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PubMed1995

Distinct expression of transforming growth factor-beta receptor subtypes on vascular smooth muscle cells from spontaneously hypertensive rats and Wistar-Kyoto rats.

1. We investigated the characteristics and expression of transforming growth factor-beta (TGFbeta) receptor subtypes on vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. 2. TGFbeta binding assays revealed that VSMC from SHR showed higher number and affinity of TGFbeta receptors than cells from WKY rats. 3. The affinity labelling with [125I]-TGFbeta1 revealed the presence of TGFbeta receptor subtypes with molecular sizes of 280, 85, 70 and 60kDa on VSMC from both strains at high cell density. The abundance of the 85kDa receptor subtype was greater in VSMC from SHR than cells from WKY rats. The 85kDa receptor subtype was not detected on VSMC from both strains at low cell density. 4. These results suggest distinct expression of TGFbeta receptor subtypes on VSMC from SHR and WKY rats, which may account for the exaggerated proliferative response of VSMC from SHR to TGFbeta.

Affinity Labels↗
PubMed1991

Characterization of neuropeptide Y (NPY) receptors in human hippocampus.

We identified a 50 kDa neuropeptide Y (NPY) receptor from human hippocampus by affinity labeling. NPY specific binding and labeling of the receptor were inhibited in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.27 nM and 0.18 nM, respectively). Peptide YY (PYY), but none of the pancreatic polypeptides, was as effective as NPY in displacing [125I]NPY. NPY fragments inhibited binding with the rank order of potency: NPY greater than NPY13-36 greater than NPY20-36 greater than or equal to NPY18-36 greater than NPY1-36 free acid greater than or equal to NPY26-36. These results demonstrate that the human hippocampal NPY receptor is a 50 kDa protein fitting the classification of a Y2 receptor subtype.

Affinity Labels↗
PubMed1983

CAP binding proteins associated with the nucleus.

Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in initiation factors as well as in preparations of crude nuclei. It was released from nuclei by washing with buffer of moderate salt concentration. Three high molecular weight cap binding proteins (approximately 120 000, approximately 89 000, approximately 80 000 dalton) were found in the nuclear fraction and were only partly released upon nuclease digestion and high salt extraction.

Affinity Labels↗
PubMed1993

Purification and characterization of a 3,5,3'-L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin.

We analyzed the nature of the thyroid hormone-binding proteins in bullfrog plasma using N-bromoacetyl-[125I]T3 as an affinity labeling probe. Polyacrylamide gel electrophoresis under nondenaturing conditions of the bullfrog N-bromoacetyl-[125I]T3-labeled plasma proteins revealed two proteins with specific binding to T3. A labeled protein that migrated faster than albumin (T-T3BP) was detected only in plasma obtained from tadpoles at stages earlier than, but not at the end of, metamorphic climax. Another protein that migrated more slowly than albumin appeared in the plasma at the late climax stage and was present in the adult stage. To study the function of T-T3BP during metamorphosis, it was purified from tadpole plasma to the single protein. The molecular mass of this protein was estimated to be 56 kilodaltons by gel filtration, but only 16 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the molecule comprised four identical subunits. The amino acid composition of T-T3BP and the amino acid sequence of its N-terminal portion were highly homologous with those of mammalian transthyretins. These molecular features indicate that T-T3BP is a homolog of mammalian transthyretins. However, in contrast to mammalian transthyretins, the affinity of bullfrog transthyretin for T3 was 360 times higher than that for T4. Scatchard analysis revealed that T-T3BP possessed a single class of T3-binding site, with a Kd of 0.67 nM at 0 C. These results suggest that bullfrog transthyretin may play an important role in transporting T3 in the blood during metamorphosis.

Affinity Labels↗
PubMed1994

The detection, quantification and partial characterisation of cathepsin B-like activity in human pathological synovial fluids.

In this study, the levels of the cysteine proteinase--cathepsin B were measured in diseased synovial fluids using a steady state fluorometric assay. Cathepsin B-like activity was shown to be present in all the samples analysed, with the rheumatoid arthritic synovial fluids possessing significantly higher concentrations (mean value ca. 416 mg/l) than the osteoarthritic fluids (mean value ca. 142.4 mg/l). In addition, upon treatment with pepsin, all of the rheumatoid arthritis samples were shown to possess additional cathepsin B-like activity, suggesting the presence of a reservoir of latent precursor molecules. By utilising a recently developed biotinylated affinity label for cathepsin B-like proteinases and sheep anti-(human cathepsin B) antibodies, used in combination with SDS-PAGE and Western blotting, the rheumatoid arthritic synovial cathepsin B was shown to exist in two forms with apparent molecular masses of M(r) 29,000 and 42,000. We propose that the former is a functionally active proteinase, whereas the latter is a pepsin activatable proform which, when cleaved by this aspartyl proteinase, is converted into a catalytically competent species of M(r) 20,000.

Affinity Labels↗
PubMed1986

The role of cell surface sialic acid in insulin receptor function and insulin action.

Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.

Adipose Tissue↗
PubMed1995

Sequence-specific covalent modification of DNA by cross-linking oligonucleotides. Catalysis by RecA and implication for the mechanism of synaptic joint formation.

Oligodeoxynucleotides (ODNs) were conjugated to chlorambucil and used as affinity labeling reagents to study joint molecule formation by the Escherichia coli recombinase recA. Chlorambucil is a bifunctional nitrogen mustard which alkylates the N-7 position of guanine in the major groove of double-stranded DNA (dsDNA). Incoming ODNs at least 30 nucleotides long cross-linked to a long homologous duplex DNA in the presence of recA and ATP gamma S. Efficient cross-linkage to the complementary recipient strand of the joint occurred preferentially at guanines positioned 5' relative to the appended chlorambucil group. The pattern of recipient strand alkylation was identical to that observed within a protein-free duplex and indicated that strand exchange had occurred prior to alkylation. Modification of the outgoing homologous strand of the joint was less efficient and spanned a 15-20 nucleotide long region offset to the 3' side of the tethered chlorambucil. Alkylation of both recipient and outgoing strands in the same joint molecule occurred with low frequency. By contrast, no affinity alkylation of the displaced strand was observed within a synthetic D-loop. These reaction patterns suggest that the incoming ODN approaches from the minor groove of the duplex to yield a poststrand exchange joint in which the major groove of the newly formed heteroduplex harbors the outgoing strand in an unpaired state. No evidence was obtained for the involvement of a triple-stranded DNA intermediate in recombination.

Affinity Labels↗
PubMed1994

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells.

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [125I]rAM revealed the presence of a single class of high-affinity (Kd 1.3 x 10(-8) M) binding sites for rAM in VSMC. The apparent Ki of rat calcitonin gene-related peptide (rCGRP) was 3 x 10(-7) M. Affinity labeling of VSMC membranes with [125I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP, rAM stimulated cAMP formation with an approximate EC50 of 10(-8) M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranolol, indomethacin, or quinacrine, but inhibited by a CGRP receptor antagonist, human CGRP[8-37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.

Adrenomedullin↗
PubMed1997

The effects of disulfiram and related compounds on equine hepatic alcohol dehydrogenase.

The reversible inhibition and the irreversible inactivation of equine hepatic alcohol dehydrogenase by disulfiram have been investigated. Disulfiram was found to be a potent competitive reversible inhibitor with KEO,I values at pH 7.0 and 10.0 of 50 microM and 30 microM, respectively. Reversible monodentate binding to the active site zinc is indicated by comparison with related compounds. Disulfiram was also found to chemically modify and inactivate the enzyme in an irreversible reaction, which proceeds via the formation of a reversible enzyme-disulfiram binary complex with a dissociation constant at pH 7.0 of 30 microM. The inactivation reaction has been studied over the pH 6.0 to 10.0 range. The dissociation constants for binding to the enzyme and the apparent first-order rate constants for inactivation have been determined as a function of pH. A pKa of 8.3 for the free enzyme has been assigned to the zinc-water ionization. Similar inhibition and affinity labelling kinetics are exhibited by diethyldithiocarbamate and by 2,2'- and 4,4'-dipyridyl disulphide, which have similar enzyme "on" velocity pKa values of 8.3 and 8.2, respectively. The enzyme is competitively protected from inactivation with disulfiram by 2,2'-dipyridyl, 1,7'-phenanthroline, acetone, and ethanol, all of which combine with the active site zinc to form binary complexes. Acetate gave mixed protection against inactivation due to an additional interaction with the anion binding site of the enzyme. In view of the effect of disulfiram on ethanol metabolism and the polyol pathway, its importance as an aversive drug are considered.

Affinity Labels↗
PubMedDate not supplied

A novel serine esterase expressed by cytotoxic T lymphocytes.

Cytotoxic T (Tc) lymphocytes recognize and lyse target cells and are thought to serve as an important defence against viral infections and possibly against neoplasms. The nature of the receptors responsible for antigen recognition by these cells is becoming clearer, but the molecular mechanisms responsible for their cytolytic activity remain largely unknown. The possibility that proteases are involved in this process has been suggested by the effects of certain inhibitors. Here we demonstrate that clones of murine Tc cells possess considerable trypsin-like esterase activity when assayed by a sensitive colorimetric assay. This activity was blocked completely by two serine esterase inhibitors, diisopropylfluorophosphoridate (DFP) and phenylmethylsulphonyl fluoride (PMSF), but not by N alpha-tosyl lysyl chloromethyl ketone (TLCK). The use of 3H-DFP as an affinity-labelling reagent demonstrated that the esterase activity resides in a protein of relative molecular mass (Mr) 28,000 (28K). A wide variety of other lymphocytes, including those from thymus, spleen and lymph node, established lines of B cells and noncytotoxic T cells, and clones of T helper cells, had about 300-fold less esterase activity than the Tc-cell clones and far smaller amounts of the DFP-reactive 28K protein. However, in thymocytes the esterase activity increased 20-50-fold and the 28K protein became more prominent 4 days after these cells had been stimulated in vitro to generate Tc cells.

Affinity Labels↗
PubMed1990

Circulating levels of insulin-like growth factor binding protein-1 (IGFBP-1) and hepatic mRNA are increased in the small for gestational age (SGA) fetal rat.

Insulin-like growth factors (IGFs) circulate in association with a family of specific binding proteins (BPs). Recently, we reported that circulating levels of IGFBP-1 and IGFBP-2 are increased in streptozotocin-diabetic adult rats and are differentially regulated in accordance with insulin and metabolic status. Since IGF BPs appear to be important modulators of IGF bioactivity in post-natal life, we asked whether serum levels of IGF BPs also might be altered in utero when the delivery of maternal nutrients is restricted and fetal growth is impaired. Bilateral uterine artery ligation or sham surgery was performed on maternal rats on d 19 of gestation (term 21.5 d). One day after ligation (d 20), fetuses were (SGA) compared to shams (3.1 +/- 1 vs 3.7 +/- 0.2 g, p less than .02) and serum levels of glucose (70 +/- 5 vs 96 +/- 6 mg/dL, p less than .01) and insulin (62 +/- 4 vs 138 +/- 14 microU/mL) also were reduced. In contrast, serum [125I]IGF-I binding activity was markedly increased in SGA litters compared to sham (65 +/- 5% maximum binding with 2.5 microL/mL SGA serum vs 14 +/- 3% for sham serum, p less than .001), and correlated with fetal weight (r = -0.539, p less than .05) and insulin (r = -0.622, p less than .05). Ligand blotting with [125I]IGF-I revealed that serum levels of IGFBP-1 (32 K) were greater in SGA than shams, while immunoblotting with specific antiserum demonstrated that levels of IGFBP-2 (34 K), the major fetal rat IGF BP, were similar in serum from SGA and shams litters. Affinity labeling and immunoprecipitation confirmed that IGF binding activity is increased in SGA, due largely to increased availability of IGFBP-1. In addition, Northern analysis of hepatic RNA revealed that the abundance of IGFBP-1 mRNA is increased in the SGA fetal rat, while hepatic mRNA for IGFBP-2 is similar in SGA and sham-operated litters. We conclude that circulating levels of IGFBP-1 and the abundance of hepatic mRNA are increased in the SGA fetal rat. IGFBP-1 may be an important modulator of IGF bioactivity and somatic growth in utero.

Affinity Labels↗
PubMed1996

Inhibition of ATP-diphosphohydrolase(apyrase) of Torpedo electric organ by 5'-p-fluorosulfonylbenzoyladenosine.

It has been shown previously that ATP is released into extracellular space from pre- and postsynaptic sources in peripheral synapses. The extracellular metabolism of ATP is likely to affect nucleotide- and nucleoside-mediated regulation of neurotransmission. The enzymes responsible for ATP breakdown are nucleotidases whose active site faces the extracellular space. ATPase and ADPase Ca(2+)-dependent activities were characterized in presynaptic plasma membrane preparation from the electric organ of Torpedo. Features described were in accordance with the presence of an ATP-diphosphohydrolase (apyrase EC 3.6.1.5) in this fraction. Active site studies using the affinity label 5'-fluorosulfonylbenzoyladenosine were performed on Torpedo apyrase. ATPase and ADPase Ca(2+)-dependent activities were inhibited with 5'-fluorosulfonylbenzoyladenosine. From this study it is concluded: (1) 5'-fluorosulfonylbenzoyladenosine binds specifically to the active site of apyrase. (2) Divalent cations accelerate the apyrase inactivation rate. (3) Divalent cations are not required for the binding of either the substrate or the inhibitor to the active site. (4) The apyrase active site is more specific for highly phosphorylated nucleotides. The results presented may be extrapolated to apyrases from other sources. The importance of this enzyme and its regulation are discussed.

5'-Nucleotidase↗
PubMed1998

Mechanism of ligand binding to E- and P-selectin analyzed using selectin/mannose-binding protein chimeras.

The mechanism of oligosaccharide binding to the selectin cell adhesion molecules has been analyzed by transferring regions of the carbohydrate-recognition domains of E- and P-selectin into corresponding sites in the homologous rat serum mannose-binding protein. Insertion of two basic regions and an adjacent glutamic acid residue leads to efficient binding of HL-60 cells and sialyl-Lewisx-conjugated serum albumin. Substitution of glycine for a histidine residue known to stabilize mannose in the binding site of wild type mannose-binding protein results in dramatic loss of affinity for mannose without decreasing binding to sialyl-Lewisx. The accumulated effect of these changes is to alter the ligand binding selectivity of the domain so that it resembles E- or P-selectin more closely than it resembles the parental mannose-binding domain. Affinity labeling using sialyl-Lewisx in which the sialic acid has been mildly oxidized has been used to verify this switch in specificity and to show that the sialic acid-containing portion of the ligand interacts near the sequence Lys-Lys-Lys corresponding to residues 111-113 of E-selectin. The binding of sialyl-Lewisx-serum albumin is inhibited dramatically at physiological and higher salt concentrations, consistent with a significant electrostatic component to the binding interaction. The binding characteristics of these gain-of-function chimeras suggest that they contain many of the selectin residues responsible for selective ligand binding.

Affinity Labels↗
PubMed1987

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation.

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.

Affinity Labels↗
PubMed1996

Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.

In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

Affinity Labels↗
PubMed1988

Introduction of nucleophiles and spectroscopic probes into antibody combining sites.

A general chemical strategy has been developed whereby antibody combining sites can be selectively derivatized with natural or synthetic molecules, such as catalytic groups, drugs, metals, or reporter molecules. Cleavable affinity labels were used to selectively introduce a thiol into the combining site of the immunoglobulin A MOPC 315. This thiol acted both as a nucleophile to accelerate ester thiolysis 60,000-fold and as a handle for selectively derivatizing the antibody with additional functional groups. For example, derivatization of the antibody with a fluorophore made possible a direct spectroscopic assay of antibody-ligand complexation. This chemistry should not only extend our ability to exploit antibody specificity in chemical catalysis, diagnostics, and therapeutics, but may also prove generally applicable to the functional modification of other proteins for which detailed structural information is unavailable.

Affinity Labels↗
PubMed1992

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.

Affinity Labels↗
PubMed1983

Present conceptions on the peptidyltransferase centre of animal ribosomes.

This review deals with results of affinity labelling experiments on proteins of the peptidyl-transferase centre (PTC) of animal ribosomes. From the studies it is concluded that ribosomal proteins L 5, L 21/23, L 32/33, and L 36 are the main candidates for the ribosomal P site and proteins L 6, L 18a, and L 28/29 for the ribosomal A site. Proteins L 10 and L 13/15 seem to be located in a more central region of the PTC. These findings are correlated with results obtained so far by using other techniques. Finally, the data are discussed, and a tentative model on the relative spatial arrangement of certain ribosomal proteins at the PTC is proposed.

Acylation↗