Preparation and properties of porcine relaxin derivatives shortened at the amino terminus of the A chain

Porcine relaxins shortened at the N terminus of the A chain were produced after protection of all amino groups with the base-labile [[(methylsulfonyl)ethyl]oxy]carbonyl (Msc) protecting group. The first two amino acids were removed by cyanogen bromide digestion whereby simultaneously a free alpha-amino group was generated in position A3. The resulting des-ArgA1,MetA2-N epsilon A7,N epsilon A16,N epsilon B8-tris [[[(methylsulfonyl)ethyl]oxy]carbonyl]relaxin. was further shortened by preparative Edman degradation. The shortest derivative obtained was des-ArgA1,MetA2,ThrA3,LeuA4,SerA5,GluA6 -N epsilon A7,N epsilon A16,N epsilon B8-tris[[[(methylsulfonyl)ethyl]oxy]carbonyl]relaxin. The deprotection of the derivatives in alkaline media resulted in crude des-A(1-2)- to des-A(1-6)-relaxins, which were subsequently purified by gel filtration on Sephadex G-50 superfine followed by either ion exchange chromatography on CM-cellulose at pH 5.1 or high-performance liquid chromatography on reversed-phase columns. During the CNBr digest, a side product was isolated that was identified as the corresponding homoserine ( [HseA2]relaxin) derivative. Shortened relaxin derivatives and [HseA2]relaxin were characterized by reversed-phase chromatography, electrophoresis, end-group determination, and amino acid composition. Circular dichroism studies revealed a distinct change in the structure of relaxins that were shortened by three and more amino acid residues. In the mouse interpubic ligament assay, des-A(1-2)-relaxin and [HseA2]relaxin were fully biologically active while the bioactivity of des-A(1-3)-relaxin dropped to about 50%. Relaxins shortened by four and more amino acid residues were biologically inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relaxin structure. Quasi allosteric effect of the NH2-terminal A-chain helix

The NH2-terminal heptapeptide in the relaxin A-chain (Arg-Met-Thr-Leu-Ser-Glu-Lys) has been replaced by chemical means with three different helix-promoting peptides (Arg-Met-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala, and the insulin segment Gly-Ile-Val-Glu-Gln). The partially protected NH2 terminally shortened relaxin derivative (N epsilon A16,N epsilon B8-bis(methyl-sulfonylethyloxycarbonyl)des-ArgA1,MetA2 , ThrA3,LeuA4,SerA5,GluA6,LysA7-B29-relaxin) has been prepared by a combination of cyanogen bromide digestion and Edman degradation of the epsilon-amino-protected derivative followed by mixed anhydride coupling with the synthetic peptides. All three derivatives have been isolated and purified by high performance liquid chromatography. Whole relaxins shortened at the NH2 terminus of the A-chain by 4 or more amino acid residues are biologically inactive in the mouse pubic symphysis assay (Büllesbach, E. E., and Schwabe, C. (1986) Biochemistry 25, 5998-6004). The introduction of the artificial peptides causes significant biological activity to reappear (about 30%). The loss of structural integrity of relaxins shortened by 4 or more residues of the A-chain NH2 terminus as observed by circular dichroism spectroscopy is largely reversed by the addition of the synthetic peptides. Our results suggest that no single amino acid in the NH2-terminal region of the A-chain is functionally important but that the presence of a helix is required for biological activity.     

Monobiotinylated relaxins. Preparation and chemical properties of the mono(biotinyl-epsilon-aminohexanoyl) porcine relaxin

Biotinylated porcine relaxins were synthesized and purified to homogeneity. Native porcine relaxin was reacted with biotinyl-epsilon-aminohexanoic acid-N-hydroxysuccinimide ester and the resulting mixture was separated by either ion exchange chromatography on CM-cellulose at pH 5.5 or reversed-phase high performance liquid chromatography. All four possible monosubstituted relaxin derivatives, with substitutions in N alpha A1, N epsilon A7, N epsilon A16, and N epsilon B8, were obtained. All derivatives were fully biologically active and comparative circular dichroism spectroscopy showed no significant differences in their secondary structure.     

Total synthesis of human relaxin and human relaxin derivatives by solid-phase peptide synthesis and site-directed chain combination

Human relaxin, a two-chain protein hormone, was synthesized by solid-phase peptide synthesis in combination with a novel thiol-protecting group strategy whereby the three disulfide bonds could be synthesized sequentially and without error. The final product was shown to be homogeneous by reversed-phase high performance liquid chromatography and electrophoresis and had the correct amino acid composition and sequence. Tryptic digestion and peptide mapping of the synthetic relaxin by reversed-phase high performance liquid chromatography resulted in a pattern identical with that produced by standard tryptic relaxin fragments synthetized by different methods. Three human relaxin derivatives containing oxidized methionine, formyltryptophan, and bis[B13,B17-citrulline]-relaxin, were produced and their biological activity and structural similarity to human relaxin was assessed. All derivatives, except those containing modified tryptophan residues, showed indistinguishable circular dichroic spectra, indicating that the modifications did not cause significant structural changes. However, only human relaxin and the tryptophan- and methionine-protected relaxin derivatives showed bioactivity. The derivative in which the two arginines in positions B13 and B17 had been replaced by the uncharged isosteric amino acid citrulline were biologically inactive. This observation confirms preliminary studies (Büllesbach, E. E. and Schwabe, C. (1988) Int. J. Pept. Protein Res. 32, 361-367) that suggested that these two conserved arginines located in the midregion of the relaxin B chain are essential for the function of the hormone.     

Preparation of N,N-bis(methylsulphonylethoxycarbonyl)insulins (author's transl)]

The preparation of N,N-bis(methylsulfonylethoxycarbonyl)insulins is described. In an aequeous buffer at pH 5.8 selectivity of the reaction of insulin with 20 equivalents of N-(methysulfonylethoxycarbonyloxy)succinimide (Msc-ONSu) leads very specifically to N alpha A 1,-N alpha B 1-(Msc)2 - insulin. The product can be isolated in a yield of 60%. Using N alpha A 1-citraconylinsulin the N alpha B 1, NEB29-(Msc)2 -insulin can be prepared in a yield of 40% based on insulin.     

A0-phenylalanyl] relaxin (porcine): an active intermediate

A [phenylalanylA0] relaxin has been isolated as a byproduct during large scale porcine relaxin preparations, using ion exchange chromatography on CM-cellulose at pH 7.8 followed by high performance liquid chromatography on reversed phase columns. The elongation at the N terminus of the A-chain has been demonstrated by amino acid and sequence analyses of the isolated and carboxymethylated relaxin-A-chain. The phenylalanyl relaxin and B29 relaxin are indistinguishable by circular dichroism spectroscopy, in mouse pubic ligament assay, and radioimmunoassay. The occurrence of phenylalanyl relaxin may be caused by an incomplete conversion of prorelaxin to relaxin.     

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.     

NA1, NA1, NA1-trimethylinsulin--an insulin analogue with a quaternary amino group at the A1 terminus

By utilizing the differing reactivity of the amino groups in aqueous organic solvents, des-GlyA1-NB1,N epsilon B29-(Msc)2-insulin was prepared. Its reaction with the phenyl ester of N,N,N-trimethylglycine in the presence of N-hydroxysuccinimide afforded the crystalline NA1,NA1,NA1-trimethylinsulin analogue. In the fat cell assay this analogue has an activity of 49% and, in the mouse convulsion assay, it is 70%.     

Inactivation of alanine racemase by beta-chloro-L-alanine released enzymatically from amino acid and peptide C10-esters of deacetylcephalothin

The reactions of a set of amino acid and peptidyl C10-esters of deacetylcephalothin (1-5) have been examined with purified enzymes in vitro. Each of the compounds examined is a substrate for the Escherichia coli TEM-2 beta-lactamase, and enzyme-catalyzed hydrolysis of the lactam bond gives release of an amino acid or a peptidyl fragment from a cephem nucleus. 7 beta-(2-Thienylacetamido)-3-[[(beta-chloro-L-alanyl)oxy]methyl]-3- cephem-4-carboxylate (4) gives time-dependent inactivation of E. coli JSR-O alanine racemase in a process that requires beta-lactamase for the initial liberation of beta-chloro-L-alanine from the cephalosporin. Alanine racemase is similarly inactivated by 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-beta-chloro- L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (1), but this inhibition requires the sequential action of both beta-lactamase and alanine aminopeptidase. Analysis of the enzymatic transformations of 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (3), monitored by high-field 1H NMR, reveals that (1) beta-lactamase releases the dipeptide beta-chloro-L-alanyl-L-alanine from 3 and (2) leucine aminopeptidase effects stoichiometric hydrolysis of the dipeptide to beta-chloro-L-alanine and L-alanine. These biochemical findings are discussed with reference to the mechanism of antibacterial action of 1 against beta-lactamase-producing, penicillin-resistant microorganisms [Mobashery, S., Lerner, S. A., & Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685].     

Naturally occurring porcine relaxins and large-scale preparation of the B29 hormone

Porcine ovaries were collected from pregnant sows under conditions designed to keep autolysis to an absolute minimum. During the extraction the tissues were never allowed to warm up to 0 degree C until submerged in 1.6 N HCl. Isolation and fractionation of the various relaxin forms became possible by application of CM-cellulose chromatography at pH 5.5 and 7.8, gel filtration, and high-performance liquid chromatography. The new isolation procedure has made it possible to isolate and identify LeuB32 relaxin. Also, [Leu-PheA0]B29 relaxin was identified and the existence of a [Leu-PheA0]B32 relaxin may be deduced from our data. Controlled digestion of B-chain-extended relaxins with carboxypeptidase A led to the large-scale production of homogeneous B29 relaxin, a suitable starting material for controlled chemical modification of porcine relaxin.     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

Synthesis of 2-acrylamidoethylglycosides of 3-O-(beta-D- glucopyranosyluronic acid)-alpha-L-rhamnose and 3-O-(alpha-L- rhamnopyranosyl)-beta-D-glucopyranuronic acid and copolymeric artificial antigens based on them

The synthesis of disaccharide repeating units, D-GlcA-(beta 1----3)-L-Rha (fragment A) and L-Rha-(alpha 1----3)-D-GlcA (fragment B), of the K54-antigenic polysaccharide from uropathogenic Escherichia coli 06:K54:H10 is described. Essential stages of the synthesis of fragment A involved the glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside followed by acetolysis of the methyl bioside obtained and further transformation into 2-(benzyloxycarbonylamino)ethyl glycoside; deprotection and, finally, conversion into 2-(acrylamido)ethyl glycoside. Selective opening of lactone ring in 2-azidoethyl 2,4-di-O-acetyl-beta-D-glucopyranoside-6,3-lactone was used for deprotection of 3-OH group in the synthesis of fragment B. Rhamnosylation of the glucuronic acid derivative thus obtained followed by transformation into 2-(acrylamido)ethyl glycoside and deprotection gave fragment B. Both fragments A and B were converted into artificial antigens of copolymer type.     

Functional Consequences of Deletions of the N Terminus of the [epsilon] Subunit of the Chloroplast ATP Synthase

The [epsilon] subunit of the chloroplast ATP synthase functions in part to prevent wasteful ATP hydrolysis by the enzyme. In addition, [epsilon] together with the remainder of the catalytic portion of the synthase (CF1) is required to block the nonproductive leak of protons through the membrane-embedded component of the synthase (CFO). Mutant [epsilon] subunits of the spinach (Spinacia oleracea) chloroplast ATP synthase that lack 5, 11, or 20 amino acids from their N termini ([epsilon]-[delta]5N, [epsilon]-[delta]11N, and [epsilon]-[delta]20N, respectively), were overexpressed as inclusion bodies. Using a procedure that resulted in the folding of full-length, recombinant [epsilon] in a biologically active form, none of these truncated forms resulted in [epsilon] that inhibited the ATPase activity of CF1 deficient in [epsilon], CF1(-[epsilon]). Yet, the [epsilon]-[delta]5N and [epsilon]-[delta]11N peptides significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO in NaBr-treated thylakoids. Although full-length [epsilon] rapidly inhibited the ATPase activity of CF1(-[epsilon]) in solution or bound to CFO, an extended period was required for the truncated forms to inhibit membrane-bound CF1(-[epsilon]). Despite the fact that [epsilon]-[delta]5N significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO, it did not block the proton conductance through CFO in NaBr-treated thylakoids reconstituted with CF1(-[epsilon]). Based on selective proteolysis and the binding of 8-anilino-1-naphthalene sulfonic acid, each of the truncated peptides gained significant secondary structure after folding. These results strongly suggest (a) that the N terminus of [epsilon] is important in its binding to CF1, (b) that CF0 stabilizes [epsilon] binding to the entire ATP synthase, and (c) that the N terminus may play some role in the regulation of proton flux through CFO.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.     

Discovery of a novel series of indoline carbamate and indolinylpyrimidine derivatives as potent GPR119 agonists

GPR119 has emerged as an attractive target for anti-diabetic agents. We identified a structurally novel GPR119 agonist 22c that carries a 5-(methylsulfonyl)indoline motif as an early lead compound. To generate more potent compounds of this series, structural modifications were performed mainly to the central alkylene spacer. Installation of a carbonyl group and a methyl group on this spacer significantly enhanced agonistic activity, resulting in the identification of 2-[1-(5-ethylpyrimidin-2-yl)piperidin-4-yl]propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (20). To further expand the chemical series of indoline-based GPR119 agonists, several heterocyclic core systems were introduced as surrogates of the carbamate spacer that mimic the presumed active conformation. This approach successfully produced an indolinylpyrimidine derivative 37, 5-(methylsulfonyl)-1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indole, which has potent GPR119 agonist activity. In rat oral glucose tolerance tests, these two indoline-based compounds effectively lowered plasma glucose excursion and glucose-dependent insulin secretion after oral administration.     

Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels

G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).     

Photodimerization in pyrimidine-substituted dipeptides.

Ten N(epsilon)-glycylornithineamide derivatives have been synthesized containing various N(alpha)-linked pyrimidine-1-ylacetyl groups which can undergo (2pi + 2pi) photodimerization on irradiation with UV light at 254 nm. The dimerization efficiency of the free and bound pyrimidine groups was compared in aqueous solution: it was dependent on the substitution of the pyrimidine ring. N(alpha),N(alpha')-bis-(uracil-1-ylacetyl)-(N(epsilon)-glycylornithineamide) and N(alpha),N(alpha')-bis-(5-bromouracil-1-ylacetyl)-(N(epsilon)-glycylornithineamide) were identified as possible candidates for optical data storage.     

Anaerobic 90Y- and 177Lu-labeling of a DOTA-conjugated nonpeptide vitronectin receptor antagonist

This study describes the discovery and development of an anaerobic formulation for the routine preparation of (90)Y and (177)Lu complexes ((90)Y-TA138 and (177)Lu-TA138) of a DOTA-conjugated nonpeptide vitronectin receptor antagonist (TA138: 3-sulfon-N-[[4,7,10-tris(carboxymethyl)1,4,7,10-tetraazacyclododec-1-yl]acetyl]-l-alanyl-N-[2-[4-[[[(1S)-1-carboxy-2[[[1,4-dihydro-7-[(1H-imidazol-2-ylamino]meth-yl]-1-methyl-4-oxo-3-quinolinyl]carbonyl]amino]ethyl]amino]-sulfonyl]-3,5-dimethylphenoxy]-1-oxobutyl]amino]ethyl]-3-sulfo-l-alaninamide). Since (90)Y-TA138 and (177)Lu-TA138 are very sensitive to radiolytic degradation, exclusion of oxygen is necessary during the radiolabeling. Using the anaerobic formulation, (90)Y-TA138 and (177)Lu-TA138 can be prepared in high yield and high specific activity. The anaerobic formulation described in this study is particularly useful for (90)Y- and (177)Lu-labeling of DOTA-conjugated small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling.     

A novel cell-permeable cromoglycate derivative inhibits type I Fc epsilon receptor mediated Ca2+ influx and mediator secretion in rat mucosal mast cells.

Type I Fc epsilon receptor (Fc epsilon RI) mediated Ca2+ uptake and secretion of rat serosal mast cells have been shown to be inhibited by disodium 1,3-bis [(2'-carboxylatochromon-5'-yl) oxy]-2-hydroxypropane (disodium cromoglycate, DSCG), which is widely employed in the treatment of allergic asthma [Foreman et al. (1977) Br. J. Pharmacol. 59, 473P-474P; Cox (1967) Nature (London) 216, 1328-1329]. This drug was also found to modify the protein phosphorylation pattern of these mast cells. [Theoharides et al. (1980) Science 207, 80-82]. We have isolated by affinity chromatography on a water-insoluble cromoglycate-carrying matrix a cytosolic enzyme recently identified as a nucleoside 5'-diphosphate kinase. In order to examine a possible intracellular activity of the drug, a cell-permeant cromoglycate derivative, 1,3-bis [[2'-[[(acetoxymethyl)oxy]carbonyl]chromon-5'- yl]oxy]-2-hydroxypropane [bis(acetoxymethyl) cromoglycate, CG/AM], has been synthesized, and its uptake and effect on the Fc epsilon RI-mediated exocytosis of mast cells was investigated. A tritium-labeled CG/AM derivative, used as radioactive tracer, was found to permeate mucosal mast cells of the rat line RBL-2H3 and accumulate intracellularly up to 40-fold its extracellular concentration following hydrolysis by cytoplasmic hydrolases. A CG/AM dose dependent inhibition of the Fc epsilon RI-induced mediator secretion was observed in RBL-2H3 cells loaded with this compound (I50 approximately 40 microM extracellular CG/AM). A similar dose-dependent inhibition was observed for both the Fc epsilon RI-mediated transient rise in the concentration of cytosolic free Ca2+ ions [( Ca2+]i) and the net Ca2+ influx, as monitored by the fluorescent indicator Quin2 and the radioactive tracer 45Ca2+, respectively. These results clearly show that cell-permeant cromoglycate inhibits the Fc epsilon RI-mediated Ca2+ influx into the cell and further underscore the dominant role of this process in the coupling of stimulus to secretion in RBL cells. Furthermore, with the identification of nucleoside 5'-diphosphate kinase as a potential intracellular target for CG activity, distinct mechanisms of action may be inferred for cell-permeant and nonpermeant forms of CG.