Histochemical and ultrastructural studies demonstrate that keratohyalin can be mobilized from fresh specimens of cattle hoof epidermis by 1 .0 M potassium phosphate buffer (pH 7.0) .Macroaggregates with histochemical characteristics identical to those of in situ keratohyalin granules (staining by Harris' hematoxylin, Congo red, diazotized sulfanilic acid, sodium alizarin sulfonate, toluidine blue, methyl green-pyronin, and acridine orange) and with similar morphological characteristics at the ultrastructural level are formed upon dialyzing the extracted keratohyalin against distilled water . Staining by basic dyes (toluidine blue, methyl green-pyronin, and acridine orange) is abolished by treating either in situ keratohyalin granules or isolated macroaggregates with ribonuclease . Electrophoresis of isolated macroaggregates on polyacrylamide gels in the presence of sodium decylsulfate results in the fractionation of a 13 member oligomeric series of ribonucleoproteins and two nonhomologous species of ribonucleoproteins . The oligomeric series can be purified by isolating "stacked" oligomers on low concentration (3 %) polyacrylamide gels . Fractionated oligomers on polyacrylamide gels and aggregates formed from purified ribonucleoproteins demonstrate histochemical characteristics identical to those of in situ keratohyalin granules.Aggregates formed from denatured ribonucleoproteins are highly disordered and are markedly different from in situ keratohyalin granules or nondenatured isolated macroaggregates at the ultrastructural level, possibly due to irreversible denaturation of the oligomers by sodium decylsulfate .

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Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.