Isolation and fractionation of nuclear components from Physarum flavicomum



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This research is designed to isolate histone from purified Physarum flavicomum plasmodial nuclei. Attempts are made to fractionate and characterize this histone in order to compare this histone with other eucaryotic systems. An isolation procedure is improved for obtaining large quantities of P. flavicomum plasmodial nuclei with a high degree of purity. While most of the mechanical and enzymatic treatments have failed to yield complete breakage of nuclei, pretreatment with dithiothreitol (DTT) has been found to be an effective method for increasing nuclei breakage. Using a high concentration of salt (2 M NaCl) at neutral pH an acidic protein is co-extracted from purified plasmodial nuclei along with the basic histone proteins. This acidic material is characterized as actin-myosin-like protein by its amino acid composition and molecular weight. SDS-gel-electrophoresis gives an estimated molecular weight of 41,000 +/- 4,000 for this actin-myosin-like protein. Different reagents are used for the extraction of the basic histone material from purified plasmodial nuclei. Acid extraction (0.4 N H2SO4) is impractical since extremely low yields are obtained. Among the different salts used, CaCl2, coupled with TCA precipitation and dilute acid re-extraction, yields the most histone material with the least amount of acidic protein contamination. Gel filtration and carboxymethyl cellulose chromatography are unsatisfactory for the fractionation of the plasmodial histone. Fractionation based on solubility differences gives fractions with very low purity. The amino acid composition of whole plasmodial histone indicates that it is somewhat similar to calf thymus and Tetrahymena and most similar to P. polycephalum histones.