STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES
溶酶体酶的结构-功能关系
基本信息
- 批准号:3941096
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
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- 关键词:
项目摘要
The lysosomal enzyme beta-hexosaminidase has been studied (A.)
at the level of it's genetic organization and (B.) by in vivo and in
vitro expression of it's cDNAs.
A. Genetic Organization. The gene encoding the beta-chain of
beta-hexosaminidase has been isolated and it's genomic
organization precisely characterized by restriction endonuclease
mapping and sequencing of the intron-exon junctions. Camparison
with the previously characterized alpha-chain gene revealed
extensive similarities with some interesting differences. Both
genes are about 40 kilobases in length, contain 14 exons and 13
introns, and have compact 3' ends and expanded 5' ends. The
respective coding sequences are interrupted at homologous
positions by 12 of the 13 introns. The most 5' intron of the two
genes, which is the only intron to occur at a non-homologous
position, interrupts both sequences at possible proteolytic
processing sites. The extensive conservation of intron positions
between the two genes demonstrates that they were derived from
a common ancestor.
B. In Vivo and In Vitro Expression. The beta-chain cDNA has
been expressed in transfected COS cells under the control of the
SV-40 late promotor. The expressed protein is enzymatically
active and undergoes proteolytic processing consistent with
transport to lysosomes. An in vitro expression system has been
developed in which beta-chain mRNA, generated by transcription
with SP6 polymerase, is translated in a rabbit reticulocyte lysate
supplemented with microsomes. Within the microsomes the
translated product assumes a native structure as indicated by
reactivity with a conformation-sensitive antibody, assembly of
the subunits into dimers and acquisition of catalytic activity. In
addition, by expressing genetically engineered forms of the beta-
cDNA, we have shown that the beta-chain contains two functional
initiator AUG codons.
已经研究了溶酶体酶β-己糖胺酶(A.)
在遗传组织水平上,(B.)通过体内和
体外表达。
A. 基因组织。 编码β链的基因
已经分离出β-氨基己糖苷酶,
以限制性内切酶为精确特征的组织
内含子-外显子连接的定位和测序。 卡姆什
先前描述的α链基因
广泛的相似性和一些有趣的差异。 两
基因长约40个碱基,包含14个外显子和13个外显子。
内含子,并且具有紧凑的3'末端和扩展的5'末端。 的
相应的编码序列在同源位置被中断,
13个内含子中的12个。 两者中最大的5'内含子
基因,这是唯一的内含子发生在一个非同源
位置,在可能的蛋白水解位置中断两个序列,
加工场地。 内含子位置的广泛保守
这两个基因之间的差异表明它们来自于
共同的祖先
B。 体内和体外表达。 β链cDNA具有
在转染的COS细胞中表达,
SV-40晚期启动子 表达的蛋白质是酶促的,
活性并经历蛋白水解加工,
运输至溶酶体。 体外表达系统已经被
其中通过转录产生的β链mRNA
用SP 6聚合酶,在兔网织红细胞裂解物中翻译
补充微粒体。 在微粒体中,
翻译后的产品采用原生结构,如
与构象敏感性抗体的反应性,
亚基转化为二聚体并获得催化活性。 在
此外,通过表达基因工程形式的β-
cDNA,我们已经表明,β链含有两个功能性的
启动子AUG密码子。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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