The characterisation of the cationic antibacterial compounds from some mammalian tissues and secretions

Abstract

Non-specific antimicrobial substances occur in mammalian tissues and secretions which are involved in the protection of the host against invading microorganisms. Among them are well researched proteins like lactoferrin and lysozyme, and systems like the myeloperoxidase- and lactoperoxidase-mediated system, and the two complement systems. Also, a number of mammalian cationic proteins have been found to have antibacterial properties. Conflicting data exist on their identity, as most of them have not been purified to homogeniety. This investigation was designed to find out if any of the cationic heterogeneous antibacterial proteins extracted from mammalian tissues and fluids by various workers owe their activity to the polyamine spermine which was found in bovine rumen by Briggs (1982). Previously Eschenbruch (1980) had isolated a low molecular weight antibacterial compound from bovine thymus, spleen and seminal plasma as well as sheep thymus and found them to behave similarly on cationic electrophoresis to the low molecular weight compound from bovine rumen. This was later identified as spermine (Briggs, 1982). Spermine was found to be associated with a substance which was responsible for absorbance at 210 nm. Initially beta-lysin from bovine serum was investigated for the presence of spermine. Beta-lysin was found not to contain spermine so further purification of the antibacterial substance was undertaken. A homogeneous cationic protein was isolated which had an estimated molecular weight of 6 100. Also, an investigation into the antibacterial seminalplasmin preparation found that no spermine was associated with this protein. However, further purification of seminalplasmin resulted in the finding that it had a lower molecular weight than that published (7 500 rather than 10 600). A lysozyme-like substance similar to that isolated by Eschenbruch (1980) was found closely associated with the seminalplasmin during the purification procedure. The antimicrobial milk cell cationic proteins were also investigated. Although spermine was not found associated with the milk cell proteins, further isolation and purification of the active components was carried out. The milk cell proteins exist in a highly aggregated state and normal chromatographic techniques and eluents proved unsuccessful in isolating the active compounds. However EDTA was found to be a useful disaggregating agent. Two antibacterial proteins were isolated and were found to have molecular weights of 11 000 and 18 000-20 000. The higher molecular weight protein had an isoelectric point greater than pH 9.5 and was able to lyse pregrown Micrococcus lysodeikticus cells. The lower molecular weight protein gave an anomalous results on isoelectric focusing and displayed no lytic activity. There did not appear to be any interaction between the two proteins as far as enhancing or inhibiting their overall antibacterial activities. An investigation into a bactericidal sheep thymus preparation resulted in the isolation of spermine and spermidine. This had been previously isolated by Eschenbruch (1980) and found to be electrophoretically similar to the bovine rumen antibacterial peptide. Further investigation of the antibacterial peptide found associated with lysozyme (which was also found to be electrophoretically similar to the bovine rumen antibacterial peptide) did not contain spermine. Isolation of the active factors using a variety of dissociating agents was not successful as the active compounds were highly aggregated. There appeared to be several antibacterial compounds present with molecular weights estimated to be below 15 000. The significance of the isolated cationic proteins are considered with regard to the non-specific defence against infections. Also the role of spermine and spermidine as a non-specific antibacterial agents is discussed along with the possibility that the cationic substances are artifacts produced through proteolysis of larger peptides or proteins as a result of the methods of extraction.