization. 4) The Ca⁺⁺ ions of which kinetics is altered by trypsin are not always contribute to chemical integrity of the membrane in the resting state. Ca⁺⁺ ion association by trypsin is purposeful whenviewed from proteolytic turnover process in the membrane. The result described may be suggestive of one phase of protein turnover taking place physiologically in the membrane. All the results were explained in terms of an increase of both anionic sites on protein molecules and the number of free Ca⁺⁺ ions after trypsinization.