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PubMed2001

Location of template on the human ribosome as revealed from data on cross-linking with reactive mRNA analogs.

In this review we summarize data on the location of template on the human ribosome that we obtained from cross-linking (affinity labeling) experiments using reactive mRNA analogs. Types of mRNA analogs, model complexes of these analogs with 80S ribosomes, and methods for analysis of the ribosomal components (proteins and rRNA nucleotides) cross-linked with the mRNA analogs are reviewed. From analysis of the cross-linking data, we suggest a scheme for the arrangement of mRNA on the human ribosome and compare the organization of the mRNA binding center on human and Escherichia coli ribosomes.

Affinity Labels↗
PubMed1987

Characteristics of hepatic receptors for somatomedin-C/insulin-like growth factor I and insulin in the developing human.

Insulin and the somatomedins (Sms) are putative regulators of cell proliferation and metabolism in the fetus. Since the liver is a potential target tissue of these hormones during fetal life, we characterized the hepatic receptors for Sm-C/insulin like growth factor I (Sm-C/IGF-I) and insulin during the second trimester of human fetal development. Membrane-enriched fetal liver homogenates specifically bound 8.9 +/- 1.5% (+/- SD) of added [125I]insulin and 5.1-7.2% of [125I]Sm-C/IGF-I. Binding of both hormones was constant from 12-20 weeks gestation and was much greater than that in adult liver membranes. Analysis of dose-response data indicated high affinity between each receptor and its respective ligand (Kd for the Sm-C/IGF-I receptor, 2.2 X 10(-10) M; Kd for insulin receptor, 5.2 X 10(-10) and 7.7 X 10(-9) M). Limited cross-reactivity (approximately 1:1,000) of insulin with the Sm-C/IGF-I receptor and Sm-C/IGF-I with the insulin receptor was found. Affinity labeling studies showed that each receptor possessed an approximately 135,000-dalton subunit which was a part of a larger disulfide-linked complex. Thus, the human fetal liver has specific receptors for Sm-C/IGF-I and insulin that are similar to those described for other tissues in terms of both hormone-binding characteristics and subunit structure, suggesting that these receptors mediate important cellular functions at this stage of fetal development.

Affinity Labels↗
PubMedDate not supplied

[Comparative analysis of affinity modification of several aminoacyl-tRNA synthetases with gamma-(p-azidoanilide)-ATP].

The inhibitory action of gamma-(p-azidoanilide)-ATP on the reactions of tRNA aminoacylation catalysed by several aminoacyl-tRNA synthetases was investigated. This compound was shown to be a competitive inhibitor with respect to ATP in the case of arginyl-, valyl-, isoleucyl-, leucyl-, threonyl-, phenylalanyl-tRNA synthetases of E. coli MRE-600 and tryptophanyl-tRNA synthetase of beef pancreas. The Ki value of this analog changes from 3 x 10(-5) up to 4 x 10(-3) M depending on the enzyme specificity. In the case of methionyland lysyl-tRNA synthetases from E. coli the non-competitive and mixed inhibition accordingly was observed. The activity of isoleucyl-, valyl-, leucyl-, threonyl-, phenyl-alanyl- and tryptophanyl-tRNA synthetases in the reaction of tRNA aminoacylation is decreased as a result of UV-irradiation of the enzymes in the presence of gamma-(p-azidoanilide)-ATP. ATP and aminoacids protect these enzymes against irreversible inactivation. These results confirm the affinity labelling of the substrate binding sites of these enzymes. However, the appreciable inactivation of enzymes with UV-irradiation in the presence of gamma-(p-azidoanilide)-ATP was not detected in the cases of hystidyl-, lysyl-, methionyl-, seryl-, tyrosyl- and phenylalanyl-tRNA synthetases of E. coli MRE-600. The data obtained enable one to suggest the difference in the structure of the amino acid activating sites of different aminoacyl-tRNA synthetases.

Adenosine Triphosphate↗
PubMed1977

Diazomethyl ketone substrate derivatives as active-site-directed inhibitors of thiol proteases. Papain.

The diazomethyl ketones of z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a mechanism different from that of chloromethyl ketones, since the pH dependence of their inactivation of papain is different, the rate increasing with decreasing pH. This relationship has been observed in other cases, such as in the reaction of azaserine with glutamine amidotransferases [Buchanan, J. M. (1973), Adv. Enzmol. Relat. Areas Mol. Biol. 39, 91], and is interpreted as an indication of reaction with a thiol group in its protonated form.

Affinity Labels↗
PubMedDate not supplied

Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

Affinity Labels↗
PubMed1992

Affinity alkylation of hamster hepatic arylamine N-acetyltransferases: isolation of a modified cysteine residue.

N-Acetyltransferases (NATs) play key roles in the detoxification and/or bioactivation of arylamines, arylhydroxylamines, arylhydroxamic acids, and hydrazines in mammalian tissues. In the present study, two hamster hepatic NATs (NAT I and NAT II) were separated, and each was purified greater than 2000-fold by sequential ammonium sulfate fractionation, DEAE anion exchange chromatography, Sephadex G-75 gel filtration chromatography, aminoazobenzene-coupled affinity chromatography, and DEAE anion exchange high performance liquid chromatography. Both NAT I and NAT II were purified to near-homogeneity. The molecular masses of NAT I and NAT II were estimated to be 30.5 kDa and 32.6 kDa, respectively. 2-(Bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide were synthesized and evaluated as affinity labels for NAT I and NAT II. Whereas Br-AAF was a highly selective inactivator of NAT II, bromoacetanilide inactivated both NAT I and NAT II in a similar fashion. Inactivation of NAT II by both Br-AAF and bromoacetanilide, and inactivation of NAT I by bromoacetanilide, followed pseudo-first-order kinetics. Relative rate constants (k(obs)/[I]) for the two compounds indicate that Br-AAF is approximately 25 times more potent than bromoacetanilide as an inactivator of NAT II. Both acetylcoenzyme A (CoASAc) and 2-acetylaminofluorene protected NAT II from inactivation by Br-AAF, and CoASAc provided protection of both NAT I and NAT II activities from inactivation by bromoacetanilide, indicating that the inactivation by both bromoacetanilide and Br-AAF is active site directed. The irreversibility of the inactivation of NATs by Br-AAF and bromoacetanilide was demonstrated by the failure to recover transacetylase activities after gel filtration of enzyme preparations that had been preincubated with Br-AAF or bromoacetanilide. Preincubation of NAT II with CoASAc significantly reduced the incorporation of [14C]Br-AAF into the enzyme, providing further evidence that the labeling is active site directed. In addition, pretreatment of NAT II with N-ethylmaleimide completely prevented the labeling of NAT II with [14C]Br-AAF, which suggests that a cysteine thiol is the target nucleophile of Br-AAF. High performance liquid chromatography analysis of the hydrochloric acid hydrolysate of [14C]Br-AAF-labeled NAT II revealed that 70% of total radioactivity is associated with S-carboxymethyl-L-cysteine, indicating that Br-AAF reacts primarily with a cysteine residue at the active site. These studies provide direct evidence that hamster hepatic NAT II contains an essential cysteine residue at the active site, and they establish the potential utility of Br-AAF for determining amino acid sequences in the active site of hamster hepatic NAT II.

2-Acetylaminofluorene↗
PubMed1989

Demonstration of two distinct forms of released low affinity-type rat IL-2 receptors.

The release of IL-2 binding proteins, derived from the 55,000 MW low affinity IL-2R (L chain), has been observed for virtually all L chain-bearing cells in either humans, the mouse or the rat. Based on the characterization of the released human L chain as a molecule 10,000 MW smaller than the cell surface receptor, either proteolytic cleavage or differential splicing of the L chain encoding mRNA have been suggested as mechanisms underlying the receptor release. Combining affinity labelling of the L chain with [125I]IL-2 and immunoprecipitation with L chain-specific monoclonal antibody (mAb) applied for the detection of soluble rat IL-2R revealed the existence of two classes of soluble receptors, one being of the same size as cell surface-expressed L chain, the other of 40,000 apparent molecular mass. These findings raise the possibility of other mechanisms of receptor release than those discussed for human L chain.

Affinity Labels↗
PubMed1988

Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I.

When G0-arrested BALB/c 3T3 cells were treated sequentially with platelet-derived growth factor and epidermal growth factor, cells became responsive to insulin-like growth factor-I (IGF-I). In these primed competent cells, 1 nM IGF-I elicited an approximately 3-fold increase in the calcium influx rate. IGF-I-induced calcium influx was relatively slow in onset and continued for at least 2 h in the presence of IGF-I. When a single Ca2+ channel current was studied by the patch-clamp technique using the cell-attached mode, inward currents with unitary conductance of 19 pS were observed in the presence of 1 nM IGF-I in the patch pipette. IGF-I-sensitive inward current was independent of membrane potential and was activated by a high concentration of insulin. Accordingly, 1 nM IGF-I caused a gradual increase in cytoplasmic free calcium concentration measured by fura2. The action of IGF-I on calcium influx was dependent on extracellular calcium, and IGF-I did not stimulate calcium influx when extracellular calcium concentration was reduced to 10 microM. Both cobalt and tetramethrin blocked the action of IGF-I on calcium influx without affecting the binding of 125I-IGF-I. In primed competent cells, IGF-I-stimulated [3H]thymidine incorporation was dependent on extracellular calcium and was attenuated by cobalt and tetramethrin. When cell-bound 125I-IGF-I was cross-linked by use of disuccinimidyl suberate, a 130-kDa protein was radiolabeled. Affinity labeling of the 130-kDa protein, presumably the alpha-subunit of the IGF-I receptor, was blocked by excess amount of unlabeled IGF-I. These results suggest that relatively low concentrations of IGF-I stimulate calcium influx in primed competent BALB/c 3T3 cells by activating a calcium-permeable cation channel via the IGF-I receptor and that calcium influx may be a critical intracellular message of the progression activity of IGF-I.

Affinity Labels↗
PubMed1998

Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue.

Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site. Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine-238 to be the site of modification. Site-directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity. Lysine-238 seems to interact with the gamma-phosphate group of ATP but is not involved in catalysis.

Adenosine Diphosphate↗
PubMed1993

Identification of the hydrophobic ligand-binding region in recombinant glutathione S-transferase P and its binding effect on the conformational state of the enzyme.

Recombinant glutathione S-transferase P (GST-P) was purified in a homogeneous state. Fatty acid analysis of the enzyme revealed that the final enzyme preparation endogenously bound fatty acids, mostly palmitic acid or stearic acid, which were difficult to dissociate from the complex. Temperature-dependent analysis by 1H NMR indicated that the molecular motion of fatty acids was strongly restrained under physiological conditions, which was significantly different from that of serum albumin. On the other hand, there existed another hydrophobic ligand-binding region in GST-P, to which 1-amino-8-naphthalenesulfonic acid and bilirubin would bind with relatively lower affinity than the endogenously bound fatty acid. The hydrophobic ligand-binding region was determined to be around 141-156 residues from the N-terminus by procedures including association of the enzyme to fatty acid-linked Sepharose and affinity labeling with fluorescent fatty acid. Furthermore, circular dichroism analysis showed that the binding of hydrophobic ligand to GST-P produced a remarkable conformational change of the enzyme, which led to states devoid of transferase activity. In addition, the hydrophobic ligand binding caused a significant fluorescence quenching of tryptophan 38, which was assumed to be located at the active center of GST-P. It could be the result of a conformational change of the active center of the enzyme.

Affinity Labels↗
PubMed1988

Biochemical characterization of the pancreatic cholecystokinin receptor: a possible marker of cell differentiation and development.

The biochemical expression of the cholecystokinin (CCK) receptor on the surface of the pancreatic acinar cell is a potential marker of the state of differentiation of that cell. In this report we review the basis for and the results of the use of affinity labeling techniques for the biochemical characterization of this macromolecular receptor assembly on the adult rat pancreatic acinar cell. A series of specially designed molecular probes are used to define the subunit structure of this receptor, based on the relationships between the sites of covalent attachment of these probes and their receptor-binding domains. We suggest that the receptor-binding domain resides on a Mr = 85,000-95,000 subunit, whereas a distinct Mr = 80,000 also exists as part of this complex.

Affinity Labels↗
PubMed1993

[Affinity modification of 40S ribosomal subparticles from human placenta with mRNA analogs--AUGUnC oligoribonucleotide analogs with an alkylating group at the 3'-end].

Using 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of AUGUn[32P]pC (mRNA analogues), affinity labelling of human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). The regions of 18S rRNA labelled with these mRNA analogues were identified. The main targets of 18S rRNA alkylation by the derivative of AUG[32P]pC were located within positions 1610-1747 and 1748-1869. The site of covalent attachment of AUGU3[32P]pC derivative to 18S rRNA was found within positions 593-673. Taking into account the data on labelling of human placenta ribosomes with the same derivatives of oligourydilates obtained previously, the conclusion was made that the arrangement of the codon U3 in the mRNA-binding centre of the initiation complex 40S.AUGU3[32P]pC derivative.eIF-2.GTP.Met-TPHK(fMet) differs from the arrangement of the same codon at the A-site of the complex imitating the pretranslocation state of ribosomes.

Affinity Labels↗
PubMed1989

Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand.

A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.

Affinity Labels↗
PubMed1999

Kinetic mechanism and ATP-binding site reactivity of p38gamma MAP kinase.

Activated p38gamma MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor substrate increased k(cat)/K(m)20-fold. AMP-PCP was competitive with ATP binding and non-competitive with phosphoacceptor substrate binding. The nucleotide binding site affinity label 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA) bound stoichiometrically at Lys-56 in the ATP site of both unphosphorylated and activated p38gamma. AMP-PCP only protected the activated enzyme from FSBA inactivation, implying that AMP-PCP does not bind unphosphorylated p38gamma. Basal ATPase activities were also observed for activated p38alpha, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases.

Adenosine↗
PubMed1997

Demonstration of an extracellular ATP-binding site in NCAM: functional implications of nucleotide binding.

A minor fraction of the total ecto-type (E-type) ATPase activity of rat synaptosomes has been detected in immunoprecipitates of the neural cell adhesion molecule, NCAM, indicating that this either is an intrinsic enzymatic activity of NCAM or of an ATPase tightly associated to NCAM [Dzhandzhugazyan & Bock (1993) FEBS Lett. 336, 279-283]. We here demonstrate ATPase activity in preparations of the lipid-anchored as well as the transmembrane NCAM isoforms immunoisolated from transfected L-cells. A fraction of the E-type ATPase activity is spontaneously released from synaptosomes. Released material was fractionated by various chromatographic procedures and an extracellular fragment of NCAM was shown to co-elute with the major part of the enzymatic activity. Furthermore, it was shown that agarose-coupled NCAM-antibodies retained 85% of the ATPase activity released from synaptosomes after treatment with phosphatidylinositol-specific phospholipase C. These findings restricted the association or expression of the enzymatic activity to the extracellular part of NCAM. An affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine, FSBA, was shown to inhibit ATPase activity of immunoisolated NCAM, and incorporation of FSBA was detected in all three major NCAM isoforms (A, B, and C). An excess of ATP prevented both inactivation of the enzyme and affinity labeling of NCAM. Thus, NCAM contains an ATP-binding site, and this site is localized extracellularly and probably has the catalytic function. Binding of the substrate or FSBA protected a proteolytic cleavage site in NCAM localized close to the membrane presumably by induction of a local conformational change in NCAM, indicating a mechanism by which ATP may regulate NCAM adhesion and adhesion-triggered processes. A possible role of this mechanism in synaptic plasticity and memory consolidation is proposed.

Adenosine↗
PubMed1990

Synthesis of alpha-MSH fragments containing phenylalanine mustard for receptor studies.

For chemical affinity labeling of the melanotropin receptor several alpha-MSH fragments containing phenylalanine mustard were synthesized in solution. Tested in the frog skin bioassay the derivatives roughly preserved the biological activity of the corresponding natural sequences. Alkylating peptides with prolonged biological activity containing phenylalanine mustard in place of arginine, phenylalanine or methionine are inhibitors of alpha melanotropin, suggesting an irreversible binding to reactive nucleophiles on the part of the MSH receptor, where the Met-Glu-His-Phe-Arg sequence is attached.

Affinity Labels↗
PubMed1982

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide.

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.

Affinity Labels↗
PubMed1999

Mapping of T7 RNA polymerase active site with novel reagents--oligonucleotides with reactive dialdehyde groups.

Oligonucleotides of a novel type containing 2'-O-beta-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions.

Affinity Labels↗