Freezing protein release from membranes

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-... 

The original experiments on PDI activity were carried out with microsomal membranes, suggesting the localization of this protein in the endoplasmic reticulum. This has since been conhrmed both by subcellular localization studies (Lambert and Freedman, 1985) and by immunochemical methods (Koch, 1987). However, these studies revealed that PDI does not behave like a typical ER membrane protein. Indeed, for some time there was a question as to whether there was more than one form of PDI due to the presence of this protein in the cytosol. This has now been discounted by studies on the latency of PDI that show that if microsomes are prepared carefully then the activity is entirely latent (Lambert and Freedman, 1985). Thus, although PDI exists as an ER protein, it is easily released from microsomes by freeze thawing, mild alkaline treatment, detergents, or sonication, suggesting that PDI is either a soluble protein or is only loosely associated with the ER membrane. 

Protein Release. Biomembranes consist of lipids and proteins. The latter may be subdivided into so-called intrinsic and extrinsic proteins (49). Intrinsic proteins supposedly are integrated into the membrane phase primarily by the hydrophobic interaction with lipids. Extrinsic proteins are attached to the membranes. Ionic interactions are believed to be important in the binding of extrinsic proteins. When these proteins dissociate from the membrane, they may be sufficiently hydrophilic to be soluble in the aqueous phase. When freeze-aggregated thylakoids are sedimented, a number of membrane proteins are found in the supernatant fluid. Among them are catalytic proteins involved in energy conservation and electron transport (42,48). The total amount of proteins released depends on freezing conditions and the solute environment, but may be as much as 5% of the total membrane protein (48). When frozen in the presence of a cryoprotective solute, at a sufficient concentration, thylakoids remain functional and do not release proteins in significant amounts. Protein release thus accompanies membrane injury and, in fact, is an indication of such injury. 

Figure 7. Electrophoretic patterns of proteins which are released from thyla-koids during freezing or exposure to 0°C in the presence of various solutes. The solute concentration before freezing is indicated under the gels. Freezing time was 3 hours at —25°C. After thawing, supernatant fluids from membranes were treated with sodium dodecylsulfate (SD) and mercaptoethanol then subjected to gel electrophoresis. Phe is sodium phenylpyruvate, Cap is sodium caprylate, lie is isoleucine. From Volger, Heber, and Berzborn (48).

The importance of muscle lysosomal enzymes to the food scientist stems from their apparent involvement in the aging of meats. For example, lysosomal cathepsins are possibly involved in the proteolytic degradation of muscle proteins (31). Lysosomal enzymes exhibit latency that is, they are retained in the lysosomal particle and released only when the particle membrane is damaged. In this regard the lysosomal enzymes are liberated and activated when the particle membranes are weakened by the postmortem drop in pH. Also, lysosomes are very subject to cryoinjury (72), and freezing and thawing of tissues such as muscle releases lysosomal enzymes resulting in autolysis. 

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