Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Endogenous inhibitor of GABAB and GABAA receptors

An endogenous inhibitor of γ-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na⁺-independent [³H]GABA bindings. Scatchard plot analysis of the [³H]GABA binding to GABA “B” receptor recognition site showed this inhibitor increased the K_d value (24.1 nM to 3.6 nM) without changing the B_max. On the other hand, Scatchard plot analysis of the [³H]GABA binding to GABA “A” receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the K_d value. These results suggest that the endogenous inhibitor functions as a modulator for GABA_B and GABA_A receptors.     

Lysophosphatidic acid and lipopolysaccharide bind to the PIP2-binding domain of gelsolin

The binding of the gelsolin P2 peptide (residues 150-169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, K_d and ΔH° were 920 nM and −2.07 kcal/mol, respectively, at pH 7.4 and 25 °C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, K_d was 177 nM and ΔH° was −7.6 kcal/mol.     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

Comparative Binding of 125I-and 99mTc-Labeled Native and Glycated Low-Density Lipoprotein to Human Microvascular Endothelial Cells-Potential for Atherosclerosis Imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with ¹²⁵I or ^99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-γ receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the K_D s using either ¹²⁵I-or ^99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that ^99mTc–g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for ^99mTc-nLDL and all the ¹²⁵I-labeled LDL forms. Binding studies using both ¹²⁵I-and ^99mTc-nLDL indicated a weak binding affinity (K_D 10^{? 7}mol/L) to human microvascular endothelial cells. The binding affinity of ¹²⁵I-g/goLDL to these cells was significantly higher (K_D 10^{? 9}mol/L) and could be increased further by preactivation of the endothelial cells using TNFα. Incubation with ^99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with ^99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with ¹²⁵I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only ¹²⁵I-labeled goLDL should be used, whereas nLDL may be applied as ¹²⁵I-or ^99mTc-labeled ligand.     

Manganese substrate complexes of phosphofructokinase studies by pulsed magnetic resonance

The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with K_d = 20 μm and ?_b ≦ 4, and perhaps, as many as 14 binding sites with K_d ~ 0.8 mm and ?_b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30–100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.     

Muscarinic receptor sites in human foetal brain

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K_d of 0.27 nM. Values of 0.17 nM min^?1 and 0.048 min^?1 for the association rate constant, K_on, and the dissociation rate constant K_off respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.     

Diazepam-Sensitive Specific Binding of Phenytoin in Rat Brain

Abstract

[³H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with K_d values of 32 ± 5 nM and 8.5 ± 1.1 μM, respectively. Similar enhancement of [³H]phenytoin binding was observed when diazepam was replaced by Ro 5–4864 (4″-chlorodiazepam) which is selective for the ‘peripheral’ type benzodiazepine binding sites. In contrast, neither the ‘central’ type receptor selective agonist clonazepam nor the antagonist Ro 15–1788 enhanced [³H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the ‘peripheral’ type binding site for its selective affinity for Ro 5–4864. However, judging from the micromolar concentrations required for the enhancement of [³H]phenytoin binding, they appear unlikely to be the same ‘peripheral’ type binding sites as measured by [³H]Ro 5–4864 binding (K_d approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

Binding of l-[3H]glutamate to repeatedly frozen-thawed rat brain membranes

l-[³H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, K_D = 697 nM; maximal binding capacity, B_max = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (K_D1 = 26 nM, B_max1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (K_D2 = 662 nM, B_max2 = 10.5 pmol/mg protein). l-[³H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Insulin receptors in isolated mouse pancreatic acini.

Specific insulin receptors were measured in isolated mouse pancreatic acini. Scatchard analyses revealed a high affinity binding site with a K_d of 1.67 nM and a lower affinity site with a K_d of 83 nM. Binding of insulin to these receptors was rapid, one-half maximal binding occurring at 2 min and maximal binding at 30 min. Insulin stimulated the uptake of the glucose analogue 2-deoxy-D-glucose; maximum effects were detected at 1.67 μM. Insulin, in contrast, had no direct effects on alpha-aminoisobutyric acid uptake. The finding of high affinity insulin receptors in pancreatic acinar cells supports the hypothesis that insulin may directly regulate specific functions in the exocrine pancreas.     

A radiolabeled-ligand-binding technique for the characterization of opioid receptors in the intact mouse vas deferens

The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90–95%) and saturable binding of d-[2-³H]alanine, 5-d-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent K_d of 1.5 nm and a B_max of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-d-alanine, 5-d-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible.