Altered Binding Affinity Research Articles

Autonomic Receptors in Human Prostate Adenomas

Radioligand receptor binding techniques were used to characterize alpha1 adrenergic, alpha2 adrenergic and muscarinic cholinergic (MCh) binding sites in human prostate adenomas obtained from men with symptomatic and asymptomatic benign prostatic hyperplasia (BPH). Prostate adenoma specimens were obtained from nine men with asymptomatic BPH undergoing cystoprostatectomy, 11 men with symptomatic BPH undergoing open prostatectomy, and 11 men with symptomatic BPH undergoing transurethral resection of the prostate (TURP). A quantitative symptoms score analysis and urinary flow rate determinations documented the absence of bladder outlet obstruction in men undergoing cystoprostatectomy and confirmed the presence of bladder outlet obstruction in men undergoing prostatectomy. The mean equilibrium dissociation constants (Kd) and the mean densities of 125I-Heat (alpha1 adrenergic) and 3H-NMS (MCh) binding sites were similar in tissue homogenates obtained from men with asymptomatic and symptomatic BPH. The mean Kd of 3H-Rauwolscine (3H-Ra) was significantly greater in the prostatectomy specimens obtained from men with symptomatic BPH compared to the specimens obtained from men with asymptomatic BPH (p < 0.05). The density of 3H-Ra (alpha2 adrenergic) binding sites was significantly greater in the prostate adenomas obtained from men with symptomatic BPH compared to the prostate adenomas obtained from men with asymptomatic BPH (p < 0.05). The difference in alpha2 adrenoceptor density was accounted for by an increased receptor density in the open prostatectomy specimens. There was no significant correlation between alpha2 adrenergic, alpha1 adrenergic, and MCh receptor densities and prostate weight or patient age. This study indicates that the development of infravesical obstruction in men with BPH is not related to upregulation or altered binding affinity of alpha1 adrenergic or MCh receptor binding sites. The significance of the observed upregulation of alpha2 adrenoreceptors in the prostate adenomas obtained from men undergoing open prostatectomy is unknown, and requires further investigation.

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6 12 . PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 μg trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1–20 min elicited a rapid 2–4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [ 3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II(β) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6 12 . As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.

A single mutation in 16S rRNA that affects mRNA binding and translation-termination

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.

Mutations that alter transcriptional activation but not DNA binding in the zinc finger of yeast activator HAPI

Transcription of eukaryotic genes requires an interaction between transcription factors that bind to the TATA box region, and transcriptional activators that bind to upstream activating sequences (UASs) or enhancers. Several yeast upstream transcriptional activators, such as GCN4, GAL4 and HAP1, seem to contain separate domains for binding to DNA and activating transcription. The expression of the cytochrome genes CYC1 and CYC7 is controlled by HAP1, which binds to dissimilar DNA sequences in UAS1 of CYC1 and the UAS of CYC7. HAP1 has a zinc-finger DNA-binding domain between amino-acid residues 1 and 148, and a highly acidic C-terminal activation domain between residues 1,308 and 1,483 (ref. 10). A mutant allele of the HAP1 gene, HAP1-18, leads to a change in Ser 63 to Arg 63, immediately adjacent to the zinc finger in the DNA-binding domain. The HAP1-18 mutation specifically abolishes the ability of the protein to bind to UAS1, but greatly increases the ability of the protein to activate transcription of CYC7. We now report that this increase in activation is mediated solely by the CYC7 UAS and the HAP1-18 protein, and also, that it is not caused by an altered binding affinity of the protein for the CYC7 UAS. Furthermore, even by substituting other amino acids at position 63 and over-expressing the resulting derivatives in vivo we were unable to increase activity at the UAS of CYC7 to the level obtained with HAP1-18. This rules out the possibility that the HAP1-18 mutation increases transcriptional activation by abolishing competition by UAS1 and UAS1-like sites for the protein. We thus conclude that HAP1-18 is a better activator of transcription than the wild-type protein when bound to the UAS of CYC7. Moreover, our findings indicate that in addition to the acidic activation domain, the zinc-finger DNA-binding domain participates directly in the activation of transcription.

Parathyroid Hormone Down-Regulates the Epidermal Growth Factor Receptors in Clonal Osteoblastic Mouse Calvarial Cells, MC3T3-E1: Possible Mediation by Adenosine 3',5'-Cyclic Monophosphate

The effects of PTH on the binding and mitogenic activity of epidermal growth factor (EGF) were studied in clonal, PTH- and EGF-responsive mouse osteoblastic cell line, MC3T3-E1. Treatment of cells with synthetic rat PTH-(1-34) at 10(-10)-10(-8) M resulted in a dose-related decrease (maximally 30% relative to the control value) in the specific binding of a biologically active, 125I-iodolabeled derivative of recombinant human EGF ([125I]iodo-EGF). The PTH-induced decrease in EGF binding was time dependent, requiring at least 4 h of PTH treatment at 37 C for a maximal effect, completely reversible after cessation of PTH exposure and specific only to biologically active PTH. Scatchard analysis of saturation binding data revealed that the PTH-induced reduction of EGF binding was accounted for by a proportional decrease in the available EGF-binding sites without an alteration in binding affinity (Kd = 0.7-0.8 nM). PTH treatment resulted ina concomitant decrease in mitotic responsiveness to EGF (maximally 40-50% of control). Both of these down-regulatory effects of PTH were closely mimicked by forskolin, cholera toxin, or (Bu)2cAMP. Addition of either colchicine or cytochalasin-B during PTH treatment completely abolished the PTH-induced reduction of EGF binding. These data indicate that PTH down-regulates EGF receptors and reduces the mitotic responsiveness to EGF, probably via a cAMP-dependent cytoskeleton-mediated mechanism(s) in osteoblastic MC3T3-E1 cells.

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits.

Chemical kindling decreases GABA-activated chloride channels of mouse brain

The repeated administration of N-methyl-β-carboline-3-carboxamide (FG 7142) to mice leads to ‘chemical kindling’, i.e. the development of seizures in response to doses which were initially insufficient to produce convulsive activity. To determine if chemical kindling produced changes in the GABA A receptor/chloride channel complex, we measured the binding of [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) to the convulsant site of the complex by quantitative autoradiography. As a measure of chloride channel function, we studied muscimol-stimulated uptake of 36Cl − by isolated brain synaptosomes. Kindling decreased the B max of [ 35S]TBPS binding in cortex but not in cerebellum or hippocampus. Kindling did not alter binding affinities in any of these brain regions. Some mice injected with FG 7142 did not kindle despite receiving the same treatment as kindled mice. These ‘injected but not kindled’ mice did not display decreased receptor binding in any of these brain areas. Muscimol-stimulated 36Cl − uptake into cortical synaptosomes was also diminished by chemical kindling. These findings suggest that a decrease in functioning GABA-regulated chloride channels may be responsible for chemical kindling with FG 7142.

Glucocorticoids increase parathyroid hormone receptors in rat osteoblastic osteosarcoma cells (ROS 17/2).

The effects of glucocorticoids on parathyroid hormone (PTH) receptors was studied using rat osteosarcoma-derived cells (ROS 17/2), which have an osteoblastic phenotype, and [125I][Nle8,Nle18,Tyr34]bovine(b)PTH-(1-34)amide as the radioligand. Treatment of cells with physiologic concentrations of hydrocortisone resulted in a time and dose-dependent increase in PTH binding. The increase in PTH binding could be observed by 10 h of exposure to hydrocortisone (2 x 10(-7) M), was maximally enhanced by 48 h, and was maintained for the subsequent 7 days of continuous exposure to the steroid. With removal of hydrocortisone, PTH receptor binding promptly returned toward control levels. The increase in PTH binding was attributed to an increase in the availability of receptor binding sites, not to altered receptor binding affinity, and was blocked by cycloheximide. PTH-stimulated adenylate cyclase was also enhanced by glucocorticoids, and a close correlation was observed between PTH binding and PTH-stimulated adenylate cyclase. However, hydrocortisone not only increased PTH binding but also enhanced the efficiency of postreceptor signaling: 5'-guanylimidodiphosphate [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activities were also increased. Thus, enhanced PTH stimulation of adenylate cyclase by glucocorticoids resulted from at least two effects--increased receptor availability and enhanced postreceptor efficiency of transmembranous signaling.

Opposite effects of insulin and catecholamines on LDL-receptor activity in human mononuclear leukocytes.

The mechanisms by which insulin and catecholamines affect low-density lipoprotein (LDL)-receptor activity were studied in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 24 h in a lipid-free medium resulted in an increase in the specific binding, accumulation, and degradation of 125I-labeled LDL. Insulin stimulated the ability of the cells to bind, accumulate, and degrade the lipoprotein with high affinity, which may be caused by an increase in the LDL-receptor number without altering binding affinity. (-)-Epinephrine inhibited the specific binding, accumulation, and degradation of 125I-LDL. This effect appears to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. (-)-Norepinephrine, the unspecific beta-adrenergic agonist (-)-isoproterenol, and the beta 2-specific agonist terbutaline mimicked the effect of epinephrine on LDL-receptor activity. Catecholamines and beta-adrenergic agonists yielded sigmoidal log-concentration effect curves. The action of epinephrine was attenuated by the beta-antagonist (dl)-propranolol. These results demonstrate that insulin stimulates and catecholamines suppress the specific binding, accumulation, and degradation of 125I-LDL in human mononuclear leukocytes. The catecholamine action appears to be mediated by beta 2-adrenergic receptors. A suppression of LDL-receptor activity resulting from deficiency of insulin and elevated plasma catecholamine concentrations in uncontrolled insulin-dependent diabetic patients may contribute to the increased levels of LDL cholesterol observed in these patients.

Differential localization of adenosine A 1 receptors in the rat hippocampus: quantitative autoradiographic study

[ 3H]Cyclohexyladenosine ([ 3H]CHA) was used to label adenosine A 1 receptors in the rat hippocampus by quantitative autoradiography, and selective lesions in the neurons intrinsic to CA1 and CA3 subfields were chemically produced to determine the cellular localization of A 1 receptors. Lesioning the CA3 subfield by intracerebroventricular kainic acid injection caused a 50% reduction in the maximal binding capacity of [ 3H]CHA in the CA3 subfield with no alteration in binding affinity. Five days after unilateral CA3 lesioning, a reduction of [ 3H]CHA binding by 10–30% of ipsi- and contralateral was observed in the dendritic fields in the CA1 region, though a significant reduction was restricted to the ipsilateral stratum radiatum. Thirty-five days after CA3 lesioning, [ 3H]CHA binding in the stratum radiatum in the CA1 subfield ipsilateral to the CA3 lesion revealed a small reduction in B max values but no alteration in B max in other sublayers of the CA1. The K d values in all regions of the hippocampus were not different from the control values. Selective CA1 pyramidal cell lesioning by transient ischemia caused a 70% reduction of [ 3H]CHA binding sites in the CA1 subfield. Neither CA1 nor CA3 lesions altered [ 3H]CHA binding in the stratum moleculare of the dentate gyrus. These results suggest that only a small population of adenosine A 1 receptors are associated with the terminals of the CA3 pyramidal cells (Schaffer collaterals and commissural fibers) in the CA1 subfield. A 1 receptors in the CA1 and CA3 subfields are predominantly located on intrinsic pyramidal cells.

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit.

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.

Repeated stress increases the density of angiotensin II binding sites in rat paraventricular nucleus and subfornical organ.

We have studied the properties of angiotensin II binding sites in the paraventricular nucleus, subfornical organ and anterior pituitary lobe of rats subjected to repeated immobilization stress. This treatment produced significant increase in the density of angiotensin II binding sites in these two nuclei without any significant alteration in binding affinity. Repeated stress did not alter angiotensin II binding properties in the anterior pituitary lobe. Our results suggest that brain angiotensin binding sites may have a role in regulation of the stress response.

Modulation of glycosaminoglycan production and antithrombin III binding by cultured aortic endothelial cells treated with 4-methylumbelliferyl-beta-D-xyloside.

The interaction between antithrombin III and heparinlike glycosaminoglycan molecules present on the vascular surface seems to play a significant role in the regulation of coagulation. We have tested the hypothesis that altered synthesis of glycosaminoglycans by endothelial cells could influence this interaction by using 4-methylumbelliferyl-beta-D-xyloside for metabolic perturbation of glycosaminoglycan production. Incubation of purified porcine 125I-antithrombin III with cultured porcine aortic endothelial cells demonstrated specific, time-dependent, saturable binding of this protease inhibitor to the endothelial cell surface with antithrombin III concentration at half-maximal binding of approximately 40 nM. This binding was displaced by heparin and was completely abolished by selective removal of heparan sulfate from cells with heparitinase, indicating that antithrombin III binds to heparan sulfate on the surface of endothelial cells. Incubation of cell cultures with beta-D-xyloside resulted in a reduction of maximum antithrombin III binding by approximately 65% with little alteration in binding affinity. beta-D-Xyloside did not affect the cellular growth or morphology. Reduction of the binding after exposure to various concentrations (33 to 500 microM) of xyloside occurred in parallel with the decrease in incorporation of both 35S-sulfate and 3H-glucosamine into cell surface heparan sulfate. Whereas the size of heparan sulfate chains derived from the cell surface was not altered by xyloside treatment, they appeared to have slightly less net negative charge and a significantly reduced proportion of the molecule with high affinity for antithrombin III in the presence of xyloside. On the other hand, secretion of free chondroitin sulfate (and dermatan sulfate) chains into the medium was markedly increased (16-fold) in the presence of xyloside, accompanied by a smaller increase in secretion of free heparan sulfate chains. There was a good correlation between conditions with decreased antithrombin III binding and an inhibition of the endothelial cell-mediated acceleration of thrombin inactivation by antithrombin III. These results suggest that beta-D-xyloside caused a dose-dependent decrease in production as well as some subtle structural alterations of cell-surface-associated heparan sulfate, which could serve as binding sites for antithrombin III on the endothelial cells and mediate enhancing the anticoagulant activity of this protein. This system may offer a potentially useful model to investigate the possible mechanisms responsible for the development of a procoagulant state involving these endothelial macromolecules.

Effect of flutamide on hepatic cytosolic methyltrienolone (R1881) binding kinetics and testosterone responsive hepatic drug and steroid metabolism in the adult male rat

Flutamide was used to investigate the mechanism involved in androgen responsive hepatic microsomal drug and steroid metabolism. We compared the antiandrogenic action of flutamide on the prostate to its effect on testosterone responsive hepatic microsomal benzo[ a]pyrene hydroxylase (BPH) and testosterone reductase (TR) activities. Male Wistar rats, castrated as adults, were treated with 5 μmoles · kg −1 · day −1 of testosterone enanthate subcutaneously for 10 days. Co-administration of increasing doses of flutamide caused a dose-dependent reduction in prostate to body weight ratios and, in the same animals, caused significant alterations in adult male hepatic microsomal BPH and TR activities. These doses of flutamide did not affect the serum testosterone levels. To test the possibility that the action of flutamide on androgen responsive hepatic microsomal drug and steroid metabolism may be similar to that occurring in the prostate, a tissue which contains an androgen receptor, we also studied the effect of flutamide on the binding kinetics of the high affinity hepatic cytosolic [ 3H]R1881 binding protein in vivo. Scatchard analysis of [Su3H]R1881 binding data revealed a reduction in the binding capacity of the hepatic cytosolic androgen binding protein in castrated animals treated with a combination of flutamide and testosterone enanthate at doses capable of maximally altering hepatic microsomal drug and steroid metabolism. No alteration in binding affinity occurred in this treatment group. However, a decreased binding affinity was found when flutamide alone was given. The binding kinetics of the hepatic cytosolic androgen binding protein were not altered in the castrated adult male with or without testosterone treatment. When flutamide was injected daily into the intact adult female rat, no effect was observed on either hepatic microsomal BPH or TR activities. Taken together, these data indicate that flutamide reduces hepatic cytosolic R1881 binding in the adult male rat, and this may explain some of the effects of this antiandrogen on testosterone-sensitive hepatic microsomal drug and steroid metabolism.

Free fatty acids alter calcium binding: a cause for misinterpretation of serum calcium values and hypocalcemia in critical illness.

FFAs are bound with calcium on the albumin molecule. We hypothesized that changes in circulating FFA levels during critical illness altered calcium-albumin binding. We found that serum from both normal subjects and critically ill patients contained an ether-extractable factor which lowered ionized calcium concentrations and increased albumin-calcium binding. This factor was found in higher concentrations in serum from ill patients. Oleic acid and palmitic acid increased albumin-calcium binding from 2-28% in a dose-dependent manner when added in vitro to calcium-albumin solutions. Scatchard analysis demonstrated that 0.1 mM oleic acid increased the number of calcium-binding sites on the albumin molecule (from three to five sites per molecule) without altering binding affinity. A similar effect was found when we performed Scatchard analyses of ether extracts in serum from three critically ill patients (number of calcium-binding sites increased from three to six). We also found that lipid infusions (during parenteral nutrition) lowered mean serum ionized calcium values in six critically ill patients [4.6 +/- 0.2 (+/- SEM) to 4.1 +/- 0.2 mg/dL; P less than 0.05]. These data support the concept that FFAs increase calcium binding to the albumin molecule. Alterations in FFA concentrations during critical illness may contribute to the poor correlation between corrected total serum calcium and ionized calcium concentrations in critically ill patients. In addition, acute elevations in circulating FFA concentrations may contribute to hypocalcemia in patients with defects in bone calcium mobilization.

Polybrominated and chlorinated dibenzo-p-dioxins: synthesis biologic and toxic effects and structure-activity relationships

A series of polybrominated dibenzo-p-dioxins (PBDDs) and 2,3,7,8-substituted polybrominated and chlorinated analogs were synthesized and their relative in vivo and in vitro activities were determined. The structure-activity relationships (SARs) for PBDDs as ligands for the 2,3,7,8-TCDD cytosolic receptor were comparable to those observed for the polychlorinated dibenzo-p-dioxins (PCDDs); the most active compounds were substituted in all four lateral positions and there was a decrease in binding affinities with a decrease in lateral substituents or an increase in non-lateral substituents. The binding affinities of 2,3,7,8-tetrachloro, 2-bromo-3,7,8-dichloro-, 2,3-dibromo-7,8-dichloro-, 2,8-dibromo-3,7-dichloro and 2,3,7,8-tetrabromodibenzo-p-dioxin were comparable using the hydroxylapatite assay and indicates that interchange of Br and Cl substituents does not significantly alter binding affinities. The in vivo effects (monooxygenase enzyme induction, thymic atrophy and body weight loss) of several PBDDs were also determined in the immature male Wistar rat. 2,3,7,8-Tetrabromodibenzo-p-dioxin was the most toxic compound in this series and exhibited a toxic potency comparable to that of 2,3,7,8-TCDD. The relative toxic potencies of the PBDDs used in this study followed the order 2,3,7,8- > 1,2,3,7,8- > 1,2,4,7,8- > 1,3,7,8- and a comparable rank order was previously observed with the corresponding PCDD isostereomers.

Diacylglycerol modulation of insulin receptor from cultured human mononuclear cells. Effects on binding and internalization.

Tumor-promoting phorbol esters alter binding of growth factors and hormones to their specific receptors. Action of diacylglycerols, endogenous phorbol ester analogues, on 125I-labeled insulin binding to its receptor from human cells was therefore investigated. A variety of 1,2-diacylglycerols and 1,3-diacylglycerols inhibited 125I-insulin binding to intact human monocyte-like (U-937) and lymphoblastoid (IM-9) cells in a dose-, time-, and temperature-dependent manner within 30 sec at 37 degrees C in a fashion analogous to that of the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate (TPA). Inhibition of insulin binding by diacylglycerols, analyzed by Scatchard plot, seems to be due to altered binding affinity of the insulin receptor. Diacylglycerol effects were reversible, were seen regardless of the order of addition of 125I-insulin and diacylglycerols, and were demonstrated only with occupied insulin receptors. Corresponding fatty acids or phospholipids did not affect specific insulin binding to the intact U-937 cells. Diacylglycerols also inhibited binding of 125I-insulin-like growth factor (IGF) I but not that of 125I-human growth hormone (HGH) to the human cells. The non-tumor-promoting phorbols (phorbol, 4-alpha-phorbol, phorbol-12,13-distearate) did not affect insulin binding to intact cells. Both diacylglycerols and TPA stimulated internalization of 125I-insulin by U-937 and IM-9 cells. The ability of diacylglycerol to mimic the effects of TPA on the insulin receptor supports the concept of diacylglycerols as endogenous phorbol diester analogues even though the sole role of protein kinase C in our system is doubtful.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line.

We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters.

Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein.

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein. A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein. Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P. Cd(II) and Co(II) can be exchanged with the intrinsic Zn(II) ion. The Cd(II) protein shows a charge-transfer band at approximately 250 nm. The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(II) complex (epsilon max = 660 M-1 X cm-1 at 645 nm), and two intense charge-transfer bands are present at 355 nm (epsilon = 2,250 M-1 X cm-1) and 320 nm (epsilon = 3,175 M-1 X cm-1). These observations are consistent with three cysteines as ligands to the Zn(II) ion in g32P. Zn(II) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A + B). Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A + B) stage to produce many proteolyzed fragments. Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P. Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations. Zn(II) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P.

Effect of picrotoxinin on benzodiazepine receptor binding.

The effect of picrotoxinin on [3H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [3H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without altering binding affinity. In cerebellar membranes, picrotoxinin did not alter [3H]flunitrazepam receptor binding.