Our long term objective is to develop and understanding of the structure, function and distribution of the covalently-crosslinked cell matrix which we have recently demonstrated to be present in skeletal muscle fibers. This cell matrix is prepared by extracting muscle with a protein solvent such as 6 M guanidine HCl and a disulfide cleaving reagent such as 5% beta-mercaptoethanol, at pH 8.5. When the remains of the extracted tissue are treated with high purity collagenase a cell matrix is released which is a coherent structure consisting of only 0.2% of the total protein of the skeletal muscle fiber yet conserving the cell's characteristic shape.
Our specific aims are to determine: (1) how widely distributed this matrix is in cells of various tissues, (2) whether there are covalent connections between adjoining cell matrixes, (3) whether the covalent crosslink is the epsilon (gamma-glutamic) lysine bond we found to be present in high molecular weight fractions of muscle cells, (4) what the polypeptide composition of the matrix is, (5) by means of X-ray analysis the conformation of the polypeptide chains of the matrix, (6) the role of the modulation of the crosslink content in the morphology and physiology of the cell and (7) the relationship between crosslink modulation, the maturing and aging of nerve tissue and abnormal states such as Alzheimer's disease. The methods we intend to use for these studies include: phase contrast microscopy, indirect immunofluorescence, scanning and transmission electron microscopy, enzymological and chromatographic techniques for measuring Glu-Lys crosslink content, peptide chemistry and immunochemical methods for evaluating the peptide composition of the matrix, X-ray crystallography to identify polypeptide folding patterns, and the use of normal and abnormal tissues to study differences in crosslink modulation. The health relatedness of Glu-Lys crosslink studies has been asserted in a number of articles, reviews and proposals for research programs. The significance of this proposal is that we plan to study the relationship between a newly discovered covalently crosslinked cell matrix and normal as well as abnormal states of development of nerve tissue.
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