E. V. Navolotskaya

Study of interaction between L-glutamate and human blood lymphocytes.

The presence of specific glutamate receptors on T lymphocytes separated from normal human blood was investigated. Upon study of T lymphocytes interaction with [3H]glutamate the last one was found to bind specifically to these cells, Kd = 2.36 x 10(-7) M. Thirteen unlabelled compounds were tested as potential glutamate competitors. It was shown that only quisqualate (a structural analogue of glutamate) and dipeptides Ala-Glu, Glu-Ala, Glu-Glu competed with [3H]glutamate in binding to a common binding site. The experiments on binding of labelled and unlabelled glutamate bulky conjugates with dextran have demonstrated that both conjugated and free glutamate interacted with the common binding sites on the outer cell membrane. By this means it was shown that T lymphocytes exposed quisqualate-sensitive glutamate receptors on the outer surface of the plasma membrane.

Synthetic β-endorphin-like peptide immunorphin binds to non-opioid receptors for β-endorphin on T lymphocytes

Abstract The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (Ki = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (Ki = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (Ki = 1.0 nM) possessed the ability to inhibit specific binding of [125I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. Kd values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

Macrophage-stimulating peptides VKGFY and cyclo(VKGFY) act through nonopioid β-endorphin receptors☆

We have synthesized two peptides, VKGFY and cyclo(VKGFY) (referred to as pentarphin (PNT) and cyclopentarphin (cPNT), respectively), and found that both peptides at 1 nM concentration increased the adhesion and spreading of murine peritoneal macrophages as well as their bactericidal activity in vitro, as shown by phagocytosis of Salmonella typhimurium virulent strain 415. PNT administered intraperitoneally at dose 20 microg/mouse on day 7, 3, and 1 prior to the isolation of macrophages also enhanced the macrophage adhesion and spreading. The receptor binding characteristics of PNT and cPNT were examined using 125I-labeled PNT. The binding of labeled PNT to peritoneal macrophages was high-affinity (K(d)=3.6 nM) and saturable. It was not inhibited by naloxone (NAL) or [Met(5)]enkephalin ([Met(5)]ENK) but completely inhibited by unlabeled cPNT (K(i)=2.6 nM), immunorphin (IMN, decapeptide SLTCLVKGFY, corresponding to the IgG heavy-chain sequence 364-373) (K(i)=3.2 nM) or beta-endorphin (beta-END) (K(i)=2.8 nM). Thus, the effects of PNT and cPNT on macrophages are mediated by NAL-insensitive receptors common for PNT, cPNT, IMN, and beta-END.

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

Abstractβ-Endorphin and the synthetic β-endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met5]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of β-endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of 125I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (Kd = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits 125I-labeled β-endorphin specific binding to naloxone insensitive receptors on T lymphocytes (Ki = 0.6 ± 0.1 nM)). Thus, β-endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.

Binding of synthetic fragments of β-endorphin to nonopioid β-endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [3H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (Kd = 2.0 ± 0.1 nM). The [3H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (Ki = 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met5]enkephalin (Ki > 10 µM). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [3H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the authors name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (Ki = 3.1 ± 0.3 nM). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 ± 0.2 nM). The specific binding of [3H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (Ki = 2.4 ± 0.2 and 2.7 ± 0.2 nM, respectively). Copyright

Synthetic peptide SLTCLVKGFY competes with β-endorphin for naloxone-insensitive binding sites on rat brain membranes

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.

Characteristics of non-opioid β-endorphin receptor

Tritium-labeled selective agonist of non-opioid β-endorphin receptor, the decapeptide immunorphine ([2H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid β-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid β-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [2H]immunorphinereceptor complex was 2.4 ± 0.1 nM) and β-endorphin (Ki of the [2H]immunorphine specific binding was 2.9 ± 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro2]pentarphine (VKPFY) (Ki values were 0.0060 ± 0.0004, 2.7 ± 0.2, 2.6 ± 0.2, and 2.8 ± 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 μM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [2H]immunorphine. Values of the specific binding of 8.4 nM [2H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 ± 44.7, 698.6 ± 28.1, 279.1 ± 15.4, and 172.2 ± 1.8 fmol/mg protein, respectively. Unlabeled β-endorphin, pentarphine, [Pro2]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [2H]immunorphine to membranes from these organs. No specific binding of [2H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (Ki = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met5]enkephalin (Ki > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.

The second life of antibodies

Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (∼75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289–292 of the CH2-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341–344), immunorphin (364–373), immunocortin (11–20), and peptide p24 (335–358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed.

Detection of nonopioid β-endorphin receptor in the rat myocardium.

Two selective agonists of nonopioid β‐endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high‐affinity naloxone‐insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [3H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β‐endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [3H]immunorphin specific binding with the membranes was inhibited by unlabeled β‐endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met5]enkephalin and [Leu5]enkephalin. Thus, β‐endorphin, immunorphin and octarphin bind to the common high‐affinity naloxone‐insensitive receptor of the rat myocardial membranes. Copyright