Bailyes E M, Shennan K I, Usac E F, Arden S D, Guest P C, Docherty K, Hutton J C
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):587-94. doi: 10.1042/bj3090587.
Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.
人激素原转化酶PC2在非洲爪蟾卵母细胞中表达，并将其特性与大鼠胰岛素分泌颗粒的2型内肽酶（先前鉴定为PC2）的特性进行了比较[贝内特、贝利耶斯、尼尔森、格斯特、卢瑟福、阿登和赫顿（1992年）《生物化学杂志》267卷，15229 - 15236页]。重组PC2与2型内肽酶具有相同的底物特异性，在人去31,32 - 胰岛素原的CA连接处（赖氨酸64、精氨酸65）切割以生成胰岛素；对人去64,65 - 胰岛素原或胰岛素原本身几乎没有活性。重组PC2在5 - 7 mM Ca²⁺中活性最高（K₀.₅ = 1.6 mM），而2型内肽酶在0.5 - 1 mM Ca²⁺中活性最高（K₀.₅ = 40 μM）。两种酶的最适pH均为5.0 - 5.5，但2型内肽酶在更宽的pH范围内有活性。发现重组PC2有两种分子形式（71 kDa和68 kDa），两者C末端均完整，但因前肽的存在而有所不同。内源性PC2由几种重叠形式组成（大小范围为64 - 68 kDa），其中约三分之二缺乏C末端免疫反应性。重组PC2和内源性PC2之间部分大小差异归因于N - 糖基化的差异。重组PC2和内源性PC2不同的翻译后蛋白水解修饰并不能解释酶所表现出的不同pH和Ca²⁺敏感性。不能排除碳水化合物对酶活性的调节作用。
