The NSILA-s receptor in liver plasma membranes. Characterization and comparison with the insulin receptor.

NSILA-s (nonsuppressible insulin-like activity, soluble in acid ethanol) is a serum peptide that has insulin-like and growth-promoting activities. We have demonstrated previously that liver plasma membranes possess separate receptors for NSILA-s and insulin and have characterized the insulin receptor in detail. In the present study we have characterized the properties and specificity of the NSILA-s receptor and compared them to those of the insulin receptor in the same tissue. Both 125I-NSILA-s and 125I-insulin bind rapidly and reversibly to their receptors in liver membranes; maximal NSILA-s binding occurs at 20 degrees while maximal insulin binding is seen at 1-4 degrees. The pH optimum for NSILA-s binding is broad (6.0 to 8.0), in contrast to the very sharp pH optimum (7.5 to 8.0) for insulin binding. Both receptors exhibit a high degree of specificity. With the insulin receptor, NSILA-s and insulin analogues compete for binding in proportion to their insulin-like potency: insulin greater than proinsulin greater than NSILA-s. With the NSILA-s receptor, NSILA-s is most potent and the order is reversed: NSILA-s greater than proinsulin greater than insulin. Furthermore, six preparations of NSILA-s which varied 70-fold in biological activity competed for 125I-NSILA-s binding in order of their potencies. NSILA-s which had been inactivated biologically by reduction and aminoethylation and growth hormone were less than 1/100,000 as potent as the most purified NSILA-s preparation. Purified preparations of fibroblast growth factor, epidermal growth factor, nerve growth factor, and somatomedins B and C were less than 1% as effective as NSILA-s in competing for the 125I-NSILA-s suggesting that these factors act through other receptors. In contrast, somatomedin A was 10% as active as NSILA-s and multiplication-stimulating activity was fully as active as NSILA-s in competing for the NSILA-s receptor. Analysis of the data suggests that there are approximately 50 times more insulin receptors than NSILA-s receptors per liver cell, while the apparent affinity of NSILA-s receptors is somewhat higher than that of the insulin receptor.     

Met-Lys-双C肽人胰岛素原基因的构建表达及分离纯化

应用 Ｐ Ｃ Ｒ 定点突变方法构建编码 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原基因,并在大肠杆菌中以包含体方式获得表达 表达产物经还原、重组、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 分离纯化,获得 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原,经胰蛋白酶与羧肽酶 Ｂ的酶解, Ｒｅｓｏｕｒｃｅ Ｔ Ｍ Ｑ 阴离子交换柱层析分离制备得人胰岛素,其放免活性、受体结合活性均与猪胰岛素相同      

Multiplication stimulating activity (MSA) can substitute for insulin to stimulate the secretion of testicular transferrin by cultured Sertoli cells

Both MSA and insulin were found to stimulate transferrin production in cultured Sertoli cells to the same extent in the absence or presence of follitropin, testosterone, or retinol. Sertoli cells were responsive to 30-fold lower concentrations of MSA than of insulin. MSA and insulin together stimulated transferrin secretion to the same extent as either hormone alone. These results, and what is presently understood about the relationship of MSA and insulin, suggest that insulin can substitute for the action of an MSA-like peptide in the stimulation of testicular transferrin secretion by Sertoli cells.     

Binding of insulin-like growth Factor ii and multiplication-stimulating activity-stimulated phosphorylation in basolateral membranes from dog kidney

Biologic actions of insulin and insulin-like growth factors (IGFs) are thought to be initiated by binding of peptides to tissues, followed by phosphorylation of specific hormone receptors. Both insulin and IGF bind to renal membranes, suggesting functional roles for these peptides in kidney. The present studies further characterize the interaction of multiplication-stimulating activity (MSA)/IGF II with its renal receptor. Specific binding of 125I-IGF II was measured in basolateral membranes isolated from proximal tubular cells of dog kidney. Binding was half-maximal at 10(-9) M MSA and was not inhibited by human growth hormone, IGF I, insulin, or anti-insulin receptor antibodies. Concentration-dependent MSA-stimulated phosphorylation of a Mr 135,000 protein band was demonstrated in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels from basolateral membrane suspensions. Insulin increased phosphorylation of this band only in the presence of MSA, while a Mr 92,000 band was consistently phosphorylated with insulin alone. The phosphorylated Mr 135,000 band which had been solubilized with detergent from basolateral membranes was immunoprecipitated using serum from a patient with anti-insulin receptor antibodies suggesting that the band is the alpha subunit of the insulin receptor. This was supported by the demonstration of covalent cross-linkage of 125I-insulin to the Mr 135,000 band. We conclude that receptor-mediated MSA-stimulated phosphorylation of isolated basolateral membranes may reflect a process by which biological actions of IGF II are mediated in vivo. Our data suggest that insulin and IGF II may interact by regulating protein phosphorylation.     

The binding of rat liver cell multiplication-stimulating activity (MSA) to human placenta and serum proteins.

Multiplication-stimulation activity (MSA) from the medium of BRL-3A rat liver cells in culture binds to cell membrane and cytosol receptors from human placenta and to serum proteins. The binding of MSA to placental cell membranes is dependent on time, temperature, pH and divalent ion concentration. MSA bound to placental cytosol receptor and serum is not displaced by insulin, whereas that bound to placental cell membranes is displaced by insulin and insulin-like peptides. The affinity of the three receptors for MSA is similar [approximately 10(8) M(-1)]. An assay using 125I-MSA and placental membrane receptor detects somatomedin-like receoptor activity (SmLRA) in unextracted sera from man and animals. A binding protein in serum that competes for 125I-MSA with receptor could not be completely separated from SmLRA by heating, acidification, charcoal treatment and gel chromatography of the serum. The relative activities of SmLRA and serum binding protein remained constant in three disorders of human growth (acromegaly, growth hormone deficiency and Laron's dwarfism) in which values of SmLRA varied widely. However, the binding protein is only partly responsible for the apparent SmLRA of unextracted serum. It is concluded that MSA is a suitable radioligand for the investigation of somatomedin disorders in man either by receptor assays or by studies of tissue receptors.     

Secretory expression of active clostripain in Escherichia coli

In this study, the clostripain gene was modified and its signal sequence was replaced with that of penicillin G acylase (PGA). The core clostripain protein fused to the PGA signal peptide was also prepared. With regard to the expression of the clostripain precursors, the majority of clostripain activity was observed in the culture media, thereby indicating that both the clostripain signal peptide and the PGA signal peptide were recognized in the E. coli secretion pathway, and the precursors successfully matured into the active form. Otherwise, the activity was rather low when the core protein was expressed, which indicates that the clostripain pro-peptide is important in the formation of the active enzyme in E. coli. Enzyme activity reached a value of 3200U/L in CGY media for high expression. The recombinant clostripain and porcine carboxypeptidase B were used in the conversion of a proinsulin fusion protein into insulin. The leader peptide (LP) and the proinsulin C-peptide appeared to have been removed simultaneously, and the final cleavage product evidenced an HPLC retention time identical to that of the insulin standard, thereby implying that the clostripain specifically cleaved the arginine residues in the LP and in the C-peptide. We have also demonstrated the possibility that the recombinant clostripain might prove useful in the production of insulin from the proinsulin fusion protein.