I. Studies on the amino acid sequencingof Abrin-a II. Studies on the antitumor activity of Butenolid derivatives

• Main Authors: Yung-Liang Chen, 陳媛孃
• Other Authors: Jung-Yaw Lin
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/74321053867287303831

博士 === 國立臺灣大學 === 生化學研究所 === 81 === Part I.雞母珠毒蛋白質(Abrin-a)胺基酸排列順序之研究:雞母珠毒蛋白 質 (Abrin-a of Abrus precatorius)係由A鏈與B鏈兩個次單元體所組成 。將 Abrin-a羧甲基化(Carboxylmethylation)後,A鏈以Trypsin, Chymotrypsin, Staphylococcus aureus V8 protease和Thermolysin水 解;B鏈則以Trypsin ,Chymotrypsin,Staphylococcus aureus V8 protease,Lysyl endopepti- dase和Thermolysin水解所得胜■訂出其胺 基酸排列順序。將此蛋白質胺基酸排列順序與從核■酸推測出的胺基酸排 列順序比較,發現二者不完全相同 ,顯示雞母珠毒蛋白質含有其他之異型( Isoforms)毒蛋白質。此外,比較雞母珠毒蛋白質與蓖麻子毒蛋白質( Ricin-D)的胺基酸排列順序發現其A鏈有 42%之相似性,而B鏈則有59%之相 似性。B鏈係由兩個單元體(Domain)所組成 ,而每個單元體則含有(a、b、 g)三個次單元體(Subdomain),每個次單元體約由40個胺基酸殘基組成。顯 示在演化上Abrin-a與Ricin-D係源自一相同的始祖。Abrin-a A chain則 與Ricin-D A chain有Glu164,Ala165,Arg167, Asn196,Trp198,Ser202保 留之胺基酸,可能與其N-glycosidase活性有關。我們利用試管內(in vitro)細胞生長抑制(Inhibition of cell growth),蛋白質生合成抑制( Inhibition of protein biosynthesis),RNA生合成抑制(Inhibition of RNA biosynthesis)以及DNA生合成抑制(Inhibition of DNA biosynthesis)系統從數十種合成化合物(Synthesized compounds)來篩選 具有抗癌活性的物質。結果發現4-acetoxybutenolide(命名為158G), erythro-di-4-butenolidyl-ether(159A)及threo-di-4-butenolidyl eth- er(159B)。這三種Butenolid衍生物具有抗癌活性,其抑制子宮頸癌 細胞( HeLa cells)百分之五十細胞生長濃度分別是158G:0.01mg/ml:159 A:0.03mg /ml;159B:0.02mg/ml。抑制子宮頸癌細胞百分之五十蛋白質生 合成之濃度分別是158G:0.05mg/ml;159A:0.91mg/ml;159B:0.89mg/ml。抑 制子宮頸癌細胞百分之五十RNA生合成之濃度分別是158G:5.01mg/ml;159 A:0.74mg/ml; 159B:0.55mg/ml。以及抑制子宮頸癌細胞百分之五十DNA生 合成之濃度分別是158G:7.42mg/ml;159A:0.25mg/ml;159B:0.15 mg/ml。 此外,以裸鼠進行這三種Butenolide衍生物的生物體內(in vivo)試驗,發 現這三種的化合物對於植入裸鼠背部的子宮頸癌具有部份治療效果。 Abrin-a is a toxic glycoprotein found in the seeds of Abrus pre- catorius.It consists of two chains,A chain and B chain. The amino acid sequence of A chain was determined by analysis of peptides obtained from the S-carboxymethylated protein by digestion with trypsin,chymotrypsin,Staphylococcus auerus V8 protease and thermolysin;while that of S-carboxymethylated B chain was determined by digestion with trypsin,chymotrypsin, Staphylococcus aureus V8 protease,lysyl endopeptidase and thermolysin.The amino acid sequence of Abrin-a is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of Abrin-a.Comparison of the amino acid sequence of Abrin-a A chain with that of Ricin-D A chain reveals 42% sequence identity; while B chain 59% . Abrin-a B chain also appears to consist of two do- mains,each domain with three subdomains(a,b and g)of about 40 amino acid residues.It suggests that Abrin-a and Ricin-D evolu- tionarily come from the same ancestor.Abrin-a A chain has Glu164, Ala165, Arg167,Asn196,Trp198 and Ser202 which are also conserved to Ricin-D A chain and may involve in catalytic sites of N-glyco- sidase activity.Three of them exhibit potent antitumor activity on HeLa cells.They are 4-acetoxy-butenolide(nomenclature as 158G) ,erythro-di-4-butenolidyl ether(159A) and threo-di-4- butenolidyl ether(159B)all belonging to butenolid derivatives.50% cell growth inhibitory concentations of 158G,159 A and 159B was determined to be 0.01mg/ml,0.03mg/ml and 0.02 mg/ ml,respectively. 50% protein biosynthesis-inhibition concentration of 158G,159A and 159B was 0.05mg/ml,0.91mg/ml and 0.89mg/ml,respectively. 50%RNA biosynthe- sis-inhibition concentration of 158G,159A and 159B was 5.01 mg/ml ,0.74mg/ml and 0.55mg/ml,respectively. 50% DNA biosynthesis-inhi- bition concentration of 158G,159A and 159B were 7.42mg/ml,0.25mg/ ml and 0.15mg/ml,respectively.This compounds showed an antitumor against the growth of cervix uteri tumor in nude mice.