Involvement of peripheral type of benzodiazepine receptor in social isolation stress-induced decrease in pentobarbital sleep in mice

Our previous studies have shown that central-type benzodiazepine (BZD) receptors (CBR) and neurosteroids capable of modulating GABA(A) receptor function are involved in the decrease of pentobarbital (PB)-induced sleep caused by social isolation stress in mice. In this study, to further clarify the mechanism underlying this decrease, we investigated the possible involvement of peripheral-type BZD receptors (PBR) which play an important role in neurosteroidogenesis in PB sleep in socially isolated mice. Socially isolated mice showed significantly shorter duration of PB-induced sleep than group-housed animals. When injected intracerebroventricularly (i.c.v.), FGIN-1-27 (FGIN, 25-100 nmol), a selective PBR agonist, and PK11195 (PK, 14-28 nmol), a PBR antagonist, and pregnenolone (PREG, 15-30 nmol), a neurosteroid precursor, dose-dependently normalized the PB sleep in isolated mice without having an effect on the group-housed animals. In contrast, pregnenolone sulfate (PS, 24 nmol), an endogenous neurosteroidal negative allosteric modulator of the GABA(A) receptor, reduced PB sleep in group-housed but not isolated mice. PS, at the same dose, significantly attenuated the effects of FGIN (100 nmol), PK (28 nmol) and PREG (30 nmol) in isolated mice, while FGIN (100 nmol), PK (28 nmol) and pregnenolone (30 nmol) significantly blocked the effect of PS (24 nmol) in group-housed mice. These results suggest that the PBR-mediated decrease in the genesis of neurosteroid(s) possessing a GABA(A) receptor agonistic profile is also partly involved in the down regulation of the GABA(A) receptor following long-term social isolation and contributes to the decrease of PB-induced sleep in isolation stressed mice.     

Genetic loss of diazepam binding inhibitor in mice impairs social interest

Neuropsychiatric disorders in which reduced social interest is a common symptom, such as autism, depression, and anxiety, are frequently associated with genetic mutations affecting γ‐aminobutyric acid (GABA)ergic transmission. Benzodiazepine treatment, acting via GABA type‐A receptors, improves social interaction in male mouse models with autism‐like features. The protein diazepam binding inhibitor (DBI) can act as an endogenous benzodiazepine, but a role for DBI in social behavior has not been described. Here, we investigated the role of DBI in the social interest and recognition behavior of mice. The responses of DBI wild‐type and knockout male and female mice to ovariectomized female wild‐type mice (a neutral social stimulus) were evaluated in a habituation/dishabituation task. Both male and female knockout mice exhibited reduced social interest, and DBI knockout mice lacked the sex difference in social interest levels observed in wild‐type mice, in which males showed higher social interest levels than females. The ability to discriminate between familiar and novel stimulus mice (social recognition) was not impaired in DBI‐deficient mice of either sex. DBI knockouts could learn a rotarod motor task, and could discriminate between social and nonsocial odors. Both sexes of DBI knockout mice showed increased repetitive grooming behavior, but not in a manner that would account for the decrease in social investigation time. Genetic loss of DBI did not alter seminal vesicle weight, indicating that the social interest phenotype of males lacking DBI is not due to reduced circulating testosterone. Together, these studies show a novel role of DBI in driving social interest and motivation.     

Enhancement of diazepam and gamma-aminobutyric acid binding by (+)etomidate and pentobarbital

Abstract: (+) Etomidate and pentobarbital enhance [³H]diazepam and [³H]γ-aminobutyric acid ([³H]GABA) binding to cerebral cortex membranes. Both (+)etomidate and pentobarbital increase the affinity of [³H]diazepam for its binding sites. In contrast, they increase the B _max of both the high- and low-affinity GABA receptor sites. The enhancement of [³H]diazepam and [³H]GABA by (+)etomidate and pentobarbital is blocked by GABA antagonists. These results indicate that hypnotic drugs such as (+)etomidate and pentobarbital, which are not structurally related, modulate diazepam and GABA binding sites via similar mechanisms.     

地西泮和戊巴比妥钠对两种小鼠镇静催眠作用强度的观察

目的：观察地西泮和戊巴比妥纳对不同种属小鼠自主活动和睡眠效应的影响,为筛选影响中枢神经系统功能的药物提供可参考的选择动物的依据。方法：分别取昆明种和ICR小鼠各40只,设对照组和地西泮给药组,每组20只,给药组ig地西泮4mg／kg,对照组给生理盐水,连续3天,末次给药后45分钟测定小鼠自主活动。另取两种小鼠各20只,分别ip戊巴比妥钠50mg／kg,观察两种小鼠的睡眠情况,记录潜伏期和睡眠时间。结果：ICR小鼠ig地西泮后,表现明显的镇静作用,自主活动的次数和对照组比较明显减少,P〈0．01;而昆明种小鼠给相同剂量的地西泮,小鼠自主活动的次数减少不明显,P〉0．05。昆明种和ICR小鼠同样ip阈上剂量的戊巴比妥钠,两者在睡眠潜伏期上无明显差异,但在睡眠时间上,则ICR小鼠的睡眠时间明显长于昆明种小鼠,P〈0．01。结论：ICR小鼠对中枢抑制药的反应性更好,适合于这类药物的筛选,尤其对作用相对较弱的中药制剂,可能提高筛选的阳性率。     

Involvement of GABA-A receptor chloride channel complex in isolation stress-induced free choice ethanol consumption in rats

The present study revealed the effect of diazepam, a benzodiazepine, and progesterone, a pregnane precursor of neurosteroids, which act via modulating GABA-A chloride channel complex on the isolation stress-induced free choice ethanol consumption in adult rats. Isolation stress for 24 hr over a period of 6 days produced a significant increase in ethanol consumption, which persisted during the 6-day recovery period. Pretreating the animals with diazepam (5 mg/kg, i.p.), or progesterone (5 mg/kg, i.p.), blocked the isolation stress-induced increase in ethanol consumption. Bicuculline (2 mg/kg, i.p.), a GABA-A receptor antagonist significantly attenuated the effect of both diazepam and progesterone on stress-induced modulation of ethanol consumption. Isolation stress also caused an increase in total fluid consumption, which was antagonised by both diazepam and progesterone. Like ethanol consumption, this effect of diazepam and progesterone on isolation stress-induced increase in total fluid consumption was attenuated by bicuculline. Neither diazepam nor progesterone produced an increase in ethanol consumption in non-stressed rats. However, unlike diazepam, progesterone administration to non-stressed rats caused a significant increase in total fluid consumption. Results of the present study thus show that GABAergic mechanisms may be playing an important role in isolation stress-induced increase in ethanol consumption.     

Involvement of purinergic P2Y1R in antidepressant-like effects of electroacupuncture treatment on social isolation stress mice

Depression is a common neuropsychiatric disorder with high incidence and disability. Electroacupuncture (EA) is effective in the treatment of depression. However, the underlying mechanisms are not fully understood. Social isolation stress during post-weaning period can impair purinergic signaling in the brain of rodents and has emerged as a major risk factor for depression. The purpose of this study was to investigate the involvement of P2Y1 receptor (P2Y1R) in the antidepressant-like effects of EA. In this study, C57BL/6 mice were randomly assigned to group-housed (GH) or social isolated (SI) groups at post-natal day 21. After 6 weeks of social isolation, EA was performed on acupoints “Bai-hui” (GV20) and “Yin-tang” (GV29), or non-acupoints for 4 weeks. The SI mice received either intracerebroventricular injection of a selective P2Y1R agonist, MRS2365 (1 nmol); or a selective P2Y1R antagonist, MRS2179 (2 μmol), before and after EA. We found that SI mice exhibited depression-like behaviors accompanied with anxiety-like behaviors. The expressions of P2Y1R were well co-localized with GFAP-positive astrocytes and increased in the prefrontal cortex and hippocampus of SI mice. After treated with MRS2179, the depression-like behaviors of SI mice were attenuated, but not with MRS2365. Meanwhile, we found that EA could attenuate social isolation caused depression- and anxiety-like behaviors, and inhibited the up-regulation of P2Y1R in the prefrontal cortex and hippocampus of SI mice. Notably, the positive effects of EA on depression-like behaviors of SI mice could be reversed by MRS2365, while MRS2365 had no effect on the anxiolytic-like effects of EA. Therefore, we provide new evidence that EA could ameliorate depression- and anxiety-like behaviors in social isolation stress mice, and P2Y1R was involved in the antidepressant-like effects of EA.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09827-1.     

Complete amino acid sequences of bovine and human endozepines. Homology with rat diazepam binding inhibitor

The complete amino acid sequences of bovine and human brain endozepines have been determined. The amino-terminal serine of both endozepines is acylated. Assignment of the first 7 residues was achieved through Edman degradation after acid-induced rearrangement and subsequent acid hydrolysis of the amino-terminal blocking group. Cleavage of endozepine by chemical and enzymatic techniques established all the fragments in an unambiguous sequence. Bovine and human endozepines are single-chain polypeptides of 86 residues, with calculated molecular weights of 9913, displaying 93% homology. A comparison between the sequences of bovine and human endozepines with the partial sequences of the functionally related diazepam binding inhibitor from rat brain reveals significant sequence homology. The reported results suggest that bovine and human endozepines as well as rat diazepam binding inhibitor belong to a new family of polypeptides which presumably take part in the modulation of gamma-aminobutyric acid-ergic transmission.     

Role of the peripheral-type benzodiazepine receptor and the polypeptide diazepam binding inhibitor in steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of (i) cholesterol transport from intracellular stores to the inner mitochondrial membrane and (ii) loading of P450scc with cholesterol. We demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR) and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis. We also showed that DBI, as the endogenous PBR ligand, stimulates cholesterol transport. In addition, DBI directly promotes loading of cholesterol to P450scc. We review herein our studies on the structure, function, topography and hormonal regulation of PBR and DBI in steroidogenic cells. Based on these data we propose a model where the interaction of DBI with PBR, at the outer/inner membrane contact sites, is the signal transducer of hormone-stimulated and constitutive steroidogenesis at the mitochondrial level. Hormone-induced changes in PBR microenvironment/structure regulate the affinity of the receptor. PBR ligand binding to a higher affinity receptor results in increased cholesterol transport. In addition, hormone-induced release (processing?) of a 30,000 M_W DBI-immunoreactive protein from the inner mitochondrial membrane may result to the intramitochondrial production of DBI which directly stimulates loading of P450scc with cholesterol. Thus, in vivo, hormonal activation of these two mechanisms results in efficient cholesterol delivery and utilization and thus high levels of steroid synthesis.     

Molecular cloning and expression of a novel human cDNA related to the diazepam binding inhibitor.

In order to isolate the unidentified autoantigens in autoimmune diabetes, a human pancreatic islet cDNA library was constructed and screened with the sera from the diabetic patients. From the library screening, one clone (DRS-1) that strongly reacted with the sera was isolated. Subsequent sequence analysis revealed that the clone was a novel cDNA related to the diazepam binding inhibitor. DRS-1 was expressed in most tissues including liver, lung, tonsil, and thymus, in addition to pancreatic islets. DRS-1 was in vitro translated and the recombinant DRS-1 protein was expressed in Escherichia coli and purified. The size of the in vitro translated or bacterially expressed DRS-1 protein was in agreement with the conceptually translated polypeptide of DRS-1 cDNA. Further studies are required to test whether or not DRS-1 is a new autoantigen in autoimmune diabetes.     

Changes in the emotionally conditioned behavior of rats under the influence of the hexapeptide fragment GLLDLK of the protein inhibitor of diazepam binding]

It is shown that suboccipital injection of 100 micrograms of the gexapeptide GLLDLK (the fragment of endogenous peptide--the inhibitor of diazepam binding) modified (for 1-3 days) the emotionally conditioned behaviour of the rats (the test of "emotional resonance"). This modification was realized in some reinforcement of different behavioural patterns and had signs of anxiety and depression. In the test "social hierarchy" the injection of GLLDLK didn't change significantly the hierarchy in the whole rat society, but in the recipient behaviour the exploratory activity has been changed, the time of grooming increased and the quantity of social contacts decreased.     

Identification of diazepam binding in intack animals.

An $\text{in vivo}$ method for labeling specific benzodiazepine (BDZ) binding sites in brain was developed using intravenously injected [³H]diazepam. Labeling of these sites is blocked by pretreatment of animals with high doses of pharmacologically active BDZs (but not by an inactive BDZ). Using this $\text{in vivo}$ binding technique, specific BDZ binding is enhanced by pretreatment of rats with the GAB?A agonist muscimol or with amino-oxyacetic acid, which increases GABA levels in brain.     

Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.     

Acute stress-induced tissue injury in mice: differences between emotional and social stress

Emotional stress affects cellular integrity in many tissues including the heart. Much less is known about the effects of social stress. We studied the effect of emotional (immobilization with or without cold exposure) or social (intermale confrontation) stress in mice. Tissue injury was measured by means of the release of enzyme activities to blood plasma: lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and alanine transaminase (ALT). Tape-immobilization increased all these activities in the plasma. AST-ALT ratio was also increased in these animals. Electrophoretic analysis of CK isoenzymes showed the appearance of CK-MB. These results indicate that the heart was injured in immobilized mice. Analysis of LDH isoenzymes and measurement of alpha-hydroxybutyrate dehydrogenase (HBDH) activity suggests that other tissues, in addition to the heart, contribute to the increase in plasma LDH activity. Restraint in small cylinders increased plasma LDH, CK, AST, and ALT activities, but to lower levels than in tape immobilization. Because the decrease in liver glycogen and the increase in plasma epidermal growth factor (EGF) were also smaller in restraint than in the tape-immobilization model of emotional stress, we conclude that the former is a less intense stressor than the latter. Cold exposure during the restraint period altered the early responses to stress (it enhanced liver glycogen decrease, but abolished the increase in plasma EGF concentration). Cold exposure during restraint enhanced heart injury, as revealed by the greater increase in CK and AST activities. Intermale confrontation progressively decreased liver glycogen content. Plasma EGF concentration increased (to near 100 nM from a resting value of 0.1 nM) until 60 minutes, and decreased thereafter. Confrontation also affected cellular integrity in some tissues, as indicated by the rise in plasma LDH activity. However, in this type of stress, the heart appeared to be specifically protected because there was no increase in plasma CK activity, and both AST and ALT increased, but the AST-ALT ratio remained constant. Habituation to restraint (1 h/d, 4 days) made mice resistant to restraint-induced tissue injury as indicated by the lack of an increase in plasma LDH, CK, AST, or ALT activities. Similar general protection against homotypic stress-induced injury was observed in mice habituated to intermale confrontation.     

Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.     

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.     

Involvement of DNA-dependent protein kinase in regulation of stress-induced JNK activation.

DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic subunit and the regulatory subunits Ku70 and Ku80. The complex is activated on DNA damage and plays an essential role in double-strand-break repair and V(D)J recombination. In addition, DNA-PK is involved in S-phase checkpoint arrest following irradiation, although its role in damage-induced checkpoint arrest is not clear. In an effort to understand the role of DNA-PK in damage signaling, human and mouse cells containing the DNA-PK catalytic subunit (DNA-PKcs proficient) were compared with those lacking DNA-PKcs for c-Jun N-terminal kinase (JNK) activity that mediates physiologic responses to DNA damage. The DNA-PKcs-proficient cells showed much tighter regulation of JNK activity after DNA damage, while the level of JNK protein in both cell lines remained unchanged. The JNK proteins physically associated with DNA-PKcs and Ku70/Ku80 heterodimer, and the interaction was significantly stimulated after DNA damage. Various JNK isoforms not only contained a DNA-PK phosphorylation consensus site (serine followed by glutamine) but also were phosphorylated by DNA-PK in vitro. Together, our results suggest that DNA damage induces physical interaction between DNA-PK and JNK, which may in turn negatively affect JNK activity through JNK phosphorylation by DNA-PK.     

Differential effects of pentobarbital and ethanol in mice

The duration of the loss of righting reflex (RR) after ethanol, 4 g/kg, intraperitoneally (i.p.), was significantly longer in “long-sleep” (LS) than in “short-sleep” (SS) mice. This effect was shown to be correlated with differences in brain sensitivities to ethanol. In contrast, pentobarbital sodium (PB), 50 mg/kg, i.p., produced a significantly longer loss of RR in SS than in LS mice. The PB concentrations in the brain were the same in both mouse strains at the time of RR recovery suggesting equal sensitivities of the central nervous systems to PB. The rates of disappearance of PB from the blood were the same in both strains, but the apparent volume of distribution of PB in the LS strain was greater than in SS mice.In addition, C57BL/6J mice were found to be more sensitive than DBA/2J mice to PB, 50 mg/kg. In contrast, C57BL mice are known to be less sensitive than the DBA strain to ethanol. The PB concentration in the brain of DBA mice at the recovery of the RR was significantly greater than in C57BL mice. The apparent volumes of distribution of PB were not different in the two strains, but the rate of disappearance of PB from the blood of C57BL mice was significantly greater than for the DBA strain. In conclusion, factors which govern the brain sensitivities of selected mouse strains to ethanol and pentobarbital may not be equivalent.     

Allosteric binding properties of a monoclonal antibody and its Fab fragment

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.