During freezing inactivation

Figure 3. Inactivation of thylakoids during freezing at various low temperatures as a function of time. Washed thylakoids were suspended in a solution containing 50 mM sucrose as a cryoprotectant and 20 mM sodium phenylpyru-vate as a cryotoxic solute. The suspensions were rapidly frozen and thawed. After thawing, photophosphorylation was determined. For experimental conditions, see notes in legend for Fig. 2...

Figure 4. The effect of different alkali metal chlorides on thylakoid function during freezing to —20°C. Washed thylakoids were suspended before freezing in a solution containing 0.1 M sucrose and alkali metal chlorides, as indicated. The sucrose served as cryoprotectant and was added to prevent freeze-inactivation of the membranes in the presence of low salt concentrations. The suspensions were slowly frozen for 3 hours at —20°C. After thawing in a water bath at room temperature, the activity of cyclic photophosphorylation was measured.

In contrast to these salts, sodium succinate increases thylakoid damage during freezing if present as the only solute. As will be discussed later, the situation becomes more complicated if significant concentrations of other solutes are also present with sodium succinate during freezing. Membrane inactivation by high concentrations of sodium succinate occurs even at 0°C (21). 

Salts of weak organic acids that are soluble in lipids are also injurious to thylakoids. Examples are the salts of phenylpyruvic add (Figure 3) and caprylic acid (58). These salts, even if present at very low concentrations, cause extensive membrane inactivation during freezing, if cryo-protectants are absent. At 0°C, moderate concentrations of these salts will slowly inactivate thylakoids. 

Amino Acids. As is true of organic acids, amino acids can either prevent inactivation of thylakoids by freezing or they can aggravate the situation. Some of them, for instance glycine, serine, glutamate, or aspartate, promote injury if present as the only major solutes during freezing. However, the same amino acids can be protective if certain other solutes are also present (58). The reason for this behavior, which is also observed with succinate, will be considered later. 

Proline, threonine, or y-aminobutyric acid can protect thylakoids against inactivation during freezing. Amino acids with apolar side chains such as phenylalanine, leucine, or valine always contribute to thylakoid inactivation during freezing. 

Protein Release in Relation to Loss of Membrane Function. When thylakoids are frozen in the presence of sucrose, membrane function is preserved. If a cryotoxic salt such as NaCl is also present, retention of membrane functionality during freezing depends on the ratio of sucrose to salt (5). Loss of cyclic photophosphorylation is the most sensitive parameter of membrane inactivation. Photophosphorylation is largely lost before significant protein release from the membranes can be detected (Figure 8). Since photophosphorylation requires membranes with un-... 

Little is known concerning mechanisms by which these proteins prevent inactivation of thylakoids during freezing, but they somehow contribute to membrane stabilization. They act with some specificity, since cryoprotective proteins from spinach not only fail to protect red blood cells during freezing but are actually injurious. 

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