Characterization of the Subunits of the Flagella of Proteus vulgaris

Joyce Y Chang,Douglas M Brown,A.N Glazer

doi:10.1016/s0021-9258(18)63645-2

Joyce Y Chang, Douglas M Brown + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)63645-2

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Abstract

Abstract A procedure is described for the purification of Proteus vulgaris flagellin. The purified protein is homogeneous on acrylamide gel electrophoresis under a variety of conditions and in the ultracentrifuge. The amino acid composition of the unmodified and oxidized flagellin is reported. The protein is totally lacking in half-cystine, histidine, and tryptophan. Molecular weight determinations, by sedimentation equilibrium in the presence of 6 m guanidine, tryptic maps, and the amino acid composition are all consistent with the conclusion that Proteus vulgaris flagellin consists of a polypeptide chain with a molecular weight of approximately 40,000. Alanine, recovered in a yield of 0.5 residue per mole of protein, was the sole amino-terminal residue, as determined by the cyanate method. The results obtained with this flagellin, in conjunction with data available on flagellins of other bacteria, lead to the conclusion that identical subunits, of about 40,000 molecular weight, having a similar number of basic residues, form the fundamental building block of bacterial flagella.

Highlights

Our studies show that the flagella of P. vulgaris, do
PuriJication of Flagellin-Flagella were isolated by differential centrifugation
Purification of P. vulgaris flagella was achieved by chromatography on DEAE-cellulose in urea

Summary

Methods

Vulgaris, Purdue strain, was obtained from the Purdue University collection, and strain SC 31a, from Dr R. The cells were grown in 0.8% nutrient broth (Difco Laboratories) on a reciprocal shaker at 37” and harvested after 16 hours. PuriJication of Flagellin-Flagella were isolated by differential centrifugation [7]. The flagella were suspended in distilled water, exhaustively dialyzed against distilled water, and lyophilized Approximately 400 mg of lyophilized material were dissolved in 30 ml of 0.005 M glycine-8 M urea at pH 9.5 and applied to a column, 3.8 x 45 cm, of DEAE-cellulose (Whatman, microgranular No DE32), equilibrated with the same buffer. A linear gradient, formed from 1500 ml of 0.005 M glycine-8 M urea at pH

Results

Discussion

Conclusion