Hamada S, Torii M, Okahashi N, Kotani S, Kato K, Kawata S, Yokogawa K, Michalek S M, McGhee J R
Microbiol Immunol. 1983;27(3):237-49. doi: 10.1111/j.1348-0421.1983.tb03586.x.
The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan--polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4: 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an alpha-linked galactose-glucose terminal linkage.
变形链球菌的血清型特异性碳水化合物部分是通过用细胞壁裂解酶温和降解纯化的细胞壁而分离得到的。血清型g变形链球菌菌株6715的细胞壁用M1酶消化，M1酶是一种从球形链霉菌菌株1829的培养上清液中纯化得到的内切-N-乙酰胞壁酸酶。细胞壁的酶解产物应用于CM Sephadex C-25柱，以从细胞壁裂解物中去除M1酶，然后进行Sephadex G-100柱色谱分析。分离出具有血清型g特异性的碳水化合物抗原，命名为M1g，以及缺乏血清型特异性的肽聚糖-多糖复合物（M1PG）。通过在120℃的盐水中将变形链球菌6715全细胞高压灭菌30分钟，也获得了纯化的血清型g抗原。提取物应用于DEAE Sephadex A-25柱，以去除核酸和磷壁酸。未结合的峰馏分被浓缩，并在Bio-Gel P-100柱上重新进行色谱分析。空体积馏分含有血清型g碳水化合物，被命名为RRg抗原。通过免疫扩散，M1g和RRg抗原与抗血清型g血清形成了一条同一带。这些抗原主要由半乳糖、葡萄糖和鼠李糖组成，其重量比约为8:4:1，而M1PG的组成糖由鼠李糖和葡萄糖组成，未检测到半乳糖。M1g还含有除苏氨酸以外的肽聚糖残基，苏氨酸是天然细胞壁肽聚糖的肽间桥成分。蜜二糖和半乳糖对M1g与抗血清型g血清之间的定量沉淀反应有明显抑制作用，这表明血清型g碳水化合物的免疫决定簇是α-连接的半乳糖-葡萄糖末端连接。
