Duckworth W C, Peavy D E, Hamel F G, Liepnieks J, Brunner M R, Heiney R E, Frank B H
Veterans Administration Medical Center, Omaha, NE.
Biochem J. 1988 Oct 1;255(1):277-84.
Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.
对胰岛素原生物活性的研究得出了大相径庭的结论。相对于胰岛素，胰岛素原的生物活性报道范围从不到1%到近20%不等。许多体外检测胰岛素原生物效能的方法都利用了分离的大鼠脂肪细胞。为了进一步研究胰岛素原与大鼠脂肪细胞的相互作用，我们制备了特异性标记的胰岛素原异构体，这些异构体在对应于胰岛素A14、A19、B16或B26残基的酪氨酸残基上进行了碘化。将它们与大鼠脂肪细胞一起孵育，并通过三氯乙酸沉淀、葡聚糖凝胶G - 50色谱法和高效液相色谱法检测其代谢情况。通过三氯乙酸沉淀法检测，几乎没有胰岛素原降解。然而，通过G - 50色谱法及随后的高效液相色谱分析，我们发现标记的胰岛素原异构体迅速且几乎完全转化为在高效液相色谱上洗脱行为与完整胰岛素原不同的物质。这种转化主要归因于从用于分离细胞的粗制胶原酶中吸附到脂肪细胞上的蛋白水解活性。发现了两种主要的转化中间体：一种在胰岛素原的23 - 24位残基（胰岛素的B链区域）处有切割，另一种在连接肽区域的55 - 56位残基处有切割。这些中间体具有与完整胰岛素原相当或更低的受体结合特性。这些发现表明，由于制备过程中受到用于分离细胞的胶原酶的蛋白水解活性污染，分离的脂肪细胞可将胰岛素原降解为中间体。因此，先前报道的脂肪细胞中胰岛素原生物活性的范围可能是由这种蛋白酶污染引起的。最后，目前的研究结果表明，需要一种灵敏的激素降解检测方法来检测分离细胞中的生物活性。
