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研究生:吳篤安
研究生(外文):WU, DU-AN
論文名稱:1.人類21-羥化功能與結構的關係2.人類含帖氧化還原蛋白基因的結構與染色體的定位
論文名稱(外文):1.STRUCTURE AND FUNCTION RELATIONSHIP OF HUMAN P450C21 2.STRUTURE AND CHROMOSOME ASSIGNMENT OF HUMAN FERREDOXIN GENE
指導教授:鍾邦柱宣立人宣立人引用關係
指導教授(外文):ZHONG, BAN-ZHUXUAN, LI-REN
學位類別:博士
校院名稱:國防醫學院
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:1990
畢業學年度:79
語文別:中文
論文頁數:71
中文關鍵詞:哺乳類細胞酵母菌一氧化碳差異光譜鐵紫質含鐵氧化還原蛋白染色體定位
外文關鍵詞:21- 羥化P450C21MAMMALIAN-CELL-LINE-COS-1YEAET-CELLFERREDOXIN-GENECHROMOSOME-ASSIGNMENT
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先天性腎上腺增生症是一種常見的遺傳疾病,大部份是因為細胞色素p450c21(21-羥
化)缺乏引起的.它的缺乏引起荷爾蒙不平衡,導致腎上腺增生、女性男性化、甚
至引起低血壓、休克.為了要了解基因變化對疾病的影響,我們根據文獻把人類P450
c21 基因以定點突變改變了Ser-268,Val-281,Cys-428 這三個胺基酸,將基因導入哺
乳類細胞(COS-1 )及酵母菌.
P450c21 基因在COS-1 表現出具有活性、50kD大小的蛋白.在COS-1的實驗中,將 Ser
-268換成Thr,Cys,Met 後,蛋白質仍維持了十足的活性,這表示268 不是重要的部位
,改變它並不影響整個蛋的的結構與功能,因此 Ser-268的突變是屬於多型性的變化
.Val-281 換成Leu, Thr後,活性降至原來的10-20 %,換成Ile 則降至60-70 %.
至於Cys-428 換成Met, Ser, Thr 後,則完全喪失活性.P450c21 與各突變種在酵母
菌和COS-1 裡的活性相同.以酵母菌微粒體來測量P450c21 動力學,原始型(l-281)
,Leu-281,Ile-281,Thr-281 的Kmojbm amyo:0.27,0.4,0.29,0.48 (μM);Vmax 值
是:40,2.5,26,2.1 (nmol/h/1 mg微粒體蛋白).顯示改變了281 號胺基酸並沒有對
基質的結合產生太多改變,但催化能力的差別就大了.
以一氧化碳差異光譜測量酵母菌微粒體鐵紫質蛋白的含量,281號突變種降低,428號
突變種則偵測不到.由於一氧化碳差異光譜是測鐵紫質與蛋白的結合,鐵紫質含量降
低代表第281與428號胺基酸與鐵紫質很有關連.由已知立體結構P450cam 的排列得知
281號胺基酸應是座落在類似I段螺旋的結構上頭,第428號胺基酸座落在L段螺旋這個
結構的附近,提供硫原子與鐵紫質的鐵原子結合,使電子的傳遞與氧化還原反應能夠
順利進行.而I段螺旋與L段螺旋與鐵紫質的結合有密切的關係,它們像三明治般的夾
住鐵紫質.
含鐵氧化還原蛋白,是個以鐵,硫為中心的蛋白,主要在類固醇組織的粒線體內層基
質用來傳遞電子至終點以進行氧化還原反應.為了要了解它的基因結構,我們篩檢
人的基因庫,共得到真基因H1,H4,H5以及兩個假基因 2,3.以內限制將含真假基因
的DNA片段切成小段和具有重複序列之genomic DNA探針雜交,挑出不雜交的片段,再
以該片段與genomic blot雜交證明它是single copy DNA,利用它當探針與
human-rodent cell hybrid之DNA 作雜交,我們將含鐵氧化還原蛋白的真基因定在第
十一號染色體,h2與h3這兩個假基因定在第二十和第二十一號染色體.
因為牛的含鐵氧化還原蛋白mRNA在基因的5''端另有一個起始點,將人含鐵氧化還原蛋
白基因的5''端約1k b的DNA定序,發現了與牛第二種含鐵氧化還原蛋白mRNA 相似的序
列,以這段的部份DNA當探針尋找第二個起始點的 mRNA,所用的方法包括篩檢人類腎
上腺與胎盤的 cDNA基因庫,Northern analysis, S1 mapping,結果都沒有發現另一
mRNA,配合起動子的資料,我們確認人的含鐵氧化還原蛋白mRNA只有一個起點.
///////
Congenital adrenal hyperlpasia is a common genetic dissease. MOre than 90%
of the cases are due to P450c21 (21-hydroxylase) deficiency. Its
deficiency results in adrenal hyperplasia, virilization and/or electrolyte
imbalance. In order to understand how the mutated gene acts on the
disease, we mutagenized the human P450c21 gene at three positions,
Ser-268, Val-281, Cys-428, and transformed the mutated genes into
mammalian cell line (COS-1) and yeast cell.
After gene transfer, the COS-1 cells expressed 50kD protein which reacted
with anti-human P450c21 antibody and catalyzed the conversion of two
substrates. We compared the enzyme activity between the wild type and
mutant proteins. The 268-mutant proteins, Thr-268, Cys-268, Met-268 were
as active as the wild type. The Leu-281 was only 10-20% activity of the
wild type; the Ile-281 60-70%, the thr-281 10-20%. As to 428-mutants,
their enzymatic function as totally absent.
The yeast also expressed human P450c21 after transformation of P450 cDNA
plasmmid. The activity was similar to that of the COS-1. Performing the
enzyme kinetics with yeast microsomal protein, we obtained the Km and
Vmax. The Km values of wild type and Leu-281, Ile-281, Thr-281 were 0.27,
0.4, 0.29, 0.48 (μ M) and Vmax were 40, 2.5, 26, 2.1 (nm01/hour/1 mg
microsomal protein) respectively.
The hemoprotein content of the wild type P450c21 was 0.076 nm01/1mg
microsomal protein measured by CO difference spectroscopy, and 281-mutant
proteins, Leu-281, Ile-281, were 0.006, 0.028, respectively, and Ser-428
was undetectable.
We suggest 268 codon is a polymorphic site since its mutation doesn''t
change the protein function. Mutations at 281 codon don''t change the
substrate binding (Km), but protein activities and hemoprotein contents
are reduced. Because hemoprotein content is closely related to the binding
ability of the P450c21 to the heme, we suggest Val-281 is heme associated.
Mutation at the Cys-428 renders total loss of activity and hemoprotein
content, we suggest this is the 5th heme-binding ligand.
Ferredoxin is an iron-sulfur protein which serves as an electron transport
intermediate for all mitochondrial cytochromes P450 involved in steroid,
vitamin D, bile acid metabolism. We have cloned and characterized the
human ferredoxin gene. The include at least on authentic genes (h5, h1,
h4) and two pseudogenes (h2 and H3).
Specific DNA fragments were isolated from the flanking region of each
ferredixin genes by hybridization of genomic DNA as a probe to exclude the
fragments with repetitive sequence. These fragments were used as a probe
to hybridize genomic blot to ascertain their single copy nature. Finally,
hybridizations of human-rodent hybrid cell DNA with the single copy probes
were performed. according to the experiment, we assigned the authentic
gene to chrmosome 11, pseudogene h2 to 20, psedogene h3 to 21.
Their are two ferredoxin mRNAs with different initiation sites in the
bovine adrenal. The 5'' end of the human ferredoxin gene has been
sequenced. There is a region homologous to the second mRNA initiating at
the 5'' end. Using the subfragments in this region as probes to look for
another set of human ferredoxin mRNA, we have screened the human placental
and adrenal cDNA libraries, Northern analysis, S2 mapping. The results
turned out to be negative. Together with promotor study, we ascertain that
the human ferredoxin mRNA has only one initiation site.
目次
圖次
表次
縮寫表
中文摘要
英文摘要
第一部份 人類P450c21之功能與結構的關係
實驗材料與方法
結果
討論
第二部份 人類含鐵氧化還原蛋白基因的結構及染色體的定位
實驗方法與材料
結果
討論
參考文獻
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