THE INSULIN-SECRETORY-GRANULE CARBOXYPEPTIDASE-H - PURIFICATION AND DEMONSTRATION OF INVOLVEMENT IN PROINSULIN PROCESSING
THE INSULIN-SECRETORY-GRANULE CARBOXYPEPTIDASE-H - PURIFICATION AND DEMONSTRATION OF INVOLVEMENT IN PROINSULIN PROCESSING
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DOI：
10.1042/bj2450575
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发表时间：
1987-07-15
影响因子：
4.1
通讯作者：
HUTTON, JC
中科院分区：
文献类型：
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作者：
DAVIDSON, HW;HUTTON, JC
在纯化的胰岛素分泌颗粒的可溶性部分中检测到羧肽酶B样酶，并与胰岛素生物合成有关。为了进一步研究这种活性的作用，我们通过凝胶过滤色谱和对氨基苯甲酰精氨酸亲和洗脱从大鼠胰岛素瘤组织中纯化了该酶。获得了42%的产率，匀浆的纯化因子为674。在还原或非还原条件下通过SDS/聚丙烯酰胺凝胶电泳对纯化的羧肽酶进行分析，结果表明它是表观Mr 55000的单体蛋白。该制剂通过高效凝胶过滤色谱法也是均匀的。这种酶与伴刀豆球蛋白A结合，表明它是一种糖蛋白。氨基酸分析或化学去糖基化和SDS/聚丙烯酰胺凝胶电泳表明，蛋白质的Mr为50000，这表明碳水化合物含量约为。9%的重量。纯化的酶能够从胰岛素原胰蛋白酶肽的C-末端去除碱性氨基酸以产生胰岛素，但不进一步降解成熟激素。它被EDTA，1，10-菲咯啉和胍基乙基巯基琥珀酸抑制，并刺激5倍的氯化钴。二乙酰基-胰岛素转化为胰岛素的最适pH在5-6的范围内，在pH 6.5以上几乎没有活性。还表达了对丹磺酰化三肽底物（丹磺酰-苯丙氨酰-亮氨酰-精氨酸; Km = 17.5 μ M）的活性，并且具有5.5的最适pH。这些性质与位于分泌颗粒中的活性是不可区分的，并且与颗粒内环境相容。胰岛素分泌颗粒羧肽酶具有牛肾上腺髓质和垂体羧肽酶H的一些特性。我们提出，我们纯化的羧肽酶是羧肽酶H的胰腺同工酶（crino羧肽酶B; EC 3.4.17.10），并且参与胰腺β-淀粉酶中胰岛素的生物合成。cell.
A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55000. The preparation was also homogenous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein of Mr of 50000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 .mu.M), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic .beta.-cell.