Lysozyme isolation

The sequence of lysozyme isolated from the urine of human leukemic patients was determined by Canfield et al. (1971), and that of human milk lysozyme was determined by Jolles and Jolles (1971). Canfield et al. 

Jolles et al. (1984) determined the 129-residue sequence of one of the three variants (variant 2) of the three c-type lysozymes isolated by Dobson et al. (1984) from bovine stomach mucosa. The sequence is given here. However, residue 98 has been altered from the original designa-don of Lys to His in accordance with the revision by R Jolles and J. Jolles (quoted by Prager and Wilson, 1988). Note (6) Work by Jolles et al. (1990) on the other bovine variants and two variants of caprine stomach mucosa lysozyme, and by Irwin and Wilson (1990) on three ovine variants, was in press at the time of writing and the following information was made available courtesy of Ellen Prager and Allan Wilson. 

Cross, M., Mangeldorf, J., Wedel, A Renkawit R. (1988). Mouse lysozyme isolation, characterization and expression studies. Proc. Natl Acad. Sci. USA, 85, 6232-6. 

The affinity labeling reagents described above have proved to be useful in studies of the active sites of lysozymes isolated from different organisms. Many of these lysozymes exhibit close similarity in their amino acid sequences, fluorescence spectra, and enzymic properties including not only the ability to digest cell walls of M. luteus, but also to catalyze transglycosylation reactions with a variety of saccharide acceptors when the cell wall tetrasaccharide or chitin oligosaccharides are used as substrates. ... 

Figure 2 Internal RMSF of residues (average over heavy atoms) determined for human lysozyme by the X-ray normal mode refinement method applied to real X-ray data (heavy curve), m comparison with results from a normal mode analysis on a single isolated lysozyme molecule (lightweight curve). (From Ref. 33.)...

One of the minor by products of the isolation procedure of the disaccharide from lysozyme digests of bacterial cell walls was the tetrasaccharide /i-GIcNAc-G 4)-/i-MurNAc-G 4)-/)-GlcNAc-(l 4)-MurNAc. This structure is readily cleaved by lysozyme, and has proved to be extremely useful in other laboratories for the study of the mechanism of action of the enzyme. 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

Lysozyme was isolated from human milk in 1961 by Jolles and Jolles, who believed that bovine milk was devoid of lysozyme. Milks of many species have since been shown to contain lysozyme and several have been isolated and characterized. Human and equine milks are an exceptionally rich source, containing 130 mg 1 1 (3000 times the level of bovine milk) and about 800 mg l-1, respectively (see Farkye, 1992). 

As discussed in section 8.2.5, lysozyme has been isolated from the milk of a number of species human and equine milks are especially rich sources. In view of its antibacterial activity, the large difference in the lysozyme content of human and bovine milks may have significance in infant nutrition. It is claimed that supplementation of baby food formulae based on cows milk with egg-white lysozyme gives beneficial results, especially with premature babies, but views on this are not unanimous. 

Metraux, J.P., Burkhart, W., Moyer, M., Dincher, S., Middlesteadt, W., Williams, S., Payne, G. Ryals, J. (1989). Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proceedings of the National Academy of Sciences (USA) 86, 896-900. 

The lysozyme mechanism (Scheme 11.21) has an acylal intermediate with a decay step which is fast compared with its rate of formation, so it is not normally possible to observe the intermediate. The acylal mechanism has recently been established by modifying both enzyme and substrate so that the intermediate can be isolated in this case, the intermediate was stabilised so much that its X-ray crystallographic structure could be determined. Substrate A in Scheme 11.21 was employed [22 ] in which the 2 -fluoro substituent reduces the reactivity at the Cl position so that any acylal enzyme formed from this substrate would have a... 

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