Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.

中文翻译：

---

牛脑突触体中的硫胺素单磷酸酯和二磷酸酯酶

神经退行性疾病伴随着硫胺素单磷酸酶和二磷酸酶活性的变化，但对这些哺乳动物酶的分子鉴定尚不完整。在这项工作中，对硫胺素衍生物显示磷酸酶活性的牛脑突触体的蛋白质部分在硫胺素-琼脂糖凝胶上进行亲和色谱。测定了用硫胺素（pH 7.4 或 5.6）、NaCl 和尿素洗脱的蛋白质级分对硫胺素单磷酸 (ThMP)、硫胺素二磷酸 (ThDP) 和结构相似的嘌呤核苷酸的磷酸酶活性。由于牛基因组的注释不足，使用所有生物体的 SwissProt 数据库通过质谱法鉴定每个部分中的蛋白质。两种带注释的细菌磷酸酶的肽，在酸性硫胺素洗脱液中鉴定出来自 DING 蛋白家族的碱性磷酸酶 L 和外切多磷酸酶。洗脱部分中碱性磷酸酶 L 和外切多磷酸酶的肽丰度分别与 ThMPase 和 ThDPase 活性相关。ThMPase 和 ThDPase 活性的洗脱曲线不同于核苷酸磷酸酶的洗脱曲线，因此表明这些酶对硫胺衍生物的特异性。在硫胺素-琼脂糖凝胶洗脱液中寻找哺乳动物 DING 磷酸酶发现 X-DING-CD4，主要由酸性硫胺素溶液 (pH 5.6) 洗脱。鉴定出的胞外多磷酸酶与具有 ThDPase 活性的腺苷三磷酸双磷酸酶表现出结构相似性。