Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin.
Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin.
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大鼠脑己糖激酶的蛋白水解解剖：胰蛋白酶有限消化过程中切割模式的测定。
DOI：
10.1016/0003-9861(84)90279-0
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发表时间：
1984
影响因子：
3.9
通讯作者：
Wilson,JE
中科院分区：
文献类型：
--
作者：
Polakis,PG;Wilson,JE
用胰蛋白酶有限地处理大鼠脑己糖激酶（ATP：d-己糖-6-磷酸转移酶; EC 2.7.1.1）导致Mr 98 K酶裂解成分子量为10 K、40 K和50 K的三个主要片段，检测到Mr 60 K和90 K的中间体。这一信息，结合完整酶和胰蛋白酶裂解产物的N-和C-末端分析，建立了胰蛋白酶裂解模式，其中T1和T2指示胰蛋白酶裂解位点;仅T1或T2裂解分别产生90 K或60 K中间体。这种切割模式的确认已提供了二维肽作图usingStaphylococcusaureusV 8蛋白酶，和抗原决定簇作图与两个单克隆抗体针对大鼠脑己糖激酶。由一种单克隆抗体识别的表位位于40 K C-末端片段内，而另一种单克隆抗体的表位位于50 K片段内。一个二维的肽映射免疫印迹技术已经允许一个更明确的定位这些表位的特定区域内这些主要的胰蛋白酶切割片段。酶的完全胰蛋白酶裂解仅发生适度（约20%）的催化活性损失，并且裂解的酶保留了完整己糖激酶的许多特性。具体地说，尽管ATP的Km一直被认为是由切割引起的，但切割对Glc的Km或Glc-6-P的Ki没有影响。此外，像完整的酶一样，切割的己糖激酶保留了以Glc-6-P敏感的方式结合线粒体外膜的能力。在非变性条件下，切割的片段通过非共价力保持结合。因此，裂解酶沉淀的速度相当于完整的酶在蔗糖密度梯度离心过程中，和迁移只有稍微快时，在非变性条件下的梯度丙烯酰胺凝胶上的电泳。
Limited treatment of rat brain hexokinase (ATP:d-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of theMr98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates ofMr60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1and T2indicate tryptic cleavage sites; cleavage atonlyT1or T2gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping usingStaphylococcus aureusV8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (~20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on theKmfor Glc or theKifor Glc-6-P, though a slight decrease inKmfor ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.
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