Two Dimensional Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically-Labeled Pancreatic Islet
Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2D
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Introduction The secretory granules of pancreatic B cells perform a specialized function in the packaging, storage, and secretion of the hormone insulin (Fig. 1) [1]. In addition to insulin and the connecting (C-) peptide, these subcellular organelles contain more than 150 polypeptides [2]. This includes the proteases involved in proinsulin-toinsulin conversion, proinsulin conversion intermediates, other polypeptide precursor proteins, minor cosecreted peptides, membrane proteins involved in cell trafficking, and ion translocating proteins involved in regulation of the intragranular environment. Most of these proteins are likely to be synthesized, transported, and packaged into nascent granules in a coordinated manner to ensure correct functioning of the granule. Insulin biosynthesis is regulated by many circulating nutrients and other factors although glucose is the most important physiologically [3]. However, it is
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_15, © Springer Science+Business Media LLC 2017
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Paul C. Guest Pancreac Islet
Beta Cell
Insulin Granule
Fig. 1 Images of (a) pancreatic islet as seen through a light microscope, (b) pancreatic beta cell visualized by electron microscopy, and (c) insulin secretory granule obtained by high power electron microscopy. Each islet contains around 5000 cells of which 70–80 % are comprised of the insulin-producing pancreatic beta cells. The granules contain a dense core of insulin hexamers which comprise around 80 % of the protein mass and this is surrounded by less dense material containing approximately 150 different granule accessory proteins [1]
not known whether just some or all of the granule constituents are affected in a similar manner. This chapter addresses this question by two dimensional gel electrophoresis (2DE) [4] of secretory granule subcellular fractions prepared from 35S-methionine-labeled rat islets, as described by Guest and coworkers [5]. The protocol employed is a 1 h pulse labeling of pancreatic islets with 35S-methionine in the presence of either low or high glucose concentrations, followed by a chase period of 3 h in nonradioactive medium containing a low glucose concentration (Fig. 2). The production of mature insulin requires cleavage of proinsulin by the endoproteases prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2) on the carboxy-terminal side of Arg31-Arg32 and Lys64Arg65, respectively, followed by the removal of the exposed basic residues by the exopeptidase carboxypeptidase H [6, 7]. This conversion is optimal in the low pH and high Ca2+ environment in the late trans Golgi network and secretory granule compartments [1]. This fits with finding that final conversion of proinsulin to insulin does not begin to occur until approximately 30 min after initial synthesis on the rough endoplasmic reticulum and transport to these compartments. Thus, the 1 h pulse labeling and 3 h chase employed here ensures t
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