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Bioen 599 F, Autumn 2000 Bioengineering Principles of Physiology |
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Lecture Material and Notes Week 6, Lecture 16: Cytoplasmic
water as a solvent Lecture theme and outline: · Statement of the problem: this issue is fundamental and seeks an answer to the question whether the quantitative information obtained from in vitro studies of components (e.g. proteins, enzymes, organelles, etc in a reduced and isolated state in ordinary dilute solutions) is applicable and relevant to living systems that are extraordinarily organized? · Dimensions at which the problems are relevant: solvent and solute molecular interactions. Existing measurements indicate the mean free path for diffusion is on the order of 100 to 1000 nanometers, which gives half times for diffusion equilibration on the order of a millisecond for most low molecular weight solutes studied. · Information available from a number of technical approaches. Explanation of methods and results: - some detail of chemisry of electrolytes, dissociation, and non-electrolytesmacroscopic diffusion - nmr properties of molecules -- spin relaxation rates - microcopic rotational and translational diffusion by NMR - fluorescence recovery after photobleaching gives diffusion coefficients and viscosity - molecular transport and electrophoretic mobility - selected examples of in vivo and in vitro enzyme and organelle activity comparisons: creatine kinase, mitochondria. · Consensus information is that the viscosity is ~ 3 centipoise. · Concept of macromolecular obstruction in a normal solvent environment. · Effects of molecular crowding on macromolecular properties and on solvent and solute properties. NO Required Reading Assignment Annotated bibliography for Lecture 16. These are for your curiosity; none of these are assigned. Physiological measurements of extra- and intra-cellular water: One of the best papers on this topic is (1) which quantifies the distribution of water in human muscle. 1. Sjogaard, G., and B. Saltin. Extra- and intracellular water spaces in muscles of man at rest and with dynamic exercise. Am. J. Physiol. 243: R271-R280, 1982. Osmotic behavior: Solutes in solution influence the physical chemical properties of the solvent. One of these is the osmotic pressure. A very interesting study was done bathing a single frog muscle cell in solutions of varying concentrations (osmotic strength). Most of the water in the cell behaved as a normal osmometer (Van't Hoff osmotic relationship) over a wide range of osmotic pressures. The osmotic behavior of the cell was that of a semipermeable bag in which the solutes are dissolved in an amount of water equal to 67% of the total cellular volume. Thus the rest of the water (~ 13% of total volume because ~20% is solids) was involved in hydrating macromolecules. (1) 1. Blinks, J. R. Influence of osmotic strength on cross-section and volume of isolated single muscle fibers. J. Physiol. 177: 42-57, 1965. NMR relaxation properties of water: Much work has been done on the fact that the NMR spin relaxation rate of water in cells is very much faster than the relaxation in pure solvent. The value and problem with this approach is in the generality and sensitivity of NMR spin state to inter-molelcular interactions. NMR interactions are extremely weak, many orders of magnitude weaker than ordinary solute binding (e.g. weak acid dissociation, or substrate binding to enzyme). Many factors influence the relaxation of the protons in water in an NMR experiment, including the concentration of oxygen which is paramagnetic. These matters might be solved by a totally quantitative treatment of the interactions of liquid water with hydration water on macromolecules and other structures and chemical components, but this has not been done to my knowledge. It is clear that the fact of rapid relaxation does not mean that cellular water is "ice like" as has been claimed, because water so organized would not give narrow line spectra. Examples of work in this area include: (1-5) 1. Belton, P. S., R. R. Jackson, and K. J. Packer. Pulsed NMR studies of water in striated muscle. I. Transverse nuclear spin relaxation times and freezing effects. BBA 286: 16-25, 1972. 2. Belton, P. S., K. J. Packer, and T. C. Sellwood. Pulsed NMR studies of water in striated muscle. II. spin-lattice relaxation times and the dynamics of the non-freezing fraction of water. BBA 304: 56-64, 1973. 3. Ceckler, T. L., and R. S. Balaban. Field dispersion in water-macromolecular proton magnetization transfer. Journal of Magnetic Resonance, Series B 105: 242-8, 1994. 4. Hazelwood, C. F., and B. L. Nichols. Evidence for the existence of a minimum of two phases of ordered water in skeletal muscle. Nature 222: 747-750, 1969. 5. Rorschach, H. E., C. Lin, and C. F. Hazelwood. Diffusion of Water in Biological Tissues. Scanning Microscopy Supplement 5: S1-S10, 1991. Diffusivity and viscosity: Muscle cells have been widely used by a number of techniques. One set of results in today's lecture and handout used radioactive tracers microelectrophoresed into mechanically "skinned" cells surrounded by mineral oil. (4) Other results used NMR spectroscopic results to measure various metabolites in several types of muscles. (1, 2, 12, 13). Some of the results I showed in lecture 16 are unpublished from Tim Moerland, Florida State Univ. The primary results from fluorescent techniques are form the labs of A. Verkman and of K. Luby-Phelps. A great variety of cells, mostly cultured, were used. (3, 5, 6, 10, 11). These are particularly elegant in methodology, both in the spatial resolution by microscopy and in biological insight and targeting by bioengineering and molecular biology. Oxygen diffusion also have been heavily investigated, as two examples show (7-9). Oxygen follows simple diffusion rules. 1. Hubley, M. J., B. R. Locke, and T. S. Moerland. The effects of temperature, pH, and magnesium on the diffusion coefficient of ATP in solutions of physiological ionic strength. Biochim Biophys Acta 1291: 115-21, 1996. 2. Hubley, M. J., R. C. Rosanske, and T. S. Moerland. Diffusion coefficients of ATP and creatine phosphate in isolated muscle: pulsed gradient 31P NMR of small biological samples. NMR Biomed 8: 72-8, 1995. 3. Kao, H. P., J. R. Abney, and A. S. Verkman. Determinants of the translational mobility of a small solute in cell cytoplasm. J Cell Biol 120: 175-84, 1993. 4. Kushmerick, M. J., and R. J. Podolsky. Ionic mobility in muscle cells. Sci. 166: 1297-1298, 1969. 5. Luby Phelps, K., P. E. Castle, D. L. Taylor, and F. Lanni. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci U S A 84: 4910-3, 1987. 6. Luby-Phelps, K., S. Mujumdar, R. B. Mujumdar, L. A. Ernst, W. Galbraith, and A. S. Waggoner. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys J 65: 236-242, 1993. 7. Mahler, M. Diffusion in an elliptical cylinder, and a numerical method for caculating time-varying diffusant sink rates, with special reference to diffusion of oxygen in the frog sartorius muscle. Math. Biosci. 73: 109-130, 1985. 8. Mahler, M., C. Louy, E. Homsher, and A. Peskoff. Reappraisal of Diffusion, Solubility, and Consumption of Oxygen in Frog Skeletal Muscle, with applications to Muscle Energy Balance. J. Gen. Physiol. 86: 105-165, 1985. 9. Meng, H., T. B. Bentley, and R. N. Pittman. Oxygen diffusion in hamster striated muscle: Comparison of in vitro and near in vivo conditions. Am J Physiol Heart Circ Physiol 263: H35-H39, 1992. 10. Olveczky, B. P., and A. S. Verkman. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles. Biophys J 74: 2722-30, 1998. 11. Partikian, A., B. Olveczky, R. Swaminathan, Y. Li, and A. S. Verkman. Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J Cell Biol 140: 821-9, 1998. 12. Penke, B., S. Kinsey, S. J. Gibbs, T. S. Moerland, and B. R. Locke. Proton Diffusion and T1 Relaxation in Polyacrylamide Gels: A Unified Approach Using Volume Averaging. J Magn Reson 132: 240-54 240-54, 1998. 13. Yoshizaki, K., Y. Seo, H. Nishikawa, and T. Morimoto. Application of pulsed-gradient 31P NMR on frog muscle to measure the diffusion rates of phosphorus compounds in cells. Biophys. J. 38: 209-211, 1982. |
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Contact the instructor at: kushmeri@u.washington.edu
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