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PubMed1981

[Affinity modification of creatine kinase from rabbit skeletal muscles using gamma-(p-azidoanilide)-ATP].

Ultraviolet irradiation of creatine kinase (adenosine 5'-triphosphate: creatine-N-phosphotransferase, EC 2.7.3.2) in the presence of gamma-(p-azidoanilide)-[14C]-ATP results in a complete enzyme inactivation and covalent binding of two moles of analog per mole of enzyme. MgADP strongly protects the enzyme against inactivation. However, in the presence of MgADP the covalent binding of 0.9 moles of analog per mole of enzyme occurs without the enzyme inactivation. The rate of enzyme inactivation and covalent modification decreases in the presence of MgATP and creatine. These results indicate that gamma-(p-azidoanilide)-ATP is an affinity label for the active site of creatine kinase.

Adenosine Triphosphate↗
PubMed1990

Gallbladder CCK receptors: species differences in glycosylation of similar protein cores.

Receptors for cholecystokinin (CCK) on gallbladder muscularis smooth muscle have different apparent sizes in man (Mr = 85,000-95,000) and cow (Mr = 70,000-85,000). In this work, these receptors were demonstrated to represent N-linked complex glycoproteins with Mr = 43,000 protein cores, based on lectin-affinity chromatography and the deglycosylation of bands affinity labeled with 125I-D-Tyr-Gly-[(Nle28,31, pNO2-Phe33)CCK-26-33] using neuraminidase, O-glycanase and endoglycosidases H and F. Similarities in the core proteins were further demonstrated by Staphylococcus aureus V8 protease peptide mapping, in which both proteins yielded similar fragment patterns. Thus, gallbladder CCK receptors present in man and cow are both N-linked complex glycoproteins, with different carbohydrate domains and similar protein cores.

Affinity Labels↗
PubMed1995

11 beta-Substituted estradiol derivatives, potential high-affinity carbon-11-labeled probes for the estrogen receptor: a structure-affinity relationship study.

In view of their possible development as carbon-11-labeled receptor-based radiotracers for imaging estrogen-responsive breast tumors, we have synthesized a series of estradiols (1), estriols (2), 11 beta-ethylestradiols (3), 11 beta-ethylestriols (4), 11 beta-methoxyestradiols (5), and 11 beta-methoxyestriols (6), differing in the type of substituent R present at the 17 alpha-position (a, -H; b, -CH3; c, -C identical to CH; d, -C identical to CCH3; e, -Ph; f, -CH = CHMe cis), and measured their binding affinity for the estrogen receptor relative to estradiol (RBA). As expected, all the derivatives having an 11 beta-ethyl substituent have good binding properties (3a-d, 4a-d, RBA (25 degrees C): 109-3000%), and among them there are several promising candidates for carbon-11 labeling. Moxestrol (RBA (25 degrees C) = 185%) and its corresponding estriol derivative (4c, RBA (25 degrees C) = 20%) were the analogs having the highest affinity in the 11 beta-methoxyestradiol (5a-f) and 11 beta-methoxyestriol (6a-e) series, respectively; other analogs (R = Me, C identical to CMe, Ph, or cis-CH = CHMe) had uniformly lower RBA values.

Binding Sites↗
PubMed1985

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.

Affinity Labels↗
PubMed1982

Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.

Affinity Labels↗
PubMed1989

Effect of porcine brain natriuretic peptide (pBNP) on human adrenocortical steroidogenesis.

Porcine brain natriuretic peptide (pBNP), purified from porcine brain, had a significant suppressive effect on aldosterone and cortisol secretions in ACTH-treated cultured human adrenal cells. Concomitantly, the intracellular cGMP formation was enhanced by pBNP treatment. A specific pBNP receptor was identified in the human adrenal tissues. Affinity labelling of 125I-pBNP showed two separate molecular weights of specific binding sites for pBNP of 140 and 67 kDa. A 125I-pBNP binding study of the human adrenal membrane fraction demonstrated the presence of high-affinity and low-capacity binding sites for pBNP. Moreover, these binding sites for 125I-pBNP were displaced by unlabelled alpha-rANP as well as pBNP. From these studies, we concluded that pBNP had suppressive effects on human adrenocortical steroidogensis, possibly via a receptor which may be shared with ANP.

Adrenal Cortex↗
PubMed1994

Calmodulin interaction with the skeletal muscle sarcoplasmic reticulum calcium channel protein.

Studies were initiated to define the equilibria of calmodulin binding to the skeletal muscle sarcoplasmic reticulum (SR) Ca(2+)-release channel protein in native SR vesicles. Calmodulin affinity-labeling experiments indicated that the major calmodulin receptor in heavy SR preparations was a protein of M(r) > 450,000, corresponding to the Ca(2+)-release channel protein. [3H]Ryanodine-binding assays indicated 10.6 +/- 0.9 pmol of high-affinity ryanodine binding per milligram of SR protein. Wheat germ calmodulin was derivatized with rhodamine-x-maleimide. The affinity and binding capacity of the channel protein in SR vesicles for the derivatized calmodulin (Rh-CaM) were determined by fluorescence anisotropy in the presence of (1) 1 mM EGTA, (2) 0.1 mM CaCl2, and (3) 0.1 mM CaCl2 plus 1 mM MgCl2. In the presence of EGTA, Rh-CaM bound to the channel protein with a Kd of 8.6 +/- 0.8 nM and a Bmax of 229 +/- 7 pmol/mg, suggesting that calmodulin binds to the channel protein at [Ca2+] comparable to that in resting muscle. In the presence of 0.1 mM CaCl2, the binding equilibrium shifted to a two-site ligand-binding model; the high-affinity class of sites had a Bmax1 of 54 +/- 7 pmol/mg and a Kd1 of 4.3 +/- 1.1 nM, while the lower affinity class of sites had a Bmax2 of 166 +/- 28 pmol/mg and a Kd2 of 239 +/- 102 nM. In the presence of both Ca2+ and Mg2+, there was a further change in the Rh-CaM/channel protein interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells.

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.

Affinity Labels↗
PubMed2002

Evaluation of caspase activity in apoptotic cells.

A family of cysteine proteases, the caspases, plays a central role in the initiation and execution phases of apoptosis. Upon activation, these enzymes cleave specific substrates and thereby mediate many of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing. Hence, the detection of activated caspases can be used as a biochemical marker for apoptosis. Here we review a set of methods available for characterizing and quantifying the activation of caspases, including immunoblotting, cleavage of synthetic substrates, affinity labeling and confocal microscopy. Each method is described in general terms and the advantages and disadvantages of each technique are discussed.

Affinity Labels↗
PubMed1999

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.

Affinity Labels↗
PubMedDate not supplied

Serum factors inhibit melanoma cell surface expression of type I and type II IGF receptors.

We have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with 125I-IGF-I, 290-300 kDa form was characterized. Using 125I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or 125I-IGF-II labeling of IGF-related peptides and alpha IR3, an antibody directed against type I receptor alpha subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8- and 5-fold respectively, without any significant change in Kd values. In both culture conditions, we found IGFBP-2, -3 -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium.

Affinity Labels↗
PubMed1995

Localization of the factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators.

The propeptide binding/activation site on the vitamin K dependent carboxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [125I]tyrosyl-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 analogs with Bpa substituents at -16, -13, -7, and -6 were synthesized. These peptides were specific photoinactivators of carboxylase and were used to label a His6-carboxylase construct produced in baculovirus-infected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies and by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consistent with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C., & Roth, D.A. (1994) J. Biol. Chem. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-third of the vitamin K dependent carboxylase.

Affinity Labels↗
PubMed1981

Inactivation of pyroglutamyl aminopeptidase by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone.

Pyroglutamyl aminopeptidase [pyrrolidone-carboxylate peptidase: EC 3.4.11.8] from Bacillus amyloliquefaciens was inactivated rapidly and irreversibly by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone (Z-PGCK). The second-order rate constant of the inactivation was 1.1 x 10(5) M-1.s-1, a value which is comparable to that of the clostripain-TLCK reaction. The D-isomer of this chloromethyl ketone derivative was almost inert toward the enzyme under the same conditions. The inactivation reaction was prevented by the presence of a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that was reversibly reactivated by 2-mercaptoethanol, failed to react with Z-PGCK. These results suggest that this chloromethyl ketone derivative reacts as an affinity label, presumably with the active site cysteinyl residue of the enzyme, as was reported for L-pyroglutamyl chloromethyl ketone.

Affinity Labels↗
PubMed1989

Identification and regulation of the eukaryotic hyaluronate synthase.

Hyaluronate synthesis is required for fibroblast detachment in mitosis and migration. It is regulated by the activity of the synthase which is localized at the inner side of plasma membranes. The synthase was identified as a 50 kDa protein by immunological cross-reaction with the streptococcal enzyme and by affinity labelling. Transformation of fibroblasts by Rous sarcoma virus activated the synthase by enhanced transcription and phosphorylation. The synthase was a natural target of pp60v-src kinase.

Affinity Labels↗
PubMed1986

Preparation of a high-affinity photolabeling reagent for the Gal/GalNAc lectin of mammalian liver: demonstration of galactose-combining sites on each subunit of rabbit hepatic lectin.

On the basis of the knowledge that the D-galactose/N-acetyl-D-galactosamine-specific lectin of rabbit liver can tolerate a large group on the C-6 hydroxyl group of a galactoside [Lee, R. T. (1982) Biochemistry 21, 1045-1050], we prepared a high-affinity photolabeling reagent for this lectin from a triantennary glycopeptide fraction of asialofetuin. The C-6 hydroxyl group of a D-galactopyranoside was converted, under mild conditions, into a primary amino group. The procedure involves conversion of the hydroxyl group to an oxo group with galactose oxidase, followed by reductive amination using benzylamine and sodium cyanoborohydride. Catalytic hydrogenolysis of the benzylamino derivative yielded the desired 6-amino-6-deoxy-D-galactoside. A 4-azidobenzoyl group was attached to the newly produced amino group to yield a photoactivatable affinity-labeling reagent. The reagent labeled the Triton-solubilized, purified hepatic lectins of rabbit and rat in a photo- and affinity-dependent manner. All the polypeptide subunits of the lectins were labeled, indicating that each subunit contains at least one D-galactose-combining site. In the case of the rabbit hepatic lectin, the minor subunit (46 kDa) was labeled more efficiently than the major one (40 kDa).

Acetylgalactosamine↗
PubMed1984

Chemical crosslinking of the mononuclear phagocyte specific growth factor CSF-1 to its receptor at the cell surface.

The cell surface receptor for CSF-1 has been identified by affinity labeling intact mouse bone marrow-derived macrophages and intact cells of the murine J774.2 and BAC1 lines, which differ in their specific CSF-1 binding capacities and biological responses. The receptor, labeled by crosslinking to 125I-CSF-1 with disuccinimidyl suberate, is a polypeptide of approximately 165,000 molecular weight, irrespective of cell type.

Affinity Labels↗
PubMed1992

Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas. Age-related expression of distinct receptor glycoforms.

Cholecystokinin/gastrin receptors in the pancreas of newborn (3-day-old) rats are of type A, as in control mature rats, revealed by pharmacological analysis of specific 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) (Ahx, aminohexanoic acid) binding. Also, by 1 day post-partum, pancreatic cholecystokinin receptors were shown to be coupled to guanine-nucleotide-binding regulatory (G) proteins. Scatchard analysis of 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) binding to pancreatic membranes from rats at different times after birth showed a slight increase in the binding capacity of cholecystokinin receptors between days 3 and 14 and a sixfold increase in 21-day-old rats, with no change in receptor affinity during development. SDS/PAGE analysis of pancreatic membranes affinity labelled with the photoactivable ligand 125I-[2-(p-azidosalicylamido)-1,3'-dithiopropionate]-labelled [Thr34,Ahx37]cholecystokinin-(31-39) identified cholecystokinin receptors of 100-135 kDa in 3-day-old rats, 96-130 kDa in 7-day-old rats, 90-125 kDa in 10-day-old rats and 85-100 kDa in 14-day-old and 21-day-old rats, as found in control adult rats. Endo-beta-N-acetylglucosaminidase F treatment yielded a core protein of 42 kDa in all developmental stages. These findings are consistent with an age-related postnatal expression of distinct glycoforms of pancreatic cholecystokinin receptors. Furthermore, it was observed that the period 2-3 weeks after birth, characterized by stabilization of the mass of the cholecystokinin receptor, precedes the dramatic increase in the receptor number.

Affinity Labels↗
PubMed1986

Epidermal growth factor receptor is increased in multidrug-resistant Chinese hamster and mouse tumor cells.

Multidrug-resistant sublines of Chinese hamster lung and mouse tumor cells selected for high levels of resistance to vincristine or actinomycin D have increased numbers of epidermal growth factor (EGF) receptors compared to control cells. Evidence for this increase was found in six of six resistant cell lines with the use of receptor binding or immunoprecipitation techniques. Levels of 125I-labeled EGF binding to intact actinomycin D-resistant cells derived from DC-3F or CLM-7 Chinese hamster lines are increased 3- to 10-fold compared to controls. Scatchard analysis of these data suggests that increased binding is a result of increased receptor number rather than altered affinity of receptor for ligand. Affinity-labeling and immunoprecipitation studies confirmed the finding of increased receptor amount in resistant hamster and mouse cells. Multidrug-resistant variants of DC-3F cells overproduce a plasma membrane glycoprotein (gp150-180) with several physicochemical properties that resemble those of EGF receptor. However, electrophoretic transfer blots with a polyclonal antibody to gp150-180 show that EGF receptor and gp150-180 are probably different molecules. Resistant cells described in this report manifest a normalized phenotype compared to transformed, tumorigenic, drug-sensitive parental cells. EGF receptor increase in resistant variants may be associated with this reverse transformation.

Affinity Labels↗