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Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.
It has been well established that heterologous antisera against whole rat kidney homogenate when injected into pregnant rats during the embryonic organogenetic period may induce abnormal embryonic development. Attempts were made to isolate the active components from soluble rat kidney extract by ammonium sulfate precipitation, anion-exchange chromatography, and concanavalin A-Sepharose 4B affinity chromatography. The glycoproteins isolated were capable of stimulating the production of potent rabbit antisera. When injected ip into the 9th day pregnant rats, these antisera induced embryonic death, congenital abnormalities, and fetal growth retardation. Eighty-four surviving fetuses were examined, all of them were malformed. The most frequently observed congenital defects were anophthalmia and microphthalmia. Attempts were made to analyze the glycoprotein fraction by discontinuous and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results indicate that the glycoproteins were of high molecular weight and could be dissociated by SDS into a multitude of molecules or subunits. Although double immunodiffusion indicated that there were one major and two minor antigens in the glycoprotein fraction, attempts to identify the antigens as to their size by analytical gel electrophoresis have not been successful. Electron microscopic study seemed to suggest that the glycoproteins might tend to aggregate to form particulates. The underlying mechanism whereby the antisera to these glycoproteins induce abnormal embryonic development is not understood. The hypotheses to explain the possible sites of teratogenic antibody interaction are discussed.
We have shown previously that periodate oxidation of collagen carbohydrate does not affect its ability to aggregate platelets. We now describe an additional characterization of periodate-modified collagen which demonstrates that collagen devoid of intact carbohydrate is fully capable of fibril formation, and we confirm its capacity to initiate platelet aggregation. Furthermore, we demonstrate that the platelet aggregating abilities of Types I, II, and III fibrillar collagen are quite similar despite differences in carbohydrate content and amino acid sequence. We also demonstrate that monomeric, pepsin-solubilized Type I human collagen is ineffective inhibiting aggregation by performed fibrils derived from the same molecule, thus establishing that the affinity of platelets for collagen depends upon prior polymerization of collagen. We interpret these and other findings to demonstrate that the hydroxylysyl glycoside regions of collagen are not highly specific sites involved in platelet-collagen interactions leading to "physiological" aggregation, and that the possibility must be considered that multiple interactions involving collagen sites of comparatively low structural specificity may be the initiating events in release of platelet ADP and the ensuing aggregation.