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Preparation and Chemical Composition of the Cell Walls of Mature Infectious Dense Forms of Meningopneumonitis Organisms

¹ Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514

ABSTRACT

Relatively large-scale production and purification of meningopneumonitis organisms was developed for chemical and immunological studies on cell walls of the infectious dense forms. By disruption of purified organisms with glass beads in a Mickle shaker, highly purified preparations of cell walls were obtained by sucrose density gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treatment. The dry-weight recovery of purified cell walls from intact organisms was about 13%. When ³²P-labeled preparations of cell walls were fractionated into acid-soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions, about 80% of the ³²P in cell wall preparations was recovered in the phospholipid fraction, which corresponded to about 3% of the total phospholipid in the intact organisms. About 7% of the ³²P in purified cell walls was recovered in the RNA and DNA fractions respectively, but this corresponds to only about 0.4% of the ³²P found in those fractions in intact organisms. From dry-weight determinations, it was calculated that the purified cell wall preparations contained only 0.6% total nucleic acids, and these are probably not true cell wall constituents. These cell walls contained 70 to 75% protein, corresponding to about 14% of the protein in intact organisms. Amino acid analysis of these protein showed the existence of all common amino acids, glucosamine, and galactosamine. However, no muramic acid was detected by the methods employed.