生物谷报道：最近科学家在一种豹蛙（Rana pipiens）的卵细胞内发现的一种分子或许能提供第一种治疗脑癌的药物。
豹蛙 Rana pipiens
该分子被称为Amphinase，它能识别肿瘤细胞表面的多糖，然后结合到其上，并且入侵到肿瘤细胞内部使RNA失去活性，最终导致肿瘤死亡。在发表于Journal of Molecular Biology上的文章中，来自英国Bath大学和美国Alfacell公司的科学家描述了对于这一分子进行的首次完全的结构和化学性质分析的结果。
尽管可能被用于多种形式的肿瘤，但是Amphinase最有希望治疗的是脑癌，目前针对脑癌只能进行复杂的手术以及化疗。科学家从北部豹蛙的卵母细胞中分离出了这种核糖核酸酶。核糖核酸酶在生物体内普遍存在，它们负责整理那些自由飘浮的RNA链，通过结合到分子上从而将其切割成小的片断。
在细胞中RNA起着关键作用的部位，核糖核酸酶被抑制分子所阻碍。但是由于Amphinase是一种两栖动物的核糖核酸酶，因此它们可以躲开哺乳动物的抑制分子，然后进攻癌症细胞。
在治疗中，这些分子将可能被注射到需要的区域。由于只和肿瘤细胞表面的多糖进行结合，因此这类分子对于其它细胞不会产生副作用。这已经是Alfacell公司从该种青蛙体内分离出的第二种抗癌核糖核酸酶了。另一种目前处于恶性间皮瘤临床试验的最后阶段，这是一种罕见且致命的肺癌，而针对其它实体癌症的临床试验处于第二、三阶段之间。（援引教育部科技发展中心）
原始出处:
Enzymatic and Structural Characterisation of Amphinase, a Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes
Umesh P. Singh1, †, Wojciech Ardelt2, †, Shailendra K. Saxena2, †, Daniel E. Holloway1, †, Eugene Vidunas2, 2, Hung-Suen Lee2, Abha Saxena2, Kuslima Shogen2, , and K. Ravi Acharya1, ,
1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
2Alfacell Corporation, Bloomfield, NJ 07003, USA
Received 21 March 2007; revised 26 April 2007; accepted 29 April 2007. Edited by M. Guss. Available online 10 May 2007.
Abstract
Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1–4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8–99.1% and 38.2–40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of kcat/KM with hypersensitive fluorogenic substrates are 104 and 102-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B1 or B2 subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 Å resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual α2–β1 loop may do so) and the B2 subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.
Keywords: Amphinase; cytotoxic RNase; X-ray crystallography; enzyme efficiency; substrate specificity
Abbreviations: Amph, Amphinase; Amph-1–4, Amphinases-1–4; CpA, cytidylyl-3′,5′-adenosine; DTT, dithiothreitol; 6-FAM-dArC(dA)2-6-TAMRA and rCA, 6-carboxyfluorescein-dArC(dA)2-6-carboxytetrarhodamine; 6-FAM-dArU(dA)2-6-TAMRA and rUA, 6-carboxyfluorescein-dArU(dA)2-6-carboxytetrarhodamine; 6-FAM-dArUdGdA-6-TAMRA and rUG, 6-carboxyfluorescein-dArUdGdA-6-carboxytetrarhodamine; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; ONC, Onconase; PEG, poly(ethylene glycol); PNGase F, peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase from Elizabethkingia (Chryseobacterium/Flavobacterium) meningsepticum; rAmph-2, recombinant Amphinase-2; RC-RNase, Rana catesbeiana ribonuclease; RC-RNase-6, Rana catesbeiana ribonuclease-6; RI, ribonuclease inhibitor protein; RNase A, bovine pancreatic ribonuclease A; rRNA, ribosomal RNA; TFA, trifluoroacetic acid; TPCK, N-p-tosyl-L-phenylalanine chloromethyl ketone; UpA, uridylyl-3′,5′-adenosine
Corresponding authors.
† U.P.S., W.A., S.K.S. and D.E.H. contributed equally to this work.
2 Present address: E. Vidunas, Wyeth Pharmaceuticals, Pearl River, NY 10965, USA.
