Cook T A, Urrutia R, McNiven M A
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.
Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):644-8. doi: 10.1073/pnas.91.2.644.
Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.
发动蛋白是一种100 kDa的微管激活型GTP酶，最初从哺乳动物大脑中分离出来，有人提出它在胞吞作用的早期步骤中起关键作用。先前对发动蛋白的一级结构、生化特性和功能作用的研究表明它具有神经元特异性。然而，使用针对代表大鼠脑发动蛋白（D100）酶区域的合成肽的抗体，我们在大鼠肝脏中鉴定出一个100 kDa的蛋白双峰，这表明发动蛋白以与脑对应物不同的异构体形式存在。然后我们开始用抗体和cDNA探针寻找独特的发动蛋白异构体。一个对应于大鼠脑发动蛋白编码基因酶区域的500 bp PCR产生的cDNA探针被用于从大鼠肝脏cDNA文库中分离出六个重叠克隆，它们共同跨越了另一个发动蛋白基因“Dyn2”的完整编码序列。序列分析表明，发动蛋白2（Dyn2）在DNA水平上与脑发动蛋白有75%的同一性，在蛋白质水平上有79%的同一性。通过Northern印迹分析和异构体特异性PCR，发现Dyn2在成年大鼠组织中普遍存在，有3.5 kb和4 kb的两种转录本；在睾丸中的水平最高。这些结果表明，发动蛋白由至少两个在哺乳动物组织中差异表达的基因编码，并且Dyn2而非脑发动蛋白的表达解释了发动蛋白在大鼠组织中的普遍分布。
