Lamango N S, Zhu X, Lindberg I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
Arch Biochem Biophys. 1996 Jun 15;330(2):238-50. doi: 10.1006/abbi.1996.0249.
Although previous efforts to produce significant quantities of purified prohormone convertase 2 from either recombinant or natural sources have been unsuccessful, our recent finding that the neuroendocrine polypeptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombinant enzyme from the conditioned medium of PC2-producing CHO cells supertransfected with cDNA coding for 21 kDa 7B2. The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligram quantities. Two protein bands of Mrs 71 and 75 kDa were observed after SDS-PAGE followed by either Coomassie staining or Western blotting with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa species to the 66-kDa form occurred during incubation at pH 5.0; the degree of conversion correlated with a dramatic increase in activity. Kms of 124 and 131 microM and Kcats of 0.49 and 0.81 s(-1) were obtained for the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AMC, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested. Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2). Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection against thermal denaturation. When coassociated 7B2 was mostly removed from activated PC2 through gel filtration, subsequent addition of recombinant 7B2 exerted a significant stabilizing effect on enzyme activity. Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretory granule protein, our data suggest a physiological role for 21 kDa 7B2 in the stabilization of PC2 activity.
尽管此前从重组或天然来源大量生产纯化的激素原转化酶2的努力均未成功，但我们最近发现神经内分泌多肽7B2对于具有酶活性的激素原转化酶2（PC2）的生物合成是必需的，这使我们能够从用编码21 kDa 7B2的cDNA进行超转染的PC2产生型CHO细胞的条件培养基中获得活性重组酶。重组酶被纯化至表观均一，回收率为40%，产量达毫克级。用考马斯亮蓝染色或用PC2抗血清进行蛋白质印迹法检测后，在SDS-PAGE上观察到71 kDa和75 kDa的两条蛋白带。在pH 5.0孵育期间，71 kDa和75 kDa的蛋白自发转化为66 kDa的形式；转化程度与活性的显著增加相关。底物Cbz-Arg-Ser-Lys-Arg-AMC和Pyr-Arg-Thr-Lys-Arg-AMC的Km分别为124和131 μM，Kcat分别为0.49和0.81 s(-1)。最适pH为5.0，该酶被h7B2(155 - 185') p-CMS和EDTA抑制，但不受其他测试抑制剂的抑制。有趣的是，观察到21 kDa 7B2与该酶以约1:100（7B2:PC2）的摩尔比共纯化。将重组21 kDa 7B2预先添加到活化的66 kDa PC2中可提供显著的抗热变性保护。当通过凝胶过滤从活化的PC2中基本去除共结合的7B2后，随后添加重组7B2对酶活性产生显著的稳定作用。观察到这种作用需要毫摩尔浓度的Ca2+和5至6之间的pH。由于这些条件类似于分泌颗粒内被认为会出现的条件，并且由于21 kDa 7B2代表一种储存的分泌颗粒蛋白，我们的数据表明21 kDa 7B2在稳定PC2活性方面具有生理作用。
