Ahmed H, Fink N E, Pohl J, Vasta G R
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
J Biochem. 1996 Nov;120(5):1007-19. doi: 10.1093/oxfordjournals.jbchem.a021493.
Selected biochemical properties, including the charge heterodispersity profile and carbohydrate specificity, of bovine galectin-1 were determined in detail. The lectin was purified through an improved purification protocol that yielded 35-40 mg/kg of wet tissue with a specific activity of 1.7-2 x 10(4) mg-1.ml. The galectin is a homodimer of approximately 14.5 kDa subunits with E(280)mg/ml of 0.65 ml.mg-1.cm-1. When stored in the presence of its carbohydrate ligand, the lectin's binding activity remained stable in a non-reducing environment even at room temperature. The optimal pH for binding to the ligand was 6.5-8.0. The overall carbohydrate specificity of the bovine galectin-1 isolated from spleen is similar to that of the galectin isolated from heart and to other mammalian galectins that exhibit "conserved" (Type I) carbohydrate recognition domains (CRDs) [Ahmed, H. and Vasta, G.R. (1994) Glycobiology 4, 545-549], but differs from those from Xenopus laevis and rat intestine domain I. The fluorescence of 4-methylumbelliferyl alpha-D-galactopyranoside was quenched on binding to bovine spleen galectin-1. Scatchard plots of data obtained at 5, 15, and 30 degrees C showed that the galectin has two sugar exothermic binding sites with association constants of 3.4 x 10(5), 1.0 x 10(5), and 0.3 x 10(5), respectively. Chemical modification studies indicated that histidine, tryptophan, carboxylic acid, and arginine, but not lysine or tyrosine, are involved in the binding to the carbohydrate ligand. On isoelectric focusing, the spleen galectin-1 appeared as six isoforms ranging from pI4.56-4.88 with main components at pI 4.63 (34.0%), 4.73 (42.6%), and 4.88 (16.6%). The galectin-1 isolated from heart yielded a quali- and quantitatively different profile with four isoforms ranging from pI 4.53-4.73, those with pIs of 4.56, 4.63, and 4.73 being common to the spleen homolog. Edman degradation of selected peptides purified from the spleen galectin-1 digest revealed amino acid sequences identical to those obtained for the heart galectin-1. This suggests that although point mutations in the subunit primary structure may not be the likely source of isolectins, as observed for X. laevis, tissue-specific co- or post-translational modifications may be the possible cause of the differences in the galectin isoform profile between bovine spleen and heart.
详细测定了牛半乳糖凝集素-1的一些生化特性，包括电荷异质性图谱和碳水化合物特异性。通过改进的纯化方案对该凝集素进行纯化，每千克湿组织可获得35 - 40毫克，比活性为1.7 - 2×10⁴毫克⁻¹·毫升。该半乳糖凝集素是由约14.5 kDa亚基组成的同二聚体，E(280)毫克/毫升为0.65毫升·毫克⁻¹·厘米⁻¹。当在其碳水化合物配体存在下储存时，即使在室温下，该凝集素在非还原环境中的结合活性仍保持稳定。与配体结合的最佳pH为6.5 - 8.0。从脾脏分离的牛半乳糖凝集素-1的总体碳水化合物特异性与从心脏分离的半乳糖凝集素以及其他具有“保守”（I型）碳水化合物识别结构域（CRD）的哺乳动物半乳糖凝集素相似[艾哈迈德，H.和瓦斯塔，G.R.（1994年）《糖生物学》4，545 - 549]，但与非洲爪蟾和大鼠肠道结构域I的不同。4 - 甲基伞形酮基α - D - 吡喃半乳糖苷与牛脾脏半乳糖凝集素-1结合时荧光猝灭。在5℃、15℃和30℃下获得的数据的Scatchard图表明，该半乳糖凝集素具有两个糖结合放热位点，其缔合常数分别为3.4×10⁵、1.0×10⁵和0.3×10⁵。化学修饰研究表明，组氨酸、色氨酸、羧酸和精氨酸参与了与碳水化合物配体的结合，而赖氨酸或酪氨酸则未参与。在等电聚焦时，脾脏半乳糖凝集素-1呈现出六种等电形式，范围从pI4.56 - 4.88，主要成分在pI 4.63（34.0%）、4.73（42.6%）和4.88（16.6%）。从心脏分离的半乳糖凝集素-1产生了质量和数量上不同的图谱，有四种等电形式，范围从pI 4.53 - 4.73，其中pI为4.56、4.63和4.73的与脾脏同源物相同。从脾脏半乳糖凝集素-1消化物中纯化的选定肽段的埃德曼降解显示其氨基酸序列与从心脏半乳糖凝集素-1获得的序列相同。这表明，虽然亚基一级结构中的点突变可能不像在非洲爪蟾中观察到的那样是同工凝集素的可能来源，但组织特异性的共翻译或翻译后修饰可能是牛脾脏和心脏之间半乳糖凝集素同工型图谱差异的可能原因。
