Steiger R F, Opperdoes F R, Bontemps J
Eur J Biochem. 1980 Mar;105(1):163-75. doi: 10.1111/j.1432-1033.1980.tb04486.x.
Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, alpha-glucosidase, inosine diphosphatase and alpha-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated. Acid proteinase and alpha-mannosidase are particle-bound and latent in cytoplasmic extracts; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral alpha-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1% Triton X-100. Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both alpha-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18-1.25 g/cm3). Neutral alpha-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at rho = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles. In intact cells, neutral alpha-glucosidase and acid phosphatase appear to be highly accessible to their substrates. It is tentatively concluded that (a) acid proteinase and alpha-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) alpha-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.
为了研究布氏锥虫消化系统的亚细胞结构，对布氏锥虫的血流形式进行了筛选，以寻找可作为质膜、鞭毛袋、溶酶体、内质网和高尔基体标志物的酶。对乙酰酯酶、酸性脱氧核糖核酸酶、酸性磷酸酶、酸性磷酸二酯酶、酸性蛋白酶、酸性核糖核酸酶、丙氨酸转氨酶、半乳糖基转移酶、α - 葡萄糖苷酶、肌苷二磷酸酶和α - 甘露糖苷酶进行了部分特性鉴定，并针对pH依赖性活性、反应与孵育时间和酶浓度的线性关系以及抑制剂和激活剂的影响对其测定方法进行了优化。研究了这些酶与颗粒物质的关联以及结构潜伏性的存在。酸性蛋白酶和α - 甘露糖苷酶在细胞质提取物中与颗粒结合且具有潜伏性；它们可被 Triton X - 100 部分激活并溶解。酸性磷酸酶、酸性磷酸二酯酶和肌苷二磷酸酶也得到了类似结果。中性α - 葡萄糖苷酶虽然部分可沉降，但不具有潜伏性，且很容易被去污剂溶解。即使在存在 0.1% Triton X - 100 的情况下，半乳糖基转移酶也牢固地与膜结合。通过差速离心和在蔗糖梯度上的密度平衡进行细胞分级分离表明，α - 甘露糖苷酶和酸性蛋白酶都与密度约为 1.20 g/cm³ 的细胞器相关。肌苷二磷酸酶、半乳糖基转移酶、酸性磷酸酶和酸性磷酸二酯酶主要作为微粒体成分沉降，在密度 1.13 至 1.15 g/cm³ 之间达到平衡。此外，肌苷二磷酸酶和半乳糖基转移酶在更高密度（1.18 - 1.25 g/cm³）下表现出相当高的活性。中性α - 葡萄糖苷酶主要在核和微粒体部分回收；其颗粒部分在ρ = 1.22 g/cm³ 处作为单一带达到平衡。乙酰酯酶和酸性脱氧核糖核酸酶在很大程度上是可溶的，而酸性核糖核酸酶没有产生明显沉降和条带图谱。在完整细胞中，中性α - 葡萄糖苷酶和酸性磷酸酶似乎很容易被其底物接触到。初步得出结论：(a) 酸性蛋白酶和α - 甘露糖苷酶是溶酶体酶，(b) 酸性磷酸酶和酸性磷酸二酯酶与鞭毛袋相关，且前一种酶的一部分可能与内质网相关，(c) 半乳糖基转移酶是高尔基体的组成成分，(d) α - 葡萄糖苷酶可作为质膜的标志物。肌苷二磷酸酶也可能源自后一种结构。
