Michael J, Carroll R, Swift H H, Steiner D F
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1987 Dec 5;262(34):16531-5.
Secretory granule-enriched fractions prepared from isolated rat islets of Langerhans, previously labeled in culture for 18 h with [3H]leucine, have been lysed and separated into pH 5.4 soluble and insoluble fractions by zonal sucrose gradient centrifugation. A high proportion of both labeled and immunoreactive rat insulins I and II were recovered in the insoluble granule core fraction in the expected ratio of approximately 60/40, respectively. Essentially equivalent amounts of the rat C-peptides on a molar basis were recovered in the granule supernatant fractions. The proportion of labeled proinsulin in the granule core fraction was less than 2% relative to insulin, while the soluble fraction contained about 8%, which probably arose mainly from disrupted proinsulin-rich noncrystalline prosecretory vesicles. Electron microscopic examination of the granule core fraction revealed large numbers of well preserved crystalline cores exhibiting typical dimensions and regular internal spacings of normal mature rat beta-granule inclusions. These results provide direct biochemical evidence that the beta-granules are nonuniform in composition with the insulin contained mainly in a crystalline state in the electron-dense central inclusions while the C-peptide is dissolved in the fluid bathing the crystalline hormone. The significance of this structural organization of the beta-granule is discussed.
从分离出的大鼠胰岛中制备富含分泌颗粒的组分，这些胰岛先前在培养物中用[³H]亮氨酸标记18小时，然后进行裂解，并通过区带蔗糖梯度离心分离成pH 5.4的可溶和不溶组分。标记的和具有免疫反应性的大鼠胰岛素I和II中，很大一部分以预期的约60/40的比例分别在不溶性颗粒核心组分中回收。在颗粒上清液组分中回收了基本上等摩尔量的大鼠C肽。相对于胰岛素，颗粒核心组分中标记的胰岛素原比例小于2%，而可溶组分中约含8%，这可能主要源于富含胰岛素原的非晶态前分泌小泡的破裂。对颗粒核心组分的电子显微镜检查显示，大量保存完好的结晶核心，呈现出正常成熟大鼠β颗粒内含物的典型尺寸和规则内部间距。这些结果提供了直接的生化证据，表明β颗粒在组成上是不均匀的，胰岛素主要以结晶状态存在于电子致密的中央内含物中，而C肽则溶解在包围结晶激素的液体中。本文讨论了β颗粒这种结构组织的意义。
