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M Chorev

Publications and source records attributed to M Chorev.

At least 73 records · Page 4Linked to original sources
PubMed1996

Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes.

The PTH and PTH-related protein (PTHrP) system consists of two hormones, at least two G protein-coupled seven-transmembrane domain receptors, and at least two intracellular signal transduction pathways for each receptor. The PTH/PTHrP receptor is present in the conventional target tissues of PTH action, namely kidney and bone. Both PTH and PTHrP bind to and activate the PTH/PTHrP receptor with equal affinity and efficacy. The newly discovered receptor, termed the human (h) PTH2 receptor, has 70% homology with the PTH/PTHrP receptor, but is found predominantly in brain and pancreas. It interacts selectively with PTH and not with PTHrP. PTH and PTHrP differ in several positions, including position 5 (Ile in PTH; His in PTHrP). To define the role of position 5 in receptor selectivity, we designed and synthesized a series of hybrid analogs containing specific elements of both the PTH and PTHrP sequences. Using human cell lines stably expressing either human receptor subtype, we evaluated the biological profile of the hybrids in assays of receptor binding and action. Both point-mutated hybrids, [Ile5]PTH-(1-34) and [His5]PTH-(1-34), bind to and stimulate cAMP accumulation and the release of cytosolic free calcium in HEK293/C-21, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH/PTHrP receptor. However, only [Ile5]PTHrP-(1-34), and not [His5]PTH-(1-34), binds to and stimulates cAMP accumulation and the release of cytosolic free calcium in HEK293/BP-16, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH2 receptor. The segmental hybrid PTHrP-(1-14)-PTH-(15-34) binds to and activates the hPTH/PTHrP receptor, but not the hPTH2 receptor, similar to the biological profile of His5-containing ligands: PTHrP-(1-34) and [His5]PTH-(1-34). Exchanging Ile5 for His5 in the segmental hybrid produces the analog [Ile5]PTHrP-(1-14)-PTH-(15-34), which interacts with both receptor subtypes. We conclude that His5 in PTHrP is the major structural determinant of receptor subtype specificity in the hPTH/PTHrP and hPTH2 two-receptor system. The mechanism of the specificity "switch" remains to be elucidated, but may result from a subtle perturbation of the bioactive conformation and/or from a direct steric hindrance at the hPTH2 receptor-ligand interface created by histidine at position 5. The hPTH2, but not the hPTH/PTHrP, receptor can discriminate between the two hormones based on the structural differences generated at position 5.

Amino Acid Sequence↗
PubMed1996

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Calcium↗
PubMed1995

Avian sarcoma leukemia virus protease linked to the adjacent Gag polyprotein is enzymatically active.

The activity of avian sarcoma leukemia virus (ASLV) protease (PR) prior to its release from the precursor protein was determined by introducing mutations at the cleavage site between PR and the adjacent upstream nucleocapsid (NC) protein. Gag DNA fragments containing these mutations were cloned into expression vectors and introduced into Escherichia coli in which the ASLV proteins were expressed. The dipeptide NC-PR containing these mutations did not undergo autoprocessing when expressed in bacterial cells and the fused proteins were devoid of enzymatic activity. However, when the whole Gag polyprotein containing these mutations was expressed in bacterial cells, other PR cleavage sites in the viral Gag polyprotein underwent normal cleavage, indicating that the release of free PR is not a prerequisite for correct processing of the ASLV Gag precursor.

Animals↗
PubMed1995

Probing the bimolecular interactions of parathyroid hormone with the human parathyroid hormone/parathyroid hormone-related protein receptor. 1. Design, synthesis and characterization of photoreactive benzophenone-containing analogs of parathyroid hormone.

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-protein-coupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in "photoaffinity scanning" of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physiocochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13)(Kb = 13 nM; Km = 2.7 nM) and [Nle8,18,L-Bpa23,Tyr34[bPTH(1-34)NH2(L-Bpa23) (Kb = 42 nM; Km = 8.5 nM). Another BP-containing analog, [Nle8,18,D-2-Nal12,Lys13(epsilon-pBZ2),L-2-Nal23 ,Tyr34]bPTH(7-34)NH2, was a potent antagonist (Kb = 95 nM; Ki = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe7, Gly12, Lys13, Trp23, and Lys26). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the "contact points" between ligand and receptor.

Adenylyl Cyclases↗
PubMed1995

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

Affinity Labels↗
PubMed1995

m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent Ka for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21 microM, a value similar to that for guanosine 5'-[beta,gamma-imido]triphosphate. m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-32P]m-AcAGTP, followed by immunoprecipitation of G-protein Gq, identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of alpha-subunits of heterotrimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.

Adenylyl Cyclases↗
PubMed1995

Conformation and interactions of bombolitin I analogues with SDS micelles and phospholipid vesicles: CD, fluorescence, two-dimensional NMR and computer simulations.

Bombolitins are five structurally related heptadecapeptides acting at the membrane level able to lyse erythrocytes and liposomes and to enhance the activity of phospholipase A2 (PLA2). In the presence of SDS or phospholipid vesicles bombolitins are able to form amphiphilic alpha-helical structures and this property seems to be the major determinant of bioactivity. In order to test the model of interaction between bombolitin I and membranes, an analogue was synthesized in which all the lysines were replaced by arginines: Ile-Arg-Ile-Thr-Thr-Met-Leu-Ala-Arg-Ile-Gly-Arg-Val-Leu-Ala-His-Val-NH2 ([Arg2,9,12, Ile10]bombolitin I). The design of this sequence allowed the synthesis of a second analogue through a specific postsynthetic dansylation at the epsilon-amino group of a lysine residue replacing the original leucine residue at position 7. The first analogue was fully characterized by CD and two-dimensional nmr in the presence of SDS or phospholipid vesicles. The peptide folds into an amphiphilic alpha-helical conformation with the helical segment spanning the central part of the sequence from Ile3 to His16. This behavior is identical to that observed for the native sequence. The replacement of lysine residues by arginine has no detectable effect on the conformational preference of the peptide chain. By CD and fluorescence spectroscopy measurements, the fluorophore-containing analogue [Arg2,9,12, Lys7 (epsilon-dansyl)] bombolitin I also folded into the alpha-helical conformation in the presence of SDS micelles or phospholipid vesicles. In particular, the dansyl fluorophore, which is located approximately in the middle of the apolar surface of the amphiphilic helix, is clearly buried in a hydrophobic environment when the peptide is bound to phospholipid vesicles. These findings support the hypothesis that the peptide helices are oriented parallel to the vesicle surface.

Amino Acid Sequence↗
PubMed1995

Conformation of parathyroid hormone antagonists by CD, NMR, and molecular dynamics simulations.

The conformation of two highly potent parathyroid hormone (PTH) antagonists was investigated in water/2,2,2-trifluoroethanol mixtures. The two peptides are derived from the sequence (7-34) of PTH and of PTH-related protein (PTHrP) and have a D-Trp replacing Gly in position 12. In the analogue derived from PTHrP, Lys11 was replaced by Leu to remove the residual agonist activity. The study was conducted by CD and two-dimensional proton magnetic resonance spectroscopy, and the nuclear Overhauser effects found were utilized in restrained distance geometry and molecular dynamics simulations. Both peptides adopt a helical C-terminal conformation, which seems more stable in the case of the PTHrP analogue. A type II' beta-turn centered around D-Trp12 and Lys13 is present in both structures.

Amino Acid Sequence↗
PubMed1995

Approach to discovering novel therapeutic agents for osteoporosis based on integrin receptor blockade.

On a global scale, osteoporosis is a major and growing public health problem. In the United States, osteoporosis is present in 24 million people (mostly women) and contributes to more than 1.3 million fractures/year. Serious morbidity and mortality result from these fractures. Current therapies for osteoporosis are few, efficacy is limited, and side effects problematic. Fundamental to the pathophysiology of osteoporosis is an imbalance between the tightly coupled processes of bone resorption and bone formation that characterize normal bone remodeling. Our laboratory is engaged in a research effort focused on elucidating the role of the osteoclast integrin in bone resorption, defining the nature of ligand-integrin interactions, and developing antagonists for cell surface adhesion molecules, particularly the alpha v beta 3 vitronectin-like integrin receptor present on the surface of human osteoclasts. Peptides containing the internal arginine-glycine-aspartic acid (RGD) motif have been shown to inhibit osteoclast-mediated bone resorption in vivo. We are now designing more potent and selective inhibitors of bone resorption as a potential new mechanism-based therapeutic approach to osteoporosis based on a novel mechanism. In an effort to rapidly identify the highest affinity ligands for the human alpha v beta 3 integrin, we have generated combinatorial peptide libraries containing substantial structural diversity. For instance, based on all possible sequence combinations of extracellular matrix proteins known to bind alpha v beta 3, we recently synthesized and chemically analyzed a library of 360,000 peptides, all of which contain RGD.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Recent developments in retro peptides and proteins--an ongoing topochemical exploration.

Main-chain peptidomimetics based on peptide-bond reversal and inversion of chirality represent important structural alterations for peptides and proteins, and are highly significant for biotechnology; these modifications have been widely applied: the D-HIV-protease dimer cleaves only all-D substrate; an all-D-hexapeptide opioid is able to produce analgesia following intraperitoneal administration. Antigenicity and immunogenicity can be achieved by metabolically stable antigens such as all-D- and retro-inverso-isomers of natural antigenic peptides. Isomers, including the retro- and retro-inverso- forms, of hybrid peptides derived from cercropin A and melittin, maintain antimicrobial activity. Therefore, an insight is provided into structure-activity relationships and the rational design of biologically important isomeric peptides.

Amino Acid Sequence↗
PubMed1995

Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production.

The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.

Alpharetrovirus↗
PubMed1995

Synthesis of potent agonists of substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of substance P.

The analogues [Glp6,Glu(OBzl)11]SP(6-11) and [Glp5,Glu(OBzl)11]SP(5-11) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH2C6H5 ester group has replaced the SCH3 group of the Met11 side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI+At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively.

Amino Acid Sequence↗
PubMed1995

Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals, OGP increases osteogenesis. Immunoreactive OGP (irOGP) in high abundance was demonstrated in normal animal serum mainly as an OGP-OGP-binding protein (OGPBP) complex. Here we show the presence of an OGP-OGPBP system in normal human serum. The total irOGP content, of which the bound peptide comprises at least 80-90%, ranged from 480-4460 mumol/L, several orders of magnitude higher than that of other regulatory polypeptides. The steady state/total irOGP ratio declined between 23 and 49 yr of age. The bound irOGP, purified by boiling, ultrafiltration, and hydrophobic high pressure liquid chromatography, was identical to OGP obtained previously from rat regenerating marrow and mouse stromal cell cultures in terms of its amino acid sequence, immunoreactivity, and mitogenicity. These data demonstrate the usefulness of our immunoassay to measure circulating OGP. More importantly, the identity of the human OGP with that of other species indicates the peptide's evolutionary conservation and, thus, its biological importance. The natural occurrence of OGP in man signifies its potential role in the prevention of bone loss and rescue of bone mass, especially in osteoporosis.

3T3 Cells↗
PubMed1995

Distribution and pharmacokinetics of a potent peptide antagonist of parathyroid hormone and parathyroid hormone-related protein in the rat.

Humoral hypercalcemia of malignancy results from the production by cancer cells of parathyroid hormone related protein that activates receptors in bone. Peptide antagonists that block parathyroid hormone receptors in vivo would be instrumental in the clinical treatment of humoral hypercalcemia of malignancy. We report the in vivo whole body distribution and blood plasma pharmacokinetics of the parathyroid hormone receptor antagonist [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide to determine parameters that are likely to affect its administration regimen. A single intravenously injected dose of [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide was rapidly cleared from blood plasma. The plasma concentration reaches a maximum at 10 min (Cmax = 1.93 +/- 0.27% of total injected CPM/ml), and the intact PTH derivative was detectable in plasma by HPLC analysis at this time. In vivo binding to plasma proteins was noncovalent. The peptide was rapidly cleared from blood by the liver, and more slowly by the kidney. Radiolabel was detected in excreted feces at 8 hr, but the preferred route of excretion was renal as judged by significant counts in excreted urine. Absorption of labeled peptide by skin and bone was sustained. Strong and sustained absorption also occurred in the vas deferens, seminal vesicle and hypothalamus. Given the rapid clearance of antagonist, multiple or sustained dosing schemes might be necessary to achieve the desired pharmacological effect. The high counts in liver at early time points after i.v. injection suggest that other routes of administration that do not bypass the hepatic first-pass effect would result in very low blood levels of drug.

Amino Acid Sequence↗
PubMed1994

Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.

Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.

Cell Line↗
PubMed1994

Pharmacological evaluation of phenyl-carbamates as CNS-selective acetylcholinesterase inhibitors.

The pharmacological and clinical properties of a novel phenyl carbamate acetylcholinesterase (AChE) inhibitor, SDZ ENA 713 are described. In animals and human subjects this compound showed superior chemical stability, oral bioavailability and a longer duration of action than physostigmine. SDZ ENA 713 produced a 10-fold greater inhibition of AChE in the hippocampus and cortex than in the heart and skeletal muscle, which explains its relatively low toxicity and freedom from cholinergic side effects. The selective effect in the cortex and hippocampus may be due to its preferential inhibition of the G1 form of the enzyme, which is present in relatively higher concentrations in these brain areas. Evidence of a selective hippocampal action was obtained in normal human subjects in whom REM sleep density was increased at doses that had no effect on plasma cholinesterase. If memory impairments in AD are related to a lack of cholinergic activity in cortical and hippocampal brain areas, SDZ ENA 713 should produce significant symptomatic improvement.

Animals↗
PubMed1993

Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge.

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01-1.0 and 1.0-100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys15]OGP-NH2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2/3 that of the native-like synthetic OGP. The [Cys15(S-NEtSucc)]OGP-NH2 and [3-I(Tyr10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr10)]OGP and [Cys15(S-NEtSucc)]OGP-NH2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.

3T3 Cells↗