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PubMed1983

Modification of TPN-dependent isocitrate dehydrogenase by the 2', 3'-dialdehyde derivatives of TPNH and TPN.

Catalytic reaction of the 2', 3'-dialdehyde analog of TPN (oTPN) with pig heart TPN-dependent isocitrate dehydrogenase in the presence of the substrate manganous isocitrate results in the formation of the dialdehyde derivative of TPNH (oTPNH). In the absence of the substrate, modification by oTPN leads to a progressive inactivation of the enzyme. The dependence of the pseudo-first order rate constants on the reagent concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification (kmax = 5.5 X 10(-2) min-1; K1 = 290 microM). Reaction of [14C]oTPN with the enzyme results in the incorporation of 2 mol of oTPN/mol of peptide chain. No appreciable protection against either inactivation or incorporation by the natural ligands TPN and TPNH was obtained, suggesting different modes of binding of the analog in the presence and absence of the substrate isocitrate. Enzymatically synthesized oTPNH has been isolated and demonstrated to act as an affinity label for a TPNH-binding site of isocitrate dehydrogenase. The inactivation process exhibits saturation kinetics (kmax = 2.67 X 10(-3) min-1; K1 = 33 microM). Protection against activity loss, as well as a decrease in incorporation from 2 to 1 eq of [14C]oTPNH bound/peptide chain was observed in the presence of 1 mM TPNH. From the TPNH concentration dependence of the inactivation rate by oTPNH, a dissociation constant of 3.4 microM is calculated for TPNH, indicating binding of the analog to a specific TPNH-binding site on the enzyme. Although dialdehyde derivatives are frequently assumed to form Schiff bases with proteins, the evidence presented suggests the formation of morpholino derivatives as the products of the covalent reaction of isocitrate dehydrogenase with the dialdehyde derivatives of TPN and TPNH. The new reagent, oTPNH, may serve as an affinity label for other dehydrogenases.

Adenosine Diphosphate↗
PubMed1988

Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies.

Neuronal acetylcholine receptors (AChRs), which bind nicotine with high affinity but do not bind alpha-bungarotoxin (alpha Bgt), have recently been immunoaffinity-purified from chicken (Whiting and Lindstrom, 1986a) and rat (Whiting and Lindstrom, 1987a) brain using monoclonal antibodies (mAbs). Here we report the characterization of nicotinic AChRs of bovine and human brain using as probes mAbs prepared to AChRs from rat brain. Both the human and bovine brain AChRs exhibit high affinity for L-nicotine (Ki = 16 nM for bovine AChR and Ki = 6.5 nM for human AChR) and other cholinergic agonists, relatively lower affinity for cholinergic antagonists, and do not bind alpha Bgt. These AChRs are affinity-labeled with bromoacetylcholine and 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA) after reduction with dithiothreitol, indicating that amino acid residues homologous to cysteines 192 and 193 of alpha subunits of Torpedo electric organ AChRs are conserved. Immunoaffinity-purified bovine brain AChR consists of 2 types of subunit, Mr 50,600 and Mr 74,400. The Mr 74,400 subunit was affinity-labeled with 3H-MBTA, indicating that it contains the ACh binding site. Thus, mAbs have proven to be excellent probes for these proteins, and have been used to show that neuronal nicotinic AChRs of chickens, rats, and cattle are macromolecules approximately 10 S in size and composed of only 2 kinds of subunits: an ACh-binding subunit and a structural subunit.

Acetylcholine↗
PubMed1999

Comparison of paclitaxel-, 5-fluoro-2'-deoxyuridine-, and epidermal growth factor (EGF)-induced apoptosis. Evidence for EGF-induced anoikis.

Epidermal growth factor (EGF), a hormone that stimulates proliferation of many cell types, induces apoptosis in some cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblotting, enzyme assays, and affinity labeling after treatment with EGF, paclitaxel, or 5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicated by disappearance of the corresponding zymogens from immunoblots, cleavage of substrate polypeptides in situ, and detection of active forms of these caspases in cytosol and nuclei using fluorogenic assays and affinity labeling. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspase-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Interestingly, caspase activation was consistently lower after EGF treatment than after paclitaxel or 5FUdR treatment. Additional experiments revealed that the majority of cells detaching from the substratum after EGF (but not paclitaxel or 5FUdR) were morphologically normal and retained the capacity to readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis. These observations not only indicate that EGF- and chemotherapy-induced apoptosis in this cell line involve the same downstream pathways but also suggest that detachment-induced apoptosis is responsible for the paradoxical antiproliferative effects of EGF.

Affinity Labels↗
PubMed1988

Characterization of a novel insulin receptor from stingray liver.

The insulin receptor from the liver of stingray, a cartilaginous fish, has characteristics which are in marked contrast to those of the mammalian insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cross-linked, affinity-labeled stingray insulin receptor shows an apparent molecular mass of 210 kDa for the intact receptor. Reduction with mercaptoethanol resulted in no alteration in the apparent size of the stingray insulin receptor. Gel filtration studies of Triton X-100 solubilized stingray insulin receptor demonstrated an apparent Stokes radius of 7.6 nm. Ultracentrifugation sucrose gradient studies of cross-linked affinity labeled stingray receptor resulted in determination of a sedimentation coefficient of 13 S. Both of these parameters were greater than simultaneously obtained data for the human insulin receptor (7.2 nm and 11 S, respectively). Unlabeled insulin competed with binding of 125I-insulin and 125I-insulin growth factor (IGF) I with a half-maximal concentration of 1 nM for either. Unlabeled IGF I and II also competed, but were 4-5-fold less potent than insulin. It was found that not only did IGF I bind to the 210-kDa material, but both insulin and IGF I stimulated phosphorylation of a 210-kDa material which was immunoprecipitable by a polyclonal insulin receptor antibody. Elution of this material from the gel followed by hydrolysis and thin layer chromatography demonstrated that the 210-kDa material was specifically phosphorylated on tyrosine only. These data suggest that the insulin receptor from stingray liver is a dimer made up of 2 identical subunits of about 210 kDa size which contain both binding regions and insulin-stimulated tyrosine kinase. Specificity studies suggest that the stingray insulin receptor may represent a phylogenetic position prior to the evolutionary divergence of insulin and the insulin-like growth factors.

Affinity Labels↗
PubMed1995

Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus.

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1982

Calcium sites in MSH stimulation of xenopus melanophores: studies with photoreactive alpha-MSH.

Photo-affinity labelling of MSH receptors on tail-fin melanophores of Xenopus tadpoles with p-azidophenylalanine 13-alpha-MSH (Pap13)-alpha-MSH) or p-azidophenylacetyl-serine1-alpha-MSH ([Apac-Ser1]-alpha-MSH) resulted in a long-lasting stimulation of the melanophores which cannot be reversed despite continuous washing. The generation of this irreversible response is inhibited when photo-affinity labelling is performed in a Ca2+-free medium or in the presence of Ca2+ antagonists. The irreversible stimulation produced in normal medium is completely reversed upon removal of Ca2+ , but is not affected by Ca2+ antagonists or melatonin. Re-addition of Ca2+ after temporary removal restores to irreversible stimulation, even in the presence of Ca2+ antagonists or melatonin. This proves that covalent alpha-MSH-receptor complexes remain fully functional despite temporary deprivation of ca2+. Racemized alpha-MSH, which binds 'tightly' to the receptor and produces a long-lasting effect, is removed from the receptor in Ca2+-free medium, but not by Ca2+ antagonists or melatonin. These results confirm earlier results showing that at least 2 Ca2+ sites are involved in alpha-MSH action, one associated with MSH-receptor binding and the other with the subsequent generation of the effect. The dual role of Ca2+ is not the result of EGTA present; it is specific (Mg2+ has no effect) and is identical for the two different photoreactive alpha-MSH derivatives.

Affinity Labels↗
PubMed1993

Binding of a substrate analogue can induce co-operative structure in the plasmin serine-proteinase domain.

Human miniplasminogen and miniplasmin were studied by n.m.r. spectroscopy and differential scanning calorimetry (d.s.c.) in order to investigate the structural properties of the serine-proteinase domain. The d.s.c. thermograms of both miniplasminogen and non-inactivated miniplasmin at pH 4.0 can be closely fitted to two transitions, at 62 +/- 2 and 72 +/- 2 degrees C, corresponding to unfolding of the kringle 5 and proteinase domains respectively. No evidence was found, under these conditions, for non-co-operative unfolding of the proteinase domain. On inactivation of miniplasmin with an affinity label, a number of additional resonances arising from residues of the proteinase domain are observed in resolved regions of the n.m.r. spectrum. A combination of variable-temperature n.m.r. and d.s.c. has shown that part of the proteinase domain undergoes a major conformational transition on heating which is distinct from the unfolding of the remainder of the proteinase domain or the kringle 5 domain. This additional transition occurs at a temperature that depends on the nature of the affinity label present and is not observed in the absence of an inactivating agent. These results provide direct evidence for the existence of a region of the proteinase domain which, under these conditions, becomes structured only in the presence of a bound substrate.

Affinity Labels↗
PubMed1997

Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution.

The mechanisms involved in the enzymatic degradation of cellulose are of great ecological and commercial importance. The breakdown of cellulose by fungal species is performed by a consortium of free enzymes, known as cellobiohydrolases and endoglucanases, which are found in many of the 57 glycosyl hydrolase families. The structure of the endoglucanase I (EG I), found in glycosyl hydrolase family 7, from the thermophilic fungus Fusarium oxysporum has been solved at 2.3 A resolution. In addition to the native enzyme, structures have also been determined with both the affinity label, 3,4-epoxybutyl beta-D-cellobioside, and the reaction product cellobiose. The affinity label is covalently bound, as expected, to the catalytic nucleophile, Glu197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose is found bound to the -2 and -1 subsites of the enzyme. In marked contrast to the structure of EG I with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar [Sulzenbacher, G., et al. (1996) Biochemistry 35, 15280-15287], the cellobiose complex shows no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the additional +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.

Binding Sites↗
PubMed1981

Chemically reactive estrogens: synthesis and estrogen receptor interactions of hexestrol ether derivatives and 4-substituted deoxyhexestrol derivatives bearing alkylating functions.

A series of chemically reactive derivatives of the nonsteroidal estrogen hexestrol have been synthesized as potential affinity labels for the estrogen receptor or as cytotoxic agents with selective activity against receptor-containing cells. These compounds are hexestrol ethers with halo ketone, halohydrin, or epoxide functions or 4-substituted deoxyhexestrols with halo ketone, benzyl halide, nitro, azide, sulfonyl fluoride, or sulfonyl azide groups. The alkylating activity of the electrophilic derivatives was measured using the colorimetric reagent nitrobenzylpyridine, the bromo derivatives being considerably more reactive than the chloro ones. Their reversible binding to the lamb uterine estrogen receptor was measured by competitive binding assays, and their irreversible reaction with receptor was measured by exchange assays that determine the rate and extent of receptor inactivation. In general, monoetherification of hexestrol or substitution of deoxyhexestrol produces compounds with relatively low affinity for the estrogen receptor (0.3-10% that of estradiol). Most of the electrophilic derivatives are rapid and effective inactivators of receptor (24-70% inactivation within 0.5-5 h at 25 degrees C). Of the photosensitive derivatives, 4-azidodeoxyhexestrol appears to be the most efficient receptor inactivator (49%). The high reactivity of these compounds toward the estrogen receptor and the lack of interference by their reaction with other cellular nucleophiles suggest that these compounds may be useful as affinity-labeling agents or as selective cytotoxic agents in intact systems.

Affinity Labels↗
PubMed1975

Essential thiols of yeast hexokinase: alkylation by a substrate-like reagent.

It is demonstrated that N-bromoacetyl-D-galactosamine acts as a substrate-like reagent for yeast hexokinases A and B, producing affinity labeling. At the order of 10(-3) M reagent concentrations, rapid inactivation of the enzyme is produced: the kinetics are consistent with dependence upon a reversible inhibitor-enzyme initial complex, with a dissociation constant of 3.8 x 10(-3) M for hexokinase B at 35 degrees, pH 8.5. The glucose analog is 30-fold less effective, presumably due to self-protection. The inactivating reaction is an order of magnitude faster than that with bromoacetate. All the alkylation of hexokinase B was shown to occur at two thiol groups per subunit, associated stoichiometrically with inactivation. Unlike the reaction there of simple alkylators, two nonessential thiols per subunit are left unattacked when this inactivation reaction is complete. Protection against the affinity alkylation is exerted by the substrates glucose, mannose, fructose, glucose 6-phosphate, fructose 6-phosphate, ATP-Mg, and ADP-Mg, in proportion to their affinities for the active center. Free ATP also protects. Mg2+ alone has no influence, and Mn2+ gives a slight acceleration, when correction is made for a slow inactivation that occurs when the enzyme is incubated at 35 degrees with Mn2+ alone. Galactose, virtually a nonsubstrate, has no influence on the affinity alkylation, but N-acetylgalactosamine, a nonsubstrate and a weak inhibitor of the enzymic reaction, has an accelerating effect. An interpretation is made in terms of binding to a site that influences the active center. This affinity label should provide a means of isolating a peptide containing active-center-related groups.

Adenine Nucleotides↗
PubMed1998

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations.

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.

Affinity Labels↗
PubMed1999

Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.

Affinity Labels↗
PubMed1993

Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis.

Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides. Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase. FSBA inactivated the amidotransferase with saturation kinetics. Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of [3H] sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of [3H]SBA. Six chymotryptic peptides modified with [3H]SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity. Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by [3H]SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465. Tyr258 was also modified by N3-AMP. Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme. However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP. Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively. Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation. These results localize residues important for catalysis in close proximity to a site for nucleotide binding. Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP. A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition. Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP. A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.

Adenosine↗
PubMed1993

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

Affinity Labels↗
PubMed1983

Characterization of the receptor for platelet-derived growth factor on human fibroblasts. Demonstration of an intimate relationship with a 185,000-Dalton substrate for the platelet-derived growth factor-stimulated kinase.

The receptor for platelet-derived growth factor (PDGF) on human foreskin fibroblasts has been characterized. The molecular weight of the PDGF-receptor complex was estimated by affinity labeling techniques to about 200,000, as determined by sodium dodecyl sulfate-gel electrophoresis performed under reducing conditions. Subtraction of the Mr of reduced PDGF (18,000 to 15,000) gives a Mr for the receptor proper of 185,000 (+/- 10,000). The mobility in sodium dodecyl sulfate-gel electrophoresis was similar whether or not reducing agents were present, suggesting that the receptor may be a single chain protein. The hydrodynamic size of the 125I-PDGF-receptor complex after solubilization with Triton X-100, corresponded to a Mr of approximately 320,000, as determined by gel chromatography. Subtraction of the Mr contributions from Triton X-100 and PDGF, respectively, gives a Mr of approximately 200,000 for the receptor itself, an estimate in good agreement with the value obtained from the affinity-labeling experiments. Several lectins were analyzed for their ability to inhibit binding of 125I-PDGF to its receptor. It was found that wheat germ agglutinin and a lectin from Crotalaria juncea were effective inhibitors and that their inhibitory effects could be neutralized by N-acetylglucosamine and galactose, respectively, suggesting that the receptor contains these sugars. The properties of the receptor were compared with those of a 185,000-Da component, being the major substrate for the membrane-bound PDGF-stimulated kinase. It was found that the 185,000-Da component behaved similar to the PDGF receptor in sodium dodecyl sulfate-gel electrophoresis, performed with or without reducing agents present. Further, the 185,000-Da component co-eluted with the PDGF receptor on a Sepharose 6B column, and had affinity for the same lectins that inhibited the binding of 125I-PDGF to its receptor. Finally, the 185,000-Da component had affinity for PDGF immobilized on Sepharose beads, suggesting that it has PDGF-binding activity. We conclude that the PDGF receptor and the 185,000-Da substrate for the PDGF-dependent kinase are intimately related and probably identical molecules.

Affinity Labels↗
PubMed1991

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.

Animals↗
PubMed1988

Structural and immunohistochemical characterization of insulin-like growth factor I and II receptors in the murine central nervous system.

The description of the cellular localization of insulin-like growth factor (IGF) receptors in the central nervous system (CNS) remains incomplete, as do the descriptions of changes in their characteristics with respect to different developmental stages. We, therefore, performed affinity labeling studies in microsomal membrane preparations of adult and fetal rat brain and liver tissues with [125I]IGF-I and [125I]IGF-II. These studies demonstrated tissue- and developmental stage-specific structural variants of type I receptor alpha-subunits as well as type II receptors. The adult rat brain type I alpha-subunit had an apparent mol wt (Mr) of 127,000, whereas those of adult and fetal rat liver measured 140,000. Fetal rat brain microsomes, however, had two types of type I receptor alpha-subunits measuring 130,000 and 120,000 Mr. The larger subunit from fetal brain consistently migrated at an apparent Mr of 3,000, greater than subunits from adult brain. Both type I and II receptors were more abundant in fetal liver and brain than in adult tissues. Affinity labeling was also performed directly to monolayers of cultured fetal brain neurons and newborn astrocytes. These studies detected both type I and II receptors on the surfaces of both types of cells. However, only the high Mr (140,000) form of the type I alpha-subunit was detected in cultured CNS cells, suggesting that expression of low Mr variant receptors is altered in vitro. Type II receptors were demonstrated by immunohistochemistry in adult rat hypothalamic neurons. However, the majority of neurons did not react with type II receptor antibody. This finding implies that only a minority of hypothalamic neurons are capable of responding to IGF-II via type II receptors. On the other hand, all astrocytes had striking type II receptor immunoreactivity. This signifies a more general biological role for this receptor in astrocytes compared with neurons. These results suggest that different tissue-, developmental stage-, and cell-specific processes are mediated by IGF receptors and suggests new directions in which to explore potential biological actions for these receptor-ligand systems in the CNS.

Affinity Labels↗
PubMed1990

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.

Adsorption↗