Abstract
We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.
Highlights
Outer membranes of Gram-negative bacteria have been studied in great detail, and the composition and synthesis of outer membrane proteins have been reviewed [1,2,3]
In this paper we report the purification by differential detergent extraction of a M, 15,000,peptidoglycan-associated lipoprotein from H. influenzae
From the workof one group of investigators [4] it appeared that the protein, designated P6 [4] or g [20] was a peptidoglycan-associated protein. They were able to demonstrate that rietmained associated with the peptidoglycan even after extraction of the protein cell wall complexwith SDS at 37 “C.Other investigators may have overlooked H. influenzae peptidoglycan-associated lipoproteins (PALs) in their characterization of outer membranes because of the stringency of their definition of peptidoglycan association
Summary
Outer membranes of Gram-negative bacteria have been studied in great detail, and the composition and synthesis of outer membrane proteins have been reviewed [1,2,3]. In this paper we report the purification by differential detergent extraction of a M , 15,000,peptidoglycan-associated lipoprotein from H. influenzae. Their results demonstrated the presence of a protein similar in size to H. influenzae PAL that remained associated with peptidoglycan until temperatureswere significantly increased in the presence of SDS (see Ref. 24, Fig. 3).
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