[Peculiarities of distribution of 125-I-insulin and 125I-insulin-like growth factor-I (IGF-I) at internalization in isolated rat hepatocytes].

One of determining conditions of formation in vertebrate phylogenesis of hormonal systems of insulin and IGF-I--peptides common by origin, similar by structure and biological action is temperature factor. In differentiation of functional roles of two related hormones and important place id ascribed to mechanism of their intracellular action. Study of formation of the two hormonal systems in vertebrate phylogenesis necessitates knowledge of peculiarities of internalization of two related hormones in mammals. On isolated rat hepatocytes at the identical maximal level of internalization of 125I-insulin and 125I-IGF-I there are revealed marked differences of dynamics of their internalization. Besides, at internalization of 125I-insulin or of 125I-HDP-I, their peculiar distribution was observed in the cell and on the plasma membrane. At 37 degrees C only two thirds of 125I-insulin relative to the bound to receptors on the membrane were immersed into cell, whereas the portion of internalized 125I-IGF-I turned out to be higher than the part located on the membrane. At 12 degrees C the decrease of 125I-insulin inside the cell and its increase on the membrane indicate indicated the interdependent label redistribution under these conditions. The shape of the 125I-iφP-I distribution at low temperature remained unchained. The pattern of established differences of internalization of 125I-insulin and 125I-IGF-I as well as different sensitivity of each of the processes to low temperature indicated that each of the peptides triggered individual mechanism of endocytosis of receptors. The high sensitivity of internalization of 125I-insulin to low temperature and the temperature lability of the process in isolated rat hepatocytes agree with the earlier stated suggestion about the lat formation of the regulatory insulin system in homoiothermal vertebrates.

[Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.

[The structural organization of binding determinants in insulin-like growth factor-I (IGF-I) molecule].

Insulin-like growth factor I (IGF-I) is a peptide related to insulin and IGF-II. These three related peptides produce similar biological effects, but each of them has its irreplaceable physiological significance in the organism. Multisided functional role of IGF-I in the organisms is due to its unique binding properties. Specifically, but with different degree of affinity, it is able to interact with three receptors (IGF-l-receptor, insulin receptor, and IGF-2-receptor) and six binding proteins (IGFBP 1-6). To interact with each of the above objects, the 1GF-I molecule contains individual structural determinants--binding domains (BD) providing strict specificity of interaction with them. Responsible for the IGF-I biological effects and binding with IGF-l-receptor is alpha-domain, for binding with insulin receptor--beta-, IGF-2--gamma-, while with all BP--delta-BD, respectively. Results of experimental study of binding domains are not a always can be estimated unanimously. The proposed by the author system of criteria for evaluation of changes in affinity of the IGF-I analogies proposed by the author allows unraveling the structural organization of each of the domains and tracing dependence on it of the peptide affinity to the particular object. This work considers composition, organization, and principle of formation of affinity of there binding IGF-I domains (alpha-, gamma-, and delta-BD). The alpha-domain includes three tyrosines from three different molecule sites (B-24, C-3 1, and A-60) disposed spatially in the direct vicinity on its one surface. The delta-domain also is considered as the domain participating in the high-affinity interaction; by composition and location in molecule it principally differs from alpha-BD, with the structural organization that so far has not been decoded. Analyzed in detail is the key significance of the N-terminal site of the B-chain--the linear site of the domain--for binding of IGF-I with BP, functional heterogeneity of its constituent residues, and the characteristic principle of formation of affinity to BP. Analysis indicates a probability of the second site of delta-BD, quite possibly not the only one, and a high sensitivity of the domain to configuration of the IGF-I molecule surface. Structural organization and peculiarities of affinity formation in the gamma-domain argammae studied the best in three related peptides; it consists of two linearly exposed sites of A-chain. Composition of the site S-1 A (Phe8, Arg9, Serl 0) provides a possibility of binding the ligand with IGF-I-receptor, while the level ofaf-finity to it depends on the composition of S-2. The S-2 composition A (Arg14, Arg15) determines the low affinity of IGF-I to the IGF-2-receptor. The clear functioning of IGF-I and elimination of mixture of functions at the level of the binding activity depend on the spatial autonomy of different nature BD, difference in structural organization of each of the domains, and a peculiarity of principles affinity in each formation case. The spatial coordination of several BD sites is the condition for transmission of the "structural sig-nal" by regulatory peptide. The performed analysis provides the direct notion of dependence of the binding ability of the IGF-I molecule that has BD of different nature on their structural peculiarities and allows using the revealed regularities at searching for BD in the newly discovered insulin-like peptides.

[The binding ability of insulin-related peptides as the clue to their functional role in phylogenesis. Introduction to the problem].

The common plan of structure of the main peptides of the vertebrate insulin family--insulin itself, IGF-I, IGF-II, and relaxin--has distinct structural features. Each of the peptides performs its characteristic function. However, overlapping of insulin and IGF-I actions and its stability in the vertebrate phylogenesis have formed the concept of their regulation of growth and metabolism as a function fixed in phylogenesis for a certain type of structures. At the same time, study of insulin-related peptides in invertebrates has revealed the wider spectrum, than in vertebrates, of biological effects; this indicated that the similarity of the total structure designs not sufficient for judging about their functional role. Functional possibilities of a regulatory peptide depend fundamentally on its capability for binding to the receptor realizing its biological action. However, the binding ability has a wider significance than merely transmission of biological signals. Thus, IGF-II when interacting with receptors realizing its biological effects, also binds to he IGF-2 receptor limiting its action and, besides, to the binding proteins (BP) modulating its action. The entire cycle of interactions occurs in the organism at different affinity levels. Meanwhile insulin interacts neither with IGF-2 receptor nor with BP. In this case, specificity and sequence of interaction with each of receptors or with protein are due not to the general design of the peptide structure, but rather to structure of individual submolecular determinants--binding domains. The leading role in disclosure of composition and structure of these domains is played by the "mutant-ligand" approach evaluating affinity of modified analogs. To analyze role of structural elements of the binding domains, the author proposes the system of estimation of affinity of the studied analogs. The present work, alongside with consideration of methodical aspects of the forthcoming analysis, is an introduction to the problem of organization of the binding domains related directly with functional role of peptides of the insulin type. The proposed analysis is due to the necessity of specification of this organization both within one molecule and in different molecules with a similar plan of structure on the basis of nor always unambiguous literature data and of clarification of principles of structure of these domains.

[Purification and ligand-receptor analysis of insulin-related peptides from pedal ganglion of the mollusc Anodonta cygnea]. / Ochistka i ligand-retseptornyi analiz insulinopodobnykh peptidov pedal'nogo gandliia molliuska Anodonta cygnea.

Six insulin-related peptides (IRPs) from pedal ganglions of the molluscs Anodonta cygnea have been isolated and purified by reverse-phase chromatography. Each peptide (designated as IRP8-IRP13) showed its own retention time on the HPLC column. The testing of IRPs in radioreceptor systems specific for insulin and insulin growth factor-I (IGF-I) showed their ability to bind to both types of receptors. The concentration of IRPs, producing a 50% inhibition of porcine 125I-insulin binding with rat liver plasma membrane receptors (IC50) for IRP 10, was 1167 nM, IRP11--833 nM, IRP13--1333 nm. IRP8, IRP9, IRP12 in the maximum concentration of 10(4) ng/ml displaced less than 50% of labeled hormone. All of the six peptides were capable of competing with human 125I-IGF-I for binding to receptors of a fraction of rat brain membranes. IRP8, IRP9 and IRP12 had close means equal to 1167 nM, 1500 nM, 1167 nM, respectively. Another group including IRP10, IRP11 and IRP13 showed a much higher activity (833, 83 and 500 nM, respectively). The results obtained from radioligand analysis revealed the predominance of IGF-I binding properties in all peptides of pedal ganglions. At the same time, apparent proximity of IRP's physico-chemical characteristics to porcine insulin, and also the revealed dose-dependent binding to both insulin and IGF-I receptors suggest a bifunctionality of mollusc peptides. The expression level of this bifunctionality may be associated with the molecular structure pecularities of individual isoforms.

Isolation and characterization of insulin in Russian sturgeon (Acipenser guldenstaedti).

Insulin was isolated from the pancreas of Chondrostean fish, the Russian sturgeon, Acipenser guldenstaedti, by acid-ethanol extraction followed by ion-exchange and reverse-phase high-performance liquid chromatographies. The amino acid sequence determined by automated Edman degradation is as follows: A-chain (21-amino-acid peptide), H-Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Pro-Cys-Ser-Leu-Tyr-Asp-Leu-Glu-As n-Tyr-Cys-Asn-OH; and B-chain (31-amino-acid peptide), H-Ala-Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Va l-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Asn-Lys-Val-OH. The sturgeon insulin appears to be identical with one of two forms of paddlefish insulin and differs from the other form by a single substitution in the A-chain, Asp15: His15. The amino acid sequence of sturgeon insulin is more similar to the amino acid sequence of mammalian insulins than of other fish insulins. Sturgeon insulin showed parallel but weaker displacement than porcine insulin and pink salmon insulin in their respective radioimmunoassays and was less potent than porcine insulin in displacing radiolabeled porcine insulin bound to partially purified rat liver plasma membranes.

[Insulin binding by receptors of the plasma membranes of the liver in hens in ontogenesis]. / Sviazyvanie insulina retseptorami plazmaticheskikh membran pecheni kur v ontogeneze.

Specific binding of 125I-insulin to the liver plasma membranes was studied in the chick embryos from the 10th day of incubation on, in chickens and adult fowl. The level of binding was the same in all cases although the insulin concentration of blood increases during ontogenesis, the number of receptors and their affinity to the hormone remaining constant. The data on insulin-receptor interactions in the liver have been compared with the earlier results of the authors obtained on the chick skeletal muscle and erythrocytes.

[Erythrocyte insulin receptors in the ontogeny of chickens]. / Insulinovye retseptory éritrotsitov v ontogeneze kur.

Specific binding of 125I-insulin by the erythrocytes was studied in the chick embryos, chicken and adult fowl. The binding was shown to decrease during ontogenesis. The maximal level of binding was observed in the chick embryos. Within a month after hatching, it decreased to the level of adult fowl due, mainly, to the decrease in the number of receptors per erythrocyte. This phenomenon can be accounted for both by the increase of insulin content in blood with age and the age changes of the population of erythrocytes. It is proved that during ontogenesis of the domestic fowl the decrease of a portion of immature erythrocytes in the peripheral blood with age accounts for the dynamics of binding observed. At the same time the affinity of hormone receptors increased but the influence of this factor on the binding pattern was not leading.