Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm^–1 band associated with S-S stretching vibrations at 72°C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72°C was to hinder broadening of the 505 cm^–1 band. HEW lysozyme (2.4 mM,pH 5) was also heated at 76°C, 80°C, and 95°C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76°C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80°C or 1 hr at 95°C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.     

Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. II. Model interpretation of ion exchange in lysozyme chromatography

In this paper we present a model for the ion exchange effects in protein adsorption. The model is applied to chromatography of lysozyme on strong cation exchanger ‘mono S’. The experimental and general thermodynamic aspects have been discussed in Part 1, the preceding paper. The main modelling assumptions are (i) the charge regulation is confined to the small layer of contact between adsorbed protein and exchanger surface, (ii) the contact layer as a whole is electroneutral and (iii) the number of protein acid/base groups and exchanger surface acid groups which participate in the ion exchange is proportional to the area of the contact layer. The model is fitted to the experimental data by adjustment of only two or three parameters. The experimental co-adsorption numbers are very well reproduced. A few conspicuous features emerge: (i) the number of protein acid/base groups and exchanger surface acid groups in the contact layer varies with the medium conditions, such that the number is higher when the interaction between protein and exchanger surface is stronger. (ii) There is indirect evidence for structural alterations in the upper layers of the exchanger surface: the adsorbed protein is probably partly ‘buried’ in the surface.     

Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

Summary The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) eggwhite lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983).In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the blobal and the local level. As in the globins, there are corresponding -helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another.Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive -helices in GEWL and three consecutive -helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices.There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 Å for Asp 86 and 4 Å for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

Amino acid sequences of stomach and nonstomach lysozymes of ruminants

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.     

分离蓝藻原生质球的新方法：青霉素－溶菌酶法

蓝藻原生质球(体)的研究,至今尚未取得决定性突破,成为细胞壁生物之中的唯一例外。这不仅表现在原生质球(体)的再生没有成功,而且分离制备方法亦存在很大问题。从1967年沿用至今的Crespi-Biggins溶菌酶法不仅在质量上不能得到真正理想的原生质球,而且数量上也达不到。由于在分离过程中细胞大量破裂,原生质球的产率很     

菜心溶菌酶的提纯及酶学性质

菜心（ＢｒａｓｓｉｃａｃａｍｐｅｓｔｒｉｓＬ．ｓｓｐ．ｃｈｉｎｅｎｓｉｓｖａｒ．ｕｔｉｌｉｓ）叶子高速捣碎后,滤液经酸碱处理,硫酸铵分步沉淀,凝胶柱层析等步骤分离纯化溶菌酶,酶比活力达３４１４．６Ｕ／ｍｇ,纯化倍数为１９７．４。菜心溶菌酶在较宽的温度或ｐＨ值范围均有活性,最适温度为６０℃,最适ｐＨ值为５．８,底物Ｋｍ值为８７μｇ／ｍＬ。该酶对热和酸碱的稳定性较高,巯基和酪氨酸残基不是该酶活性中心的必需基团。     

卡介苗对枯否细胞生物特性影响的免疫细胞化学研究

卡介苗(BCG)是增强肝枯否细胞(KC)抗肝癌细胞作用的最佳免疫制剂之一。本实验是给大鼠BCG后,应用溶菌酶免疫细胞化学法显示KC,以图象分析系统测定KC的数量、分布和面积,以细胞分光光度计测定KC溶菌酶活性;分离KC体外受BCG作用后,用ELISA法测定RC的Fc受体表达。结果显示:BCG使KC数量增多一倍,尤以肝小叶周边带为显著;KC体积明显增大,细胞面积增大55.71％,周边带KC面积增大一倍上;细胞溶菌酶光密度增大64.35％;Fc受体表达增强64.82％。上述结果是BCG增强KC抗肝癌细胞作用有关细胞学机制。     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.