Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies were carried out in order to characterize specific insulin binding sites in the rat pituitary gland. Binding of labeled insulin by pituitary microsomes reached equilibrium after 4 h at 4 degrees C and remained stable over 16 h; at 25 degrees C the plateau was reached in 20 min. Equilibrium binding data analysis of competitive displacement of bound 125I-iodo insulin by unlabeled insulin yielded a non-linear Scatchard plot. At 25 degrees C the Kd for the high affinity component was 2.8 +/- 0.1 X 10(-9) M and the receptor concentration was 260 +/- 80 fmol/mg of microsomal protein. A Kd value of 4.6 +/- 0.4 X 10(-8) M and a binding capacity of 800 +/- 200 fmol/mg microsomal protein were obtained for the low affinity sites. Insulin binding to microsomes was enhanced 2.7 times by increasing the ionic strength of the incubation medium with 2 M NaCl, and was abolished when the microsome preparation was preincubated with trypsin prior to binding measurements. Other hormones, such as bovine thyrotropin, ovine follitropin, human somatotropin and ovine prolactin did not interact with the insulin receptor. Proinsulin displaced the labeled hormone in direct proportion to its insulin-like biological activity.
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PMID:Specific binding sites for insulin in the rat pituitary gland. 390 55

Covalent linking of two photoactivatable insulin derivatives, B2-(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin and B29-(2-nitro,4-azidophenylacetyl)-insulin to viable rat adipocytes gives a system, which contains a fixed stoichiometry between hormone and receptor. The biological signal of prolonged lipogenesis has been used to study several aspects of insulin binding and action: the role of the site of the crosslink between insulin and receptor, recognition of bound photoinsulin by anti-insulin antibodies, the half-life of the biologically active complex, the pH-dependence of the biological signal, and the possible role of internalization. Furthermore, the effect of trypsin on the insulin receptor, as well as the insulin-receptor complex, has been investigated and a refined model of the receptor is presented.
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PMID:Biological action and fate of photoaffinity-labelled insulin-receptor complexes. 390 15

Using 10-15 day neonatal rabbit brain cells, we studied the internalization (n = 6) and intracellular degradation (n = 8) of specifically bound 125I-insulin. In addition we investigated the association between the internalization of the specifically bound 125I-insulin and the metabolic effects of insulin such as glucose (n = 13) and amino-acid (leucine) uptake (n = 6). Phenylarsine oxide (10 microM), an agent that inhibits the internalization of the insulin receptor (n = 6) decreased the specifically bound 125I-insulin in the intact and trypsin-resistant (inside) part of the brain cells by 50% (p less than 0.05). On the other hand chloroquine (100 microM), a lysosomotropic agent that interferes with the intracellular degradation of the insulin receptor (n = 8) increased two-fold the 125I-insulin specifically bound to the intact and trypsin resistant part of the cells (p less than 0.05). Both these agents did not alter the time-dependent basal glucose uptake by the brain cells. Glucose alone regulated its own uptake (n = 4) whereas 1 X 10(-6) M insulin did not augment the glucose uptake (n = 11+13) above basal. Similarly leucine regulated the leucine uptake (n = 4) but insulin did not alter this basal uptake by the brain cells (n = 6). In summary we observed no associated glucose or leucine uptake along with the presence of internalization and intracellular degradation of specifically bound 125I-insulin in the brain cells.
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PMID:Internalization of the neonatal brain insulin receptor. 408 93

Immobilized insulin, prepared by coupling insulin directly to agarose or through hydrocarbon "connecting arms," was demonstrated to be capable of firmly binding intact adipocytes and their ghosts. Various lines of evidence indicate that the insulin receptor on the plasma membrane, in addition to the insulin coupled to the agarose, was responsible for the observed binding. This evidence includes: (a) the finding that increasing the "arm" length increased the binding capacities of insulin-Sepharose affinity chromatographic columns, (b) specific inhibition and reversal by insulin and antiserum to insulin of the binding, as compared to lesser effects by other peptide hormones, (c) the indication that only the plasma membrane sacs, not the other cellular contaminants in the crude ghosts, are capable of binding, and (d) the impairment and restoration of trypsin-sensitive membrane binding sites that are also required for insulin biosensitivity. These findings support the idea that the insulin receptor is the trypsin-sensitive site. By use of the differential buoyant densities of the various cell-bead complexes that resulted from the interaction of adipocytes with insulin-Sepharose, a new procedure was developed to demonstrate and study the binding. These complexes could also be demonstrated by interference contrast microscopy. Binding readily occurred under conditions favorable for insulin stimulation of the cells. By coupling tracer amounts of [(125)I]insulin to Sepharose or insulin-Sepharose, the effects of anti-insulin antisera, free insulin, and other peptide hormones and supplemental factors on the buoyant-density distribution of the complexes could be measured, as well as the effects of other ligands coupled to Sepharose.
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PMID:Affinity binding of intact fat cells and their ghosts to immobilized insulin. 435 4

Spheroidal aggregates formed from trypsin-dissociated 14-day embryonic chicken hearts after 48 hr of rotation on a gyratory shaker. Intracellularly recorded resting membrane potentials of aggregates bathed in 1.3 mM K+ balanced salt solution had a mean (+/- SD) of 64 +/- 4 mV. After a stable potential was achieved, addition of 1-100 nM sodium bovine insulin caused a slow hyperpolarization of up to 19 mV after 4-5 min, followed, in some cases, by a further, more rapid, shift to a potential near EK. Equivalent hyperpolarizations were observed when insulin was added in the presence of 10 mM ouabain, indicating that enhanced Na+,K+ pump activity was not responsible for the change in membrane potential. The concentration of insulin that produced half-maximal hyperpolarization (2 nM) corresponded to the association constant of a high-affinity insulin receptor, suggesting that binding to this class of receptors led to the change in membrane potential. Steady-state current-voltage curves from current clamp experiments suggested that insulin produced an increase in slope conductance at potentials near rest by inducing an outward current with an apparent potential negative to -90 mV.
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PMID:Ouabain-resistant hyperpolarization induced by insulin in aggregates of embryonic heart cells. 624 86

The peripheral cycle AMP phosphodiesterase from rat liver plasma membranes binds with high affinity (2.4 nM) to a single class of receptor sites on the liver plasma membrane. These receptor sites appear to be proteins, as they are trypsin- and heat-labile. The sensitivity of these sites to denaturation by trypsin and heat is a first-order process. The presence of Ca2+ (5 mM) increases the affinity of these sites for the enzyme, but does not alter their total number. The receptor sites and the cyclic AMP phosphodiesterase occur in similar numbers, at around 2 pmol/mg of plasma-membrane protein. It is proposed that the peripheral, liver plasma-membrane cyclic AMP phosphodiesterase is attached to a specific site on the insulin receptor and that the binding of insulin to the receptor site triggers a conformational change in the enzyme such that the enzyme can be phosphorylated and activated by an endogenous cyclic AMP-dependent protein kinase.
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PMID:The insulin-stimulated cyclic AMP phosphodiesterase binds to a single class of protein sites on the liver plasma membrane. 627 57

A model for the minimum subunit composition and stiochiometry of the physiologically relevant insulin receptor has been deduced based on results obtained by affinity labeling of this receptor in a variety of cell types and species. We propose that the receptor is a symmetrical disulfide-linked heterotetramer composed of two alpha (apparent Mr = 125,000) and two beta (apparent Mr = 90,000) glycoprotein subunits in the configuration (beta-S-S-alpha)-S-S-(alpha-S-S-beta). The disulfide or disulfides linking the two (alpha-S-S-beta) halves (class I disulfides) exhibit greater sensitivity to reduction by exogenous reductants than those linking the alpha and beta subunits (class II disulfides). When the class I disulfides are reduced by addition of diothiothreitol to intact cells, the receptor retains its ability to bind insulin and to effect a biological response. The beta subunit contains a site at about the center of its amino acid sequence that is extremely sensitive to proteolytic cleavage by elastaselike proteases, yielding a beta 1 fragment (Mr = 45,000) that remains disulfide linked to the receptor complex and a free beta 2 fragment. Binding of insulin to the receptor complex appears to result in the formation or stabilization of a new receptor conformation as evidenced by an altered susceptibility of the alpha subunit to exogenous trypsin. A receptor structure with high affinity for insulinlike growth factor (IGF) I and low affinity for insulin in fibroblast and placental membranes has also been affinity labeled. It exhibits the same structural features found for the insulin receptor, including two classes of disulfide bridges and beta subunits highly sensitive to proteolytic cleavage. These recent observations identifying the presence of distinct insulin and IGF-I receptors that share similar complex structures suggest that these hormones may also share common mechanisms of transmembrane signaling.
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PMID:Subunit structure and dynamics of the insulin receptor. 628 75

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.
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PMID:Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells. 629 88

Using the technique of radiation inactivation we have previously shown that the insulin receptor behaves as if it is composed of at least two functional components: a binding component (Mr approximately equal to 100,000) and an affinity regulatory component (Mr approximately equal to 300,000). The interaction between the affinity regulator and binding component results in a decrease in the affinity of the receptor for insulin. To examine in more detail the interaction between this "affinity regulator" and the binding component we have studied the insulin receptor by radiation inactivation under conditions which alter receptor concentration or receptor affinity. Liver membranes of ob/ob mice exhibit a decrease in insulin binding when compared to their lean litter mates which is due to a decrease in receptor concentration. When studied by radiation inactivation, however, there was no detectable change in the interaction or size of the two receptor components. By contrast, under circumstances in which the affinity of the receptor was increased (treatment with high salt, high pH, 1 mM dithiothreitol, 1-5 micrograms/ml of trypsin), the interaction between the regulatory and binding components was either decreased or absent, i.e. there was no increase in binding with irradiation. Conversely, conditions which produce a decrease in receptor affinity resulted in an increase in the interaction between the regulatory and binding components. The changes in receptor affinity and interactions of the two components produced by either high salt or pH were reversible. Partial purification of the solubilized receptor on lectin affinity columns resulted in the apparent removal of the affinity regulator, i.e. receptor affinity was increased. In this state, radiation inactivation studies revealed a monoexponential decay indicating no interaction between binding and regulatory components. Taken together, these results suggest that the affinity regulator is a membrane protein which is both trypsin-sensitive and has disulfide bond(s) essential for its function. The interaction between the affinity regulator and binding component is not via a covalent bond and the two components appear to be separated by lectin chromatography. The interaction between these components appears to be altered in most states associated with altered receptor affinity.
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PMID:Characterization of a membrane regulator of insulin receptor affinity. 634 82

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.
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PMID:Hormone-induced conformational changes in the hepatic insulin receptor. 634 41


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