Stabilization and inactivation of biological membranes during freezing in the presence of amino acids

Abstract

As a contribution to the understanding of frost tolerance of cells and organisms the effect of amino acids on thylakoid membranes during freezing was investigated. Freezing inactivates photophosphorylation of thylakoids by irreversibly altering essential membrane properties. Washed thylakoids, frozen in the presence of some amino acids such as proline, threonine, γ-aminobutyric acid or lysine·HCl, were protected against inactivation by freezing. Membrane-toxic compounds such as inorganic salts reduced the protection. Other amino acids such as glycine, alanine, serine, hydroxyproline, sodium or potassium aspartate or glutamate were unable to prevent the alteration of washed membranes by freezing. In fact, membranes protected by sucrose or by cryoprotective amino acids became inactivated during freezing when an excess of these amino acids was also present. Although individually unable to provide protection, in certain combinations with one another these amino acids became protective. Even in combination of amino acid with a membrane-toxic inorganic salt such as NaCl was protection observed. For effective protection a suitable ratio between membrane-toxic compound and amino acid had to be maintained. Departure from this ratio to either side resulted in inactivation. A third group of amino acids, among them phenylalanine, tyrosine, valine, leucine, isoleucine, methionine and arginine·HCl, did not prevent freeze inactivation of thylakoid membranes either in the absence or in the presence of inorganic salts. Membranes protected against inactivation by a cryoprotectant such as sucrose became inactivated during freezing if one of these amino acids was also present. Membrane inactivation during freezing is due to the accumulation in the unfrozen part of the system of potentially membrane-toxic compounds such as inorganic salts or amino acids possessing apolar side chains, and in some instances perhaps also to eutectic solidification of the complete system. Protective compounds protect during freezing partly by colligative action, i.e. by their unspecific ability to reduce the concentration of toxic solutes below the limit of toxicity. Further, specific interaction between cryoprotectants and membranes plays an important role in membrane preservation during freezing. The results indicate that the accumulation of potentially toxic cell components, such as inorganic but also organic cell compounds, which become concentrated up to toxic levels during extracellular freezing, are the cause of injury during freezing of frost-sensitive cells. In frost-hardy cells, protection is provided by compounds that reduce non-specifically the concentration of toxic substances or that protect specifically by membrane stabilization.