Diffusion of protease inhibitors in the muscle cell
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Date
2003-05-12
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Abstract
Proteases often exist in fish muscle, arising from parasites, gut leaching, or merely normal physiology. Their activity may cause undesired softening of the meat during storage or slow cooking. Several food-grade inhibitors exist which can overcome this softening if properly delivered to the intracellular sites where proteases are located. Translational diffusion of enzyme inhibitor proteins through different subcellular compartments and intracellular architectures in the muscle cell is thus a mandatory step to effectively block the activity of such proteases.
The analyses of both macromolecule spatial distribution and fluorescence recovery after photobleaching (FRAP) by laser scanning confocal microscopy (LSCM) were used to measure the translational diffusion of fluorescein isothiocyanate (FITC)-labeled protease inhibitors into intact muscle fibers of halibut. Diffusion coefficients (D) of α-2 macroglobulin (750KDa), soy bean trypsin inhibitor (21 KDa) and cystatin (12 KDa) were measured in both muscle fibers and dilute aqueous solutions. On the time scale of the observation (35min), cystatin and soy bean trypsin inhibitor diffused through the cell membrane (sarcolemma) and sarcoplasm, but considerably more slowly (>10-fold) than in dilute aqueous solution. A hypothesis/review paper is presented which supports the theory that structuring of water within cells is a primary cause of reduced diffusivity of proteins into and within cells. α-2 macroglobulin did not diffuse at all into muscle cells within the time frame of the experiment, but did completely penetrate the cell after overnight exposure.
The present study thus shows a clear dependence of D on protein inhibitor size in intact skeletal muscle fibers. Low molecular weight protease inhibitors like cystatin can therefore be effectively diffused into intact fish muscle cells to minimize proteolytic activity and meat softening.
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Keywords
protease inhibitors, diffusion, cystatin, soybean trypsin inhibitor, macroglobulin, muscle fiber, protein, FRAP, confocal
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Degree
PhD
Discipline
Food Science
