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PubMed1989

Inter-relationship between immunoglobulin idiotype and metatype.

Allogenic anti-metatype (Met) and anti-idiotype (Id) reagents were elicited to the liganded and nonliganded states, respectively, of a high affinity murine monoclonal anti-fluorescein IgM antibody. Through comparisons of the relative immunogenicity and specificity patterns of the resulting antibody reagents, interpretations regarding the relationship between metatopes and idiotopes were rendered. Anti-Id specificity was measured in terms of the degree of ligand inhibition to two different forms of the fluorescyl hapten (i.e. free ligand and conjugated to a macromolecule). Idiotypic analysis of 18-2-3 H and L chains (immunoglobulin heavy and light chains) demonstrated that recognition of 18-2-3 Id determinants required recombination of H and L chains. Anti-Met reagents were evaluated relative to the liganded and nonliganded states of the IgM antibody. Binding studies indicated anti-Met specificity for liganded or affinity labeled Mab (monoclonal antibody) 18-2-3, but not for nonliganded 18-2-3 or the fluorescein ligand. The affinity labeled metatypic state provided the optimum immunogen yielding an antibody reagent which was rendered specific for the liganded state upon absorption with appropriate immunoglobulin reagents. Antibodies specific for affinity labeled 18-2-3 did not react with liganded 4-4-20, an IgG2a monoclonal anti-fluorescein antibody of similar high affinity but unrelated idiotypically. Results were discussed in terms of intrasite, proximal-site and distal-site epitopes.

Animals↗
PubMed1988

Molecular forms of cathepsin B in rat thyroid cells (FRTL5): comparison with molecular forms in liver (Hep G2) and insulin-secreting cells (HIT T15).

A radiolabelled peptide chloromethyl ketone (125I-tyrosyl-L-alanyl-L-lysyl-L-arginine chloromethyl ketone) was used to affinity-label proteinases in rat thyroid cells (FRTL5). Two major proteins of 34 kDa and 32 kDa were affinity-labelled. Inhibitor competition studies demonstrated that both proteins were cysteine proteinases. Over the range pH 5-8, they exhibited maximum activity against the affinity probe at pH 5. They were soluble rather than membrane-bound and were both glycosylated. The 32 kDa proteinase but not the 34 kDa proteinase was immunoprecipitated using an anti-rat liver cathepsin B antibody. The data suggested that these proteinases were molecular forms of cathepsin B. The affinity-labelled proteins in the thyroid were compared with those in an insulin-secreting cell line (HIT T15) and a liver cell line (Hep G2). Two molecular forms of cathepsin B of Mr 39,000 and 33,000 were identified in the insulin-secreting cell line and a single form of Mr 34,000 in the liver cell line. These molecular forms of cathepsin B may reflect the different functions and compartmentation of cathepsin B in these cells.

Animals↗
PubMed1990

[3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor.

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed1992

The effect of oxidation/reduction on the charge heterogeneity of the human glucocorticoid receptor.

In order to investigate the molecular basis for the charge heterogeneity which has been reported for the glucocorticoid receptor, we have analyzed the [3H]dexamethasone mesylate ([3H]DM)1 affinity labeled receptor from HeLa S3 cells by high resolution two-dimensional gel analysis. The [3H]DM labeled glucocorticoid receptor from HeLa cells exists as a population of 5-6 isoforms which range over approx. 0.6 pI units but have similar molecular weights. This heterogeneity is apparently the result of modification(s) of a single gene product since the affinity labeled receptor from Chinese hamster ovary (CHO) cells transfected with the human glucocorticoid receptor cDNA displays the same pattern of heterogeneity on two-dimensional gels. Since previous one-dimensional gel studies from our laboratory showed that the structure of the [3H]DM labeled glucocorticoid receptor from HeLa cells is highly susceptible to sulfhydryl group modification, we investigated the potential role of the same modifications in the apparent charge heterogeneity of the glucocorticoid receptor. Treatment of the affinity labeled receptor with iodoacetamide which alkylates free sulfhydryl groups and irreversibly prevents the formation of intra- or intermolecular disulfide bonds, reproducibly resulted in the appearance of 5-6 discrete isoforms of the receptor protein. Treatment with dithiothreitol, a reversible reducing reagent, resulted in detection of 3 to 4 isoforms of the glucocorticoid receptor. In marked contrast, treatment with sodium tetrathionate, which induces intramolecular disulfide bond formation, resulted in only one detectable isoform of the [3H]DM labeled glucocorticoid receptor. These data demonstrate that the oxidation/reduction state of sulfhydryl groups within the receptor protein can account for much of the charge heterogeneity of this ligand dependent transcription factor.

Affinity Labels↗
PubMed1988

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors.

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.

Affinity Labels↗
PubMed1991

Ontogeny of hepatic type I insulin-like growth factor receptors in the rat.

We studied the pre- and postnatal developmental regulation of the hepatic type I IGF receptor in the rat. Fetal rat liver membranes bound IGF-I throughout the latter part of gestation (d 17 to 21). After birth, binding diminished rapidly, reaching barely detectable levels by the 13th postnatal d. However, the presence of type I IGF receptors was readily demonstrated by affinity-labeling throughout the immediate postnatal period and in adult rats. Furthermore, IGF-I-dependent autophosphorylation of type I receptors could be seen in both fetal and adult liver membranes. Fasting for 48 h in adult rats led to a 2- to 3-fold increase in affinity-labeled type I IGF receptors. In contrast, nutrient deprivation to the fetus, via maternal fasting, did not alter fetal hepatic IGF-I binding or affinity-labeling of the type I receptor. These results support a role for the IGF and the type I IGF receptor in the autocrine/paracrine regulation of hepatic growth through the latter stages of gestation in the rat. The demonstration of enzymatically active type I IGF receptors in adult liver, and their increased expression in fasted adult rats is consistent with an autocrine/paracrine role for hepatic IGF-I in the adult.

Affinity Labels↗
PubMed1992

Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.

Affinity Labels↗
PubMed1988

The molecular basis of opioid receptor function.

An extensive body of pharmacological data demonstrates the existence of at least three opioid receptor subtypes mediating the diverse effects of opioids. Distinct binding and activity profiles of highly selective ligands, variable sensitivity to naloxone antagonisms, and selective protection and inactivation experiments strongly suggest that mu-, delta-, and kappa-opioid receptors represent recent discrete molecular entities. Purification and affinity labeling of receptor subunits are beginning to provide confirmation of this concept. The delta-opioid receptor affinity labeled and purified to homogeneity from NG108-15 cells comprises a glycoprotein subunit of Mr58,000 with one mol ligand bound/mol protein. Antibodies to this protein recognize native receptor in detergent solution and selectively bind to the Mr58,000 protein on immunoblots of partially purified preparations. Purification of the mu-opioid receptor from bovine striatum reveals a glycoprotein of Mr 65,000 which demonstrates opioid binding activity. Purification and affinity-labeling studies from other laboratories suggest a smaller size of Mr 58,000 for the mu-receptor however. The kappa-opioid receptor from guinea pig brain exhibits a unique mobility on sucrose density gradient centrifugation but has not been characterized in purified form. The primary structure of the opioid receptors, although unknown at present, will most likely reflect structural features of other inhibitory receptors coupled to G-proteins, with seven transmembrane helices and a large third cytoplasmic loop. Biochemical evidence clearly demonstrates the coupling of opioid receptors to Gi, accounting for opioid inhibition of adenylyl cyclase in neuronal cell culture and brain. Opioid inhibition of adenylyl cyclase has been reconstituted in IAP-treated NG108-15 cell membranes with a Gi preparation from brain. Electrophysiological evidence suggests that mu- and delta-opioid receptors can couple to a G-protein which mediates activation of inwardly rectifying potassium channels, perhaps to the same Gk mediating muscarinic potassium channel activation in heart. kappa-Opioid receptors are coupled to inhibition of voltage-dependent calcium channels in several neuronal systems. In NG108-15 cells opioid inhibition of calcium conductance is IAP sensitive and can be reconstituted with G-proteins purified from brain. Differences in the primary structure of mu-, delta-, and kappa-opioid receptors, as well as possible novel opioid receptor subtypes, will be defined by molecular cloning of recombinant DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Activation of CPP32 during apoptosis of neurons and astrocytes.

Members of the interleukin-1 beta-converting enzyme (ICE)/CED-3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED-3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum-free media. We show that specific inhibitors of ICE/CED-3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell-free system. Cell extracts from neurons induced to undergo apoptosis contained ICE/ CED-3 protease activity. To determine which member of the ICE/CED-3 family was activated in neurons and astrocytes during apoptosis, we developed a novel affinity-labeling technique that labeled the active site cysteine and identified a 17-kDa subunit of the activated protease. The affinity-labeled 17-kDa protease subunit shares antigenic and molecular mass identity with the processed form of CPP32 on immunoblots, suggesting that CPP32 may be the principal effector in the apoptotic pathway in neurons and astrocytes. In time-course experiments, the activation of CPP32 preceded the detection of PARP cleavage and DNA laddering, suggesting that processing of CPP32 is a very early event in apoptosis of neurons and astrocytes and may be involved in the proteolytic action on specific cellular targets. The affinity-labeling technique developed and used in this report with neural cells allows for the sensitive detection, purification, and identification of ICE/CED-3 proteases that may be activated in other cells types under a variety of conditions, including certain diseased states.

Amino Acid Chloromethyl Ketones↗
PubMed1990

Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I.

Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.

Adenosine Triphosphate↗
PubMedDate not supplied

The efficiency of covalent labeling of the pancreatic cholecystokinin receptor using a battery of crosslinkable and photolabile probes.

Affinity labeling is a powerful method for biochemical characterization of hormone receptors, dependent on approximation of reactive groups on ligand and receptor. In this work, we have compared the efficiency of covalent labeling of the rat pancreatic cholecystokinin (CCK) receptor by decapeptide probes with differing photolabile moieties sited at their amino-terminus, mid-region, or carboxyl-terminus, or chemically crosslinkable via their amino-terminus. Each labeled the same M(r) = 85,000-95,000 plasma membrane glycoprotein with a protein core of M(r) = 42,000. Affinity labeling this band through the amino-terminus of the decapeptide, 125I-D-Tyr-Gly [(Nle28,31)CCK-26-33], was inefficient using bifunctional chemical reagents, m-maleimidobenzoyl-N-hydroxy-succinimide ester (0.07% of total incubated radioactivity, representing 0.4% of specifically bound counts) or disuccinimidyl suberate (0.02, 0.2%) or a photolabile carbene precursor (0.06, 0.2%). A benzophenone at this locus yielded more efficient labeling of this band (0.09, 11.8%), but high levels of nonspecific labeling. Probes attached through residues within the receptor-binding domain were particularly useful. Photolabile derivatives of phenylalanine at the carboxyl-terminus of this domain yielded better incorporation (4-nitro-Phe33: 0.23, 1.4%; 4-azido-Phe3: 0.67, 6.0%). A 6-nitro-Trp30 derivative in the middle of this domain gave similarly efficient labeling (0.08, 3.5%) despite being a less potent pancreatic secretagogue. These studies clearly demonstrate that the efficiency of covalent labeling of a receptor can be markedly affected by the nature and site of crosslinking chosen.

Affinity Labels↗
PubMed2000

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane.

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.

Affinity Labels↗
PubMed1996

Lack of functional correlation between gamma-glutamyl transpeptidase and amino acid transport in the lactating mouse mammary gland.

Gamma-glutamyl transpeptidase (EC 2.3.2.2) in lactating mouse mammary gland was inhibited by affinity labelling of the tissue with 6-diazo-5-oxo-L-norleucine. Amino acid (L-alanine, L-methionine) uptake by the affinity labelled mammary gland tissue and the control tissue was measured in vitro. Uptake of amino acids by the affinity-labelled tissue was comparable to that of control tissue. These findings suggest that gamma-glutamyl cycle is not involved in amino acid uptake by the mammary gland.

Amino Acids↗
PubMed1990

Identification of disulfide-linked transforming growth factor-beta 1-specific binding proteins in rat glomeruli.

We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type consists of a group of proteins that bind TGF-beta 1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions, four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000 and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the 90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000 and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta 1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously described type III TGF-beta receptor.

Affinity Labels↗
PubMed1988

Peptidyl sulfonium salts. A new class of protease inhibitors.

The possibility has been examined that peptidylmethyl sulfonium salts might affinity label proteases by an alkyl transfer from sulfur to an active center residue. The synthesis of a number of agents of this type is described as well as initial results of their effect on cysteinyl proteases, papain and cathepsin B. These are readily inactivated by reagents in which the peptidyl portion contains features that promote binding to the proteases such as a penultimate phenylalanine residue. Irreversible inactivation ensues by transfer of the peptidyl portion, not methyl groups. Peptidylmethyl sulfonium salts lose a proton to form an ylide structure which may be the prevalent form at physiological pH values. The ylide may also be the active affinity labeling form of the reagent since the rate of inactivation of cathepsin B increases with pH. In contrast, the action of another affinity labeling reagent for cathepsin B, benzyloxycarbonyl-Phe-AlaCHN2, a diazomethyl ketone, is relatively independent of pH.

Affinity Labels↗
PubMed1998

Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia.

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

Amino Acid Chloromethyl Ketones↗
PubMed1981

Indium-111 tropolone, a new high-affinity platelet label: preparation and evaluation of labeling parameters.

Platelets were isolated with a new neutral, lipid-soluble metal complex of indium-111 and tropolone. Unlike oxine, which must be dissolved in ethyl alcohol, tropolone is soluble saline. Platelet labeling with In-111 tropolone can be performed in both acid-citrate-dextrose (ACD)-plasma and ACD-saline media within two hours' time. Labeling efficiency has been 80-90% in ACD-saline and 60-70% in the ACD-plasma medium. Optimum concentrations for the labeling of platelets with In-111 tropolone were 5 micrograms/ml in ACD-saline and 10 micrograms/ml in ACD-plasma, using a 15-min incubation at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. Seven parameters of platelet labeling were studied: concentration of tropolone, citrate, plasma proteins, and calcium ions; also platelet density, temperature, and pH of incubation medium. Their effects on the mechanism of platelet labeling with lipid-soluble tracers are discussed.

Affinity Labels↗