Risk of cardiovascular events appeared high beyond the first year post-MI, indicating a need for prolonged surveillance, particularly in patients with additional risk factors.
Previous methods which have been used for the preparation of protein A, such as thermal release of protein A with Iysozyme from the bacteria, have given a heterogenous product in a low yield. The present publication reports the isolation procedure of protein A from Staphylococcus aareus digested by lysostaphin for 2 h at 37 "C. 450-500 mg of protein A was obtained from 300 g of wet bacteria. The preparation is homogenous as seen from its disc electrophoretic pattern.Amino acid composition was determined and based on a molecular weight of 42000. The number of amino acid residues found was 378 f 6. Less than 0.2O/, hexosamines was present.The ultraviolet spectrum and the absorption coefficient, = 1.65 of protein A is given. Less than 0.1 mol amino terminal amino acid per mol protein was detected which indicates that the protein has a blocked N-terminal amino acid. The protein was slowly digested with a mixture of carboxypeptidase A and B and 2 mol lysine/mol protein was released.Precipitation of protein A with normal human y-globulin gave an IgG: protein A molar ratio of 2.1 : 1 within the equivalence zone.The results are discussed with regard to the subunit structure of the protein and the type of attachment of protein A to the peptidoglycan part of the cell wall.
Staphylococcus aureus, strain Cowan I, contains 1.7 protein A and its isolated cell walls contain 6.7O/,.Removal of contaminating cytoplasmic membrane fragments from the cell walls does not decrease the content of protein A. Extraction of cell walls with trichloroacetic acid releases teichoic acid but not protein A. Extractions with high concentrations of LiCl do not remove protein A. Only bacteriolytic enzymes can release the protein.Protein A isolated from bacteria after lysozyme digestion contains peptidoglycan constituents. More effective bacteriolytic enzymes such as lysostaphin release peptidoglycan fragments from this protein A preparation.We conclude from these results that protein A is a cell wall component of X. aureas and is covalently linked to the peptidoglycan structure.
To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc‐part of human IgG were isolated by affinity chromatography on IgG‐Sepharose 4B and purified by ion‐exchange chromatography on phosphocellulose.
From a partial tryptic digest of pure protein A at 30°C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein‐A‐containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A.
All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc‐reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.
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