EUROPEAN  TISSUE  REPAIR  SOCIETY

2. Molecular Structure-Function Relationship

In general the functionality of biomacromolecules, particularly for proteins, relies on the spatial arrangement of chemically reactive side-groups. Particularly for enzymes, molecular activity is governed by the conformational state. Induction of reversible changes in the 3-D molecular structure that are induced through reversible changes in primary structure (i.e., phosphorylation) is a typical strategy for controlling intracellular enzyme activity.

At physiological temperatures, macromolecules in solution normally assume many different conformations over time; most of them are irrelevant to the functionality of the molecule. The molecular free energy difference between an active conformation and an inactive one is usually quite small, typically ranging between 5-10 kcal/mol. (Tsong, 1999) Alteration of molecular conformation beyond physiological range is denaturation, which usually results from exposure to a large physicochemical stress. (Lee, 1996)

Molecular structure is also critical for the function of large supramolecular assemblies like the self-organized lipid bilayer membranes.(Arnold, 1990) The most basic functionality of the cell's membranes is to restrict and regulated transmembrane ionic transport. Ionic compartmen-talization, as permitted by the cell membrane, is essential for maintenance of cell viability. The energy required to move solvated ions across a pure phospholipid bilayers in an aqueous environment approaches 80-100 kBT, indicative of the strong impediment to passive ion-diffusion across the lipid bilayer. (Parsegian, 1969) However, cell membranes are typically 20-30% protein, which renders the cell membrane approximately 106 times more conductive to ions than the pure lipid bilayer. (Powell, 1986) Structural integrity of cell membrane is essential for making possible the transmembrane physiological ionic concentration gradients at a metabolic energy cost that is affordable. Despite the effectiveness of the membrane barrier, approximately 40-85% of the metabolic energy expended by cells is used to maintain normal transmembrane ion gradients. Cellular wounds involving disruption of the membrane structure quickly leads to cell necrosis.

3. Mechanisms of Cellular Repair

Healing of cellular wounds occur by several distinct molecular repair and replacement mechanisms. Molecular chaperones are continually active in cells folding and refolding proteins. Following cell injury, the production of a certain subset of these proteins, the heat-shock proteins, is unregulated for the purpose of limiting damage, stabilizing membranes and refolding proteins. GroEL and GroES are heat shock proteins that assist with denatured protein refolding by combining to form an ATP utilizing complex which refolds proteins.

Sealing of permeabilized cell membranes is an important naturally occurring process in many cell types. Fusigenic proteins, natural cellular macromolecules that induce membrane sealing, are active under normal conditions to seal porated cell membranes following exocytosis, egg fertilization, etc. They act to create a low energy pathway for flow of phospholipid across the defect or to induce fusion of transport vesicle to plasma membranes. Like chaperones, biological systems have evolved macromo-lecular structures that are designed to repair membrane after severe damage. For example, antifreeze proteins, found in many animals but particularly in cold water fish, absorb to cell membranes and are thought to induce sealing after membrane disruption. (Rubinsky et al., 1992)

The action of these fusigenic proteins indicates that it is possible to seal damaged cell membranes by interactions with macromolecular templates. Serum macromolecules are known to prevent lysis of liposomal bilayer systems. (Lelkes and Friedmann, 1984) This has also been accomplished using synthetic surfactants, such as poloxamer 188 at sub-CMC concentration of 0.1 mM to seal cells against loss of calcein dye post-electroporation. (Lee et al., 1992) Other have reported similar observations following heat mediated cell membrane poration. (Merchant et al.,1998)