Stentz F B, Harris H L, Kitabchi A E
Endocrinology. 1985 Mar;116(3):926-34. doi: 10.1210/endo-116-3-926.
We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.
我们研究了培养的人成纤维细胞中的胰岛素降解活性（IDA），并评估了各种胰岛素加工抑制剂对IDA的影响。为了评估胰岛素降解的三种酶（中性蛋白酶、微粒体谷胱甘肽胰岛素转氢酶和溶酶体酸性蛋白酶）的作用，我们将匀浆后的成纤维细胞亚分级为膜（和细胞核）胞质溶胶、线粒体、微粒体和溶酶体。发现超过90%的IDA存在于含有中性蛋白酶的胞质溶胶部分。完整成纤维细胞中的IDA被1 mM N-乙基马来酰亚胺完全抑制，被0.5 mM丹磺酰尸胺（75%）、0.5 mM氯喹（48%）、1 mg/ml杆菌肽（32%）和抑肽酶（30%）部分抑制。利多卡因（5 mM）和胰高血糖素（10^(-6)M）表现出约15%的抑制作用，不可抑制的胰岛素样活性的抑制作用最小（7%）。对亚分级成分的类似抑制剂研究表明，N-乙基马来酰亚胺（100%）、胰高血糖素（30%）、氯喹（41%）、不可抑制的胰岛素样活性（30%）、利多卡因（25%）、丹磺酰尸胺（16%）和杆菌肽（11%）对胞质溶胶酶有抑制作用。将硫酸铵分级的胞质溶胶酶与A14-125I-胰岛素在37℃孵育，早在1分钟时就产生了两个中间峰。这些峰可以通过高效液相色谱法鉴定，但不能通过分子筛色谱法鉴定。与完整胰岛素相比，这些中间体与抗胰岛素抗体的免疫沉淀性较低，与肝膜制剂的受体结合性也较低。将A14-125I-胰岛素与胞质溶胶酶进一步孵育导致这些峰以及胰岛素减少，并形成125I-碘酪氨酸峰。我们得出结论，人成纤维细胞能够以时间和温度依赖性方式代谢细胞相关的A14-125I-胰岛素。这个过程受到各种胰岛素加工抑制剂的抑制。大部分IDA由可溶性中性蛋白酶组成，其性质与其他更纯化的中性胰岛素蛋白酶制剂相似。这个部分与完整的成纤维细胞类似，将胰岛素降解为两种中间体，其分子量与完整胰岛素相似，但比完整胰岛素更具亲水性，与抗胰岛素抗体和肝膜的结合亲和力更低。
