Alarcón C, Cheatham B, Lincoln B, Kahn C R, Siddle K, Rhodes C J
EP. Joslin Research Laboratory, Joslin Diabetes Center, Brigham and Women's Hospital, Boston, MA 02215.
Biochem J. 1994 Jul 1;301 ( Pt 1)(Pt 1):257-65. doi: 10.1042/bj3010257.
The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.
胰岛素原受体在翻译后于一个精氨酸 - 赖氨酸 - 精氨酸 - 精氨酸位点通过有限的蛋白水解作用被切割，以产生其成熟的α亚基和β亚基形式。一种35S标记的胰岛素原受体底物制剂以及一种模拟胰岛素原受体加工位点（IRP肽）周围及包括该位点的氨基酸序列的15聚体肽底物，揭示了一种内肽酶活性，该活性可催化大鼠肝脏亚细胞组分中胰岛素原受体的切割。在最佳条件下，该组分对正常35S标记的胰岛素原受体底物的加工是定量的。与先前的体内观察结果相似，该组分无法加工一种35S标记的胰岛素原受体变体底物（其中四碱基切割位点的精氨酸 - 1被丙氨酸 - 1取代），这表明这种内肽酶活性具有生理相关性。胰岛素原受体/IRP肽加工的生化特性表明，这种大鼠肝脏内肽酶活性具有较宽的pH范围（在pH 5.5至10.0之间最大活性> 70%），最适pH为8 - 10。它是一种依赖Ca(2+)的活性，在0.5至5 mM Ca2+之间活性最大，在50至90 microM Ca2+之间被激活至最大活性的一半。内蛋白水解活性不受丝氨酸、半胱氨酸或天冬氨酸蛋白酶的基团特异性抑制剂或1,10 - 菲咯啉的抑制；然而，EDTA和1,2 - 环己二胺四乙酸确实抑制了该活性，但这是由Ca2+螯合引起的。IRP肽底物测定法能够测量该内肽酶活性的表观Km为22 microM，Vmax为18.6 pmol/min。这些生化特性表明胰岛素原受体加工内肽酶活性是Kex2相关前蛋白转化酶家族的一个合理成员。免疫印迹检测到弗林蛋白酶和PACE4蛋白（该家族的两个成员）存在于含有胰岛素原受体加工活性的大鼠肝脏亚细胞组分中。由于胰岛素原受体加工内肽酶活性的生化特性大多类似于弗林蛋白酶活性，因此胰岛素原受体的蛋白水解成熟很可能在肝脏中由弗林蛋白酶催化。
