Kinetics of reduction of ferrioxamine B by chromium(II), vanadium(II), and dithionite

Kinetic studies of the reduction of ferrioxamine B (Fe(Hdesf)⁺) by Cr(H₂O)₆²⁺, V(H₂O)₆²⁺, and dithionite have been performed. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the rate is ?d[Fe(Hdesf)⁺]/dt = k[Fe(Hdesf)⁺][M²⁺]. For Cr(H₂O)₆²⁺, k = 1.19 × 10⁴ M^?1 sec^?1 at 25°C and μ = 0.4 M, and k is independent of pH from 2.6 to 3.5. For V(H₂O)₆²⁺, k = 6.30 × 10² M^?1 sec^?1 at 25°C, μ = 1.0 M, and pH = 2.2. The rate is nearly independent of pH from 2.2 to 4.0. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the activation parameters are ΔH^≠ = 8.2 kcal mol^?1, ΔS^≠ ?12 eu and ΔH^≠ = 1.7 kcal mol^?1, ΔS^≠ = ?40 eu (at pH 2.2) respectively. Reduction by Cr(H₂O)₆²⁺ is inner-sphere, while reduction by V(H₂O)₆²⁺ is outer-sphere. Reduction by dithionite follows the rate law ?d[Fe(Hdesf)⁺]/dt =kK^{$\text{1}\text{2}$}[Fe(Hdesf)⁺][S₂O₄^2?]^{$\text{1}\text{2}$} where K is the equilibrium constant for dissociation of S₂O₄^2? into SO₂^? radicals. The value of k at 25°C and μ = 0.5 is 2.7 × 10³ M^?1 sec^?1 at pH 5.8, 3.5 × 10³ M^?1 sec^?1 at pH 6.8, and 4.6 × 10³ M^?1 sec^?1 at pH 7.8, and ΔH^≠ = 6.8 kcal mol^?1 and ΔS^≠ = ?19 eu at pH 7.8.     

Histamine-induced suppressor factor (HSF): further studies on the nature of the stimulus and the cell which produces it.

Guinea pig lymphocytes are stimulated by histamine to produce a soluble factor with immunosuppressive properties. This factor, termed histamine-induced suppressor factor or HSF, abrogates the production of migration inhibitory factor (MIF) and proliferative response to specific antigen. In the present study we have determined the lymphocyte subpopulation which elaborates HSF, the lymphoid tissue source, the kinetics of its generation in relation to immunization, and the nature of the histamine receptor involved in modification of the release of HSF. HSF activity could be detected in populations of cells from spleen and lymph nodes prior to active immunization of the donor, but not in cells from the donor's blood or thymus. Following immunization with ortho-chloro benzoyl-bovine γ-globulin in complete Freund's adjuvant (CFA), more HSF activity was detected in cells from the donor's spleen and lymph nodes. The peak response was seen 2 weeks postimmunization when significant amounts of HSF also were made by cells from the blood and thymus. Concentrations of T-cell-enriched and B-cell-enriched populations were tested for their ability to make HSF. We found that T-cell-enriched, but not B-cell-enriched populations, made significant amounts of HSF. Cells from the lymph nodes of immunized donors were chromatographed over affinity columns made of insolubilized conjugates of histamine with albumin. The nonretained cells were unable to generate HSF, whereas HSF activity was detected in the cells that were retained by the columns. This finding strongly suggests that the HSF-producing cells have receptors for histamine. Cells from CFA-immune lymph nodes were incubated with H₁ (2-methyl histamine) and H₂ (4-methyl histamine) agonists to determine their relative potency and, therefore, the nature of the histamine receptors on these cells that were modifying HSF release. Although both agonists could induce generation of HSF when high concentrations (10^?3M) were used, only the H₂ agonist stimulated production or release of HSF at lower concentrations (10^?5M). These HSF-producing cells appear to be selectively sensitive to H₂ agonists and likely have a predominance of H₂ receptors. Allergic mediators other than histamine were studied to determine their ability to allow elaboration of HSF-like activity from CFA-immune lymph node cells. Serotonin (10^?3M), slow-reacting substance of anaphylaxis (100 units/ml), eosinophil chemotactic factor (tetrapeptide; 10^?5M), and prostaglandin E₁ (10^?4M) were unable to induce HSF-like activity in lymph node cells from donors immunized with CFA. Furthermore, other agents which raise intracellular levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) such as isoproterenol and cholera toxin, as well as the dibutyryl form of cyclic AMP itself, were also unable to generate HSF-like activity. Thus, histamine is unique among the allergic mediators in stimulating elaboration of the suppressive substance. These findings also suggest that the ability of histamine to stimulate HSF may not reside in the conventional pathway linked to cAMP accumulation, but rather to an as yet undefined pathway of cell activation. A model is presented which further implicates histamine as a modulator of cellular immune reactions.     

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Alkaline phosphatase and maltase activity in the embryonic chick intestine in culture. Influence of thyroxine and hydrocortisone.

In strips of duodenum from 14-day chick embryos explanted into defined medium, alkaline phosphatase (ALP) activity accumulated in the tissue at a faster rate than in vivo for about 48 hr, but failed to increase thereafter. The addition of thyroxine (T4) to the medium at 10^?8M or less both enhanced the early accumulation and elicited a very large increase in ALP activity comparable to that normally occurring during the last 2 days in ovo. Activity with phenylphosphate (PhP) was more strongly affected than that with β-glycerophosphate (βGP), so that the high $\text{PhP}\text{β}\text{GP}$ ratio attained in vivo at 18 days was reached after 24 hr in T4-supplemented medium. Hydrocortisone (HC) evoked APL activity only slightly above that in unsupplemented medium and only during the first 48 hr in culture, but it precociously elevated $\text{PhP}\text{β}\text{GP}$ ratio to the normal maximum. In the presence of T4 or HC, maltase activity rose in explanted strips at the same rate as in the intact duodenum, but it lagged in unsupplemented medium. Assay of the medium revealed, however, that under all conditions of culture a large amount of both maltase and ALP activity had been released from the tissue. This effect was especially pronounced in the presence of T4, so that explants and medium together accumulated ALP and maltase equivalent to the high peaks of activity found in the intact duodenum at hatching. With 10^?8M T4, ALP activity began to rise above that of control explants after 8 hr, with accumulation in the medium beginning about 4 hr later. Combining 2 × 10^?6M HC with a range of T4 concentrations produced greater than additive effects, particularly with ALP, but did not lead to enhanced retention of either enzyme in the tissue strips. Prolactin, pentagastrin, and insulin were without effect alone, but the latter inhibited the effects of both T4 and HC.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.     

ICI 125,211: a new gastric antisecretory agent acting on histamine H2-receptors.

$\text{In}$$\text{vitro}$, ICI 125,211 competitively antagonized the action of dimaprit on guinea pig atrium with an apparent dissociation constant of 1.5 × 10^?8M (pA₂ = 7.8). $\text{In}$$\text{vivo}$, the histamine dose-response curve in conscious gastric fistula beagles was shifted rightward in parallel without change in the maximal response by intravenous infusions of ICI 125,211 at doses of 0.01 and 0.03 umol/kg/hr (estimated pA₂ = 7.3). Our data show that this new drug is at least 10x more potent than cimetidine as an inhibitor of gastric secretion in the dog. ICI 125,211, which is an orally effective antisecretory agent in man and devoid of antiandrogenic activity, is the most potent selective H₂-blocker described to date.     

Properties of histamine-induced suppressor factor in the regulation of lymphocyte response to PHA in mice

Splenic T lymphocytes release a suppressor factor into the culture supernatant when incubated for 24 hr with histamine (10^?4M). Histamine-induced suppressor factor (HISF) inhibits lymphocyte response to PHA; it is released by T lymphocytes (either nylon-nonadherent or nylon-adherent lymphocytes) and not by B-cells or macrophages; its production is not observed after depletion of histamine receptor-bearing lymphocytes and is blocked by the H2 receptor antagonist (cimetidine) but not by the H1 receptor antagonist (diphenhydramine). Gel filtration by Sephadex G100 chromatography indicates that HISF had an approximate MW of 45,000 to 68,000. Its inhibitory activity was removed by passage over a histamine RSA-Sepharose column, but not by passage over rabbit anti-mouse Ig-Sepharose column; it was blocked by prostaglandin synthetase inhibitor (PGS) (Ro 205720) indicating that this activity is mediated by a prostaglandin (PG) synthesis.     

Effects of angiotensin I and II and their interactions with some prostanoids in perfused human umbilical arteries

In perfused human umbilical arteries both angiotensin I and II induced vasoconstriction with a monophasic response. Angiotensin I and II induced vasoconstrictions at doses ≥10^?8M and 10^?9 M respectively. Captopril inhibited the angiotensin I response while the angiotensin II receptor blocker Sar¹-Ala⁸ AII inhibited the effect of both angiotensins. PGI₂ attenuated the angiotensin II response in a dose dependent pattern. PGE₂ and PGF_2α in concentrations below the critical levels for creating pressure responses $\text{per se}$, also attenuated the angiotensin II response. The cyclooxygenase inhibitor indomethacin potentiated the angiotensin II response indicating that endogenous production of prostanoids is of importance in the modulation of angiotensin effects.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Effect of histamine and histamine antagonists on natural and antibody-dependent cellular cytotoxicity of human lymphocytes in vitro

The in vitro effect of histamine and its antagonists, cimetidine and clemastine fumarate, on natural killer (NK) and antibody-dependent cellular Cytotoxicity (ADCC) activities of human lymphocytes was investigated. The histamine 1 (H1) antagonist, clemastine fumarate, and the histamine 2 (H2) antagonist, cimetidine, but not histamine alone, inhibited the NK and ADCC activities of lymphocytes when added directly to the mixture of effector and target cells in a ⁵¹Cr-release assay. This inhibition was proportional to the concentration of drugs added and was observed at various effector to target ratios against several targets. H1 and H2 antagonists also inhibited NK activities of T cells as well as Percoll-separated, NK-enriched effector cells. The inhibition was significantly reversed by histamine. In target binding assays, clemastine fumarate and cimetidine also decreased the target binding capacity of effector lymphocytes. Further, PBL precultured with histamine (10^?3–10^?4M) for 24 hr showed a significant decrease in their NK and ADCC activities. In coculture experiments, PBL precultured with histamine suppressed the NK activity of normal autologous effector lymphocytes. PBL precultured with histamine showed an increased number of OKT8⁺ cells, as estimated using monoclonal antibodies. The suppression of Cytotoxicity was not due to either direct toxicity, steric hindrance, crowding, or cell death, but by functionally viable suppressor cells. An immunoregulatory role for histamine in NK and ADCC reactions is proposed.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.