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PubMed1994

Isolation of cell populations from the mare corpus luteum: comparison of mechanical and collagenase dissociation.

Corpora lutea were obtained from mares at days 3, 10 and 14 after ovulation, and examined histologically. The morphology of isolated luteal cells obtained by either mechanical or collagenase dissociation of the tissue was examined and the cells stained to detect the steroidogenic enzyme delta 5, beta-hydroxysteroid dehydrogenase. The ratio of large:small cells was significantly higher for cells obtained from mechanically dissociated luteal tissue than for cells obtained by collagenase dissociation (P < 0.01). Cells obtained by both mechanical and collagenase dissociation secreted progesterone, although neither cell population responded to exogenous gonadotrophin with an increase in progesterone secretion. Homogenates of equine luteal tissue bound 125I-labelled human LH with high affinity and specificity, and the specific activity and binding affinity of luteal LH receptors did not change significantly from day 3, to days 10 and 14 after ovulation. However, mechanically dissociated cells on days 10 and 14 bound significantly more LH than did collagenase-dissociated cells on these days (P < 0.05). These results indicate that (i) collagenase dissociation of mare luteal tissue yields a population of cells that is unrepresentative of the corpus luteum, and (ii) the mare corpus luteum is not responsive to LH in vitro at the stages examined.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Radiologic diagnosis of traumatic occipitovertebral dissociation: 2. Comparison of three methods of detecting occipitovertebral relationships on lateral radiographs of supine subjects.

OBJECTIVE: The purpose of this study was to describe a method for recognizing acute traumatic occipitoatlantal dissociation that uses the basion-axial interval and basion-dental interval and to compare the accuracy of this method with the accuracies of two other methods: the Powers ratio and the x-line method. MATERIALS AND METHODS: Lateral radiographs of the cervical spine of 37 patients in whom the diagnosis of occipitoatlantal dissociation had been made on the basis of the relationship of the basion to the tip of the dens, the Powers ratio, and/or the x-line method were reviewed. Retrospectively, the occipitoatlantal junction of each was reassessed by using the the Powers ratio, the x-line, and the basion-axial interval-basion-dental interval methods. Independently, the neurologic findings at admission and the final neurologic diagnosis at discharge were obtained from the hospital records and were compared with the radiologic findings to determine the degree of radiologic-clinical correlation. RESULTS: Three groups of patients were identified by analyzing the basion-axial and basion-dental intervals of the occipitovertebral junction and related clinical findings. Twenty-three patients (group 1) had frank occipitoatlantal dislocation. Eight patients (group 2) had incomplete occipitoatlantal dissociation, which was defined as occipitoatlantal subluxation. The remaining six patients (group 3) had normal radiologic and clinical findings. Four patterns of occipitovertebral dissociation were identified: purely anterior (4/31, 13%), purely distracted (6/31, 19%), concomitantly anterior and distracted (20/31, 65%), and purely posterior (1/31, 3%). Regardless of the magnitude or direction of occipitoatlantal dissociation, the basion-axial interval-basion-dental interval method correctly identified the abnormality and the type of each. Positive clinical correlation was found in 13 (57%) of the 23 patients in group 1 and in 100% of the eight and six patients in groups 2 and 3, respectively. Neither the Powers ratio nor the x-line method could be applied in 17 (46%) of 37 cases, either because the opisthion could not be detected on the radiographs or because fusion of the posterior arch of C1 had not occurred. In the remaining 20 patients in whom the Powers ratio and the x-line method were applicable, the type of occipitoatlantal dissociation was correctly identified by the Powers ratio in 12 (60%) and by the x-line method in four (20%). Neither the Powers ratio nor the x-line method was applicable in three (50%) of the six patients in whom analysis by the basion-axial interval-basion-dental interval method and clinical findings showed no occipitoatlantal abnormality. Normal occipitovertebral anatomy was correctly identified by the Powers ratio in the remaining three patients (50%) and by the x-line method in two (33%). CONCLUSION: Direct measurement of occipitovertebral skeletal relationships altered by occipitoatlantal dissociation using the basion-axial and basion-dental intervals provides the most accurate radiologic assessment of this injury.

Atlanto-Occipital Joint↗
PubMed1980

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols.

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).

Animals↗
PubMed1981

Regulation of cAMP-dissociation kinetics in lactating rat mammary gland.

The cAMP-dissociation kinetics of rat mammary gland cytosols are dependent upon the temperature of cAMP association. Dissociation rates (measured at pH 6.5, 24 degrees C) were biphasic (k = 0.08-0.23 min-1 and k = 0.02 min-1) and monophasic (k-1 = 0.02 min-1) after 0 degrees C and 24 degrees C association, respectively. The temperature-dependent change from an initial fast rate to an initial slow rate was observed at all concentrations of cAMP tested from 1 to 1000 nM. When the slow-dissociating site was associated with non-radioactive 8-bromo-cAMP, the dissociation rates of [3H]-cAMP from the remaining dissociating site was slow (k = 0.02 min-1) and fast (k = 0.05 min-1) at 24 degrees C and 0 degrees C associating rate can be converted to the slow-dissociating rate by warming. When 0.2 M sodium thiocyanate was added to the association mixture at 24 degrees C, biphasic dissociation rates of k = 0.23 min-1 and k = 0.02 min-1 were observed, suggesting that the chaotropic salt blocks the interconversion of rates. The data are consistent with the model for cAMP-dependent protein kinase which exhibits two binding sites with different affinities. The type II enzyme from mammary gland cytosol exhibits in addition the phenomenon of temperature-dependent interconversion of the two binding affinities.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Dissociation of enzyme oligomers: a mechanism for allosteric regulation.

Most enzymes exist as oligomers or polymers, and a significant subset of these (perhaps 15% of all enzymes) can reversibly dissociate and reassociate in response to an effector ligand. Such a change in subunit assembly usually is accompanied by a change in enzyme activity, providing a mechanism for regulation. Two models are described for a physical mechanism, leading to a change in activity: (1) catalytic activity depends on subunit conformation, which is modulated by subunit dissociation; and (2) catalytic or regulatory sites are located at subunit interfaces and are disrupted by subunit dissociation. Examples of such enzymes show that both catalytic sites and regulatory sites occur at the junction of 2 subunits. In addition, for 9 enzymes, kinetic studies supported the existence of a separate regulatory site with significantly different affinity for the binding of either a substrate or a product of that enzyme. Over 40 dissociating enzymes are described from 3 major metabolic areas: carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism. Important variables that influence enzyme dissociation include: enzyme concentration, ligand concentration, other cellular proteins, pH, and temperature. All these variables can be readily manipulated in vitro, but normally only the first two are physiological variables. Seven of these enzymes are most active as the dissociated monomer, the others as oligomers, emphasizing the importance of a regulated equilibrium between 2 or more conformational states. Experiments to test whether enzyme dissociation occurs in vivo showed this to be the case in 6 out of 7 studies, with 4 different enzymes.

Allosteric Regulation↗
PubMed1984

The dissociation rate of unlabelled dopamine antagonists and agonists from the dopamine-D2 receptor, application of an original filter method.

A filtration technique was developed to measure the dissociation rate of unlabelled drugs from membrane receptors. Receptor preparations saturated with unlabelled drug were adsorbed to glass fibre filters positioned on a filtration apparatus. Dissociation of the drug from the receptor sites was achieved by repeatedly applying small buffer samples on the filter, this was followed by short incubation on the filter with a [3H]ligand. Rat striatal membrane preparations adsorbed on filters retained the same dopamine-D2 receptor binding properties as the tissue in aqueous suspension. Stereospecific [3H]haloperidol binding was maximal with 20 mg tissue per filter and 5 min incubation, KD = 2.8 nM and Bmax = 24 fmoles/mg tissue was found. The dissociation from the dopamine-D2 receptor for 18 dopamine antagonists and 3 agonists followed first order reaction kinetics. Half-lifes of dissociation ranged from 3.5 min for azaperone to 233 min for metitepine. There was no strict relationship between dissociation half-life and apparent equilibrium binding affinity or lipophilicity of the drugs. Half-life of receptor dissociation appeared neither to be a primary determining factor in the duration of pharmacological action of the drugs. The importance of the drug receptor dissociation rate for binding experiments in vitro as well as for chronic drug treatment is discussed.

Animals↗
PubMedDate not supplied

[Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures].

The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P. aeruginosa K-2 was studied. The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate. The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate. The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium. The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant. In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.

Carbon↗
PubMed2003

[Carpal instability with scapho-lunate dissociation treated using the flexor carpi radialis and scaphoid-trapezoid ligament repair: foundations, technique and results of preliminary series].

Carpal instability with scapho-lunate dissociation is still attributed to rupture of the so-called scapho-lunate ligament. Actually, this structure is not a ligament but a loose capsule allowing very different flexion of the scaphoid (92 degrees) and the lunate (20 degrees). As reconstruction of the scapho-lunate "ligament" has often been less than satisfactory we searched for another technique. Sections of the scapho-lunate "ligament" on cadaver specimens never produce scapho-lunate dissociation. This dissociation can not occur because the scaphoid is maintained in the radial facette. It was observed that the only way to produce scapho-lunate dissociation is to section the scapho-trapezo-trapezoid ligament allowing flexion of the scaphoid and dorsal dislocation out of the radial facette. The scapho-trapezo-trapezoid ligament is not well known and is not described in anatomy text books because it is hidden by the flexor carpi radialis tendon. It is confluent with the scaphoid and the trapezoid. This produces a radial (scapho-trapezoid) column which acts like a true external pillar maintaining the height of the carpus and preventing carpal collapse. Finally, dissociation of the proximal pole from the semi-lunate can only occur by posterior displacement. After experimenting the technique on cadaver specimens, we developed a reconstruction method for the palmar scapho-trapezoid ligament using a band of the flexor carpi radialis tendon, applied in 38 patients. The flexor carpi radialis band measured 7 cm and was left attached to the base of the second metacarpal then passed in a tunnel bored anteriorly to posteriorly in the distal pole of the scaphoid. The band was then drawn dorsally while maintaining the scaphoid in place, and sutured to the postero-ulnar border of the radius. The height of the carpus was restored as was scaphoid movement over the lunate. The reduction persisted at mid- and long-term and prevented carpal collapse and joint destruction. Among the 38 operated patients, 33 remained pain free and 5 complained of minor pain under stressful conditions. All were satisfied. Anatomic research and clinical experience has demonstrated that the scapho-trapezoidal ligament is the key to scapho-lunate dissociation and its correction. Currently, this operation is the only way to achieve easy and persistent correction of carpal instability with scapho-lunate dissociation.

Biomechanical Phenomena↗
PubMed2005

[The application of the dissociate bone flap in treatment of the macrosis depressed skull fracture on the frontal orbit part].

OBJECTIVE: To study the reconstructive effect of the dissociate bone flap to repair the macrosis depressed skull fracture on the frontal and orbit part. METHODS: The coronal scalp flap was elevated and dissociate bone flap was expanding to the 2cm width beside the edge of depressed skull fracture. The first step was to extract the dissociate bone flap and make there is an area for operating . Then extract free bone fragments, and elevate the depressed orbital lamina and use the biological glue to stick it to its position. The free fragments extracted were stacked into a whole one and it to its position in use of the biological glue on the dissociate bone flap. The uneven inner table should was smoother with bon-wax. The prosthetic dissociate bone flap was put back on its position and fixation. RESULTS: From January 2000 to December 2004, 17 cases of the macrosis depressed skull fracture on the frontal and orbit part undertaken plastic surgery by the dissociate bone flap to treat the macrosis depressed skull fracture and obtained excellent curative effect. CONCLUSIONS: Using dissociate bone flap to treat the mocrosis depressed skull fracture on the frontal and orbit part can avoid the complication of the traditional operation, and make the method become a plastic surgical operation.

Adult↗
PubMed1975

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...

Adenosine Triphosphate↗
PubMed2006

[Dissociated horizontal deviation--a diagnostic problem in strabismus].

Dissociated horizontal deviation (DHD) is one of the four components of the dissociated strabismus complex which also includes dissociated vertical deviation, dissociated torsional deviation and manifest-latent or latent nystagmus. These dissociated deviations may co-exist with concomitant strabismus (exotropia, esotropia, hyperopia). Sometimes it is difficult to make the difference between dissociated and non-dissociated component of the deviation. We report 3 cases of strabismus and DHD with special emphasis on the diagnostic difficulties and the therapeutic possibility. A careful clinical examination with additional measurement tests makes the difference between DHD and associated tropias. The reversed fixation test is a decisive test for the diagnosis of DHD. The therapeutic approach depends on the type of DHD (uni or bilateral, latent or manifest), the magnitude of the deviation, the fixation behavior and the association of the DHD with exotropia, esotropia or orthotropia.

Adolescent↗
PubMed1991

Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure.

Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.

Calcium↗
PubMedDate not supplied

[Dissociation of Penicillium vitale catalase under the effect of urea and acid pH].

Conditions are studied for Penicillium vitale catalase dissociation into subunits under the effect of urea (0.7-8.5 M) and acid pH (5.0-2.0). In 8.0 M urea (pH 5.0) a molecule of the P. vitale catalase dissociates with formation of the components, the sedimentation coefficient of which is 2.4+/-0.2S, the molecular weight is 153000+/-2800. Dissociation at pH 2.2 in 4.0 M urea results in formation of components with the sedimentation coefficient 2.2+/-0.3 S. The catalase molecule dissociation under the mentioned conditions is accompanied by a loss in the enzyme activity. The results obtained show that dissociation of the P. vitale catalase molecule requires more rigid conditions than dissociation of the animal catalase. At the same time the catalase after dissociation in 8.0-8.5 M urea loses its ability to reassociation with a decrease in the urea concentration up to 0.08 M.

Catalase↗
PubMed1990

The dissociation and degradation of internalized insulin occur in the endosomes of rat hepatoma cells.

We have studied the intracellular processing of insulin in the rat hepatoma cell line Fao. Fao cells internalized cohorts of surface-bound 125I-insulin or 125I-insulin-like growth factor II within 3-5 min. Degraded 125I-insulin-like growth factor II did not appear in the medium until 20-30 min after uptake, consistent with a time course of lysosomal delivery. In contrast, internalized insulin was completely degraded within 7-10 min. The half-times for dissociation and degradation of internalized insulin were identical at 37 degrees C (3 min), suggesting that the two processes occurred in the same compartment. Subcellular fractionation of Fao cells showed that a pulse of internalized insulin was largely intact after 3 min and associated with a light membrane fraction devoid of lysosomal markers. After an additional 4 min, the amount of insulin in this compartment decreased by 40%, with an increase in degraded insulin in the cytosol; no transfer of intact insulin to lysosomes or the cytosol was detected. The relationship between insulin-receptor dissociation and insulin degradation was further studied with inhibitors of insulin processing. Monensin blocked both dissociation and degradation of internalized insulin, as did incubation of the cells at 20 degrees C, suggesting that both endosomal acidification and endosomal fusion were required for insulin processing. At 25 degrees C, dissociation (+ t 1/2 = 12.9 min) preceded degradation (+ t 1/2 = 15.8 min). Inhibitors of lysosomal proteases were without effect on the half-time for either process. In contrast, bacitracin, an inhibitor of insulin degradation, caused a 2-fold increase in the half-times for both dissociation and degradation. Thus, intracellular insulin dissociation and degradation are tightly coupled endosomal processes in Fao cells, and insulin degradation facilitates the dissociation of insulin from its receptor inside the cell.

Animals↗
PubMed1975

The molecular dissociation of ferrihemoglobin derivatives.

Measurement of the dissociation constants of ferrihemoglobin by light scattering indicates that the quaternary structure is altered by the type of heme ligand. Fluoromethemoglobin and aquomethemoglobin, high spin derivatives with weak ligands, have tetramer-dimer dissociation constants of 80 and 50 muM, respectively. For low spin cyanmethemoglobin the dissociation constants were 1 muM (pH 6.0) and 3 muM (pH 9.0) under the general conditions of 0.1 ionic strength and 25 degrees. Of the ferrihemoglobins studied, alkaline methemoglobin (pH 9.0) has the lowest dissociation constant (0.2 muM). Dissociation constants of mixtures of alkaline and fluoromethemoglobin were significantly higher than that of the alkaline form alone. At pH 9.0 the 55 and 78% fluoride-bound derivatives had tetramer-dimer dissociation constants of 0.7 and 2 muM, respectively. The cyanmethemoglobin quaternary conformation was found to be less affected by pH than the fluoromethemoglobin and aquomethemoglobin conformations. Measurement of the dissociation constant (0.2 muM) for aquomethemoglobin-inositol hexaphosphate indicates stabilization of the tetramer by this organic phosphate. The extent of stabilization by inositol hexaphosphate does not appear to be that found for deoxyhemoglobin as suggested by Perutz (Perutz, M. F. (1972) Nature 237, 495-499) even though inducement of higher spin and iron-heme plane displacement may occur.

Calorimetry↗
PubMed1986

Dissociation of dimers of human hemoglobins A and F into monomers.

Dissociation of alpha beta and alpha gamma dimers of human hemoglobins (Hb) A and F into monomers was studied by alpha chain exchange (Shaeffer, J. R., McDonald, M. J., Turci, S. M., Dinda, D. M., and Bunn, H. F. (1984) J. Biol. Chem. 259, 14544-14547). Unlabeled carbonmonoxy-Hb A was incubated with trace amounts of preparatively purified, native, 3H-alpha subunits in 10 mM sodium phosphate, pH 7.0, at 25 degrees C. At appropriate times, free alpha monomers were separated from Hb A tetramers by anion exchange high performance liquid chromatography. Transfer of radioactivity from the alpha chain pool into Hb A was measured, yielding a first order dimer dissociation rate constant, k2 = (3.2 +/- 0.3) X 10(-3) h-1. The Arrhenius plot of k2 was linear between 7 and 37 degrees C, yielding an enthalpy of activation of 23 kcal/alpha beta dimer. As the chloride concentration was raised from 0 to 0.2 M, the dissociation rate increased 3-fold; with higher salt concentrations, however, the rate gradually returned to baseline. This rate was not altered by raising the pH from 6.5 to 7.2, but as pH was further raised to 8.4, kappa 2 increased about 3-fold. Hb F, which has an increased stability at alkaline pH, dissociated into alpha and gamma monomers 3 times more slowly than Hb A. Moreover, the dimer-monomer dissociation of Hb F was characterized by a significantly reduced pH dependence. These results demonstrate that both alpha beta and alpha gamma dimers of Hb A and Hb F dissociate reversibly into monomers under physiologic conditions. The differential pH dependence for dimer dissociation between Hb A and Hb F suggests that specific amino acid replacement at the alpha 1 gamma 1 interface confers increased resistance to alkaline denaturation.

Chlorides↗
PubMed1988

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene.

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

Animals↗
PubMed1979

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.

Ethylmaleimide↗