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PubMed1977

gamma-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport.

gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.

Affinity Labels↗
PubMed1991

Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base.

Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.

Acetyl-CoA C-Acetyltransferase↗
PubMed1987

Identification of insulin receptors on rat Sertoli cells.

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75-80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of Kd = 1.8 X 10(-9) M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10-50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30-40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of Mr greater than 300,000 or Mr = 130,000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of Mr 130,000 may represent the alpha-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.

Affinity Labels↗
PubMed1991

Comparison of the physicochemical properties of type I and type II iodothyronine 5'-deiodinase.

The 5'-deiodination of thyroxine is catalyzed by two enzymes which differ in their tissue distribution, substrate specificities, sensitivity to the inhibitor, propylthiouracil, and response to thyroid status. By using the affinity label, N-bromoacetyl-L-thyroxine, both isoenzymes have been found to have substrate binding subunits of approximately 27 kDa. In this study, we compared the substrate binding subunits and hydrodynamic properties of the type I and the type II isozymes using the affinity label, N-bromoacetyl-L-thyroxine, to identify the enzymes. High resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the substrate binding subunit of the type I enzyme had an Mr of 27,000, while that of the type II enzyme had a slightly higher Mr of 29,000. This difference was not accounted for by glycosylation. Partial staphylococcal V8-protease digests of the substrate binding subunit of the type I enzyme yielded fragments of 14.6, 13.7, and 7.0 kDa, while V8-protease digests of the substrate binding subunit for the type II enzyme produced fragments of 28.0, 25.1, 19.0, 9.5, 7.2, and 5.8 kDa. Unique cyanogen bromide fragmentation patterns were also observed for the two substrate binding subunits. Sedimentation coefficients of the detergent-soluble type I and type II holoenzymes were 3.67 and 5.22 S, respectively, as determined by sucrose density centrifugation. The type I enzyme behaved as a globular protein, whereas the type II enzyme showed sedimentation properties typical of asymmetric integral membrane proteins. The Stokes radii were 3.78 and 4.97 nm, respectively. From these data, the calculated Mr for detergent-solubilized type I 5'-iodothyronine deiodinase was 55,400 and for the type II enzyme was 198,700. These data indicate that the two isozymes of iodothyronine 5'-deiodinase are multimeric, differ in holoenzyme size and subunit composition, and that their substrate binding subunits are distinct.

Affinity Labels↗
PubMed1993

Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.

Anti-peptide antibody to the human glucocorticoid receptor (GR) was produced and used to demonstrate that a subset of the GR population resides in the plasma membrane of human leukemic cells. Characterization of the antibody with intracellular GR (iGR) showed its ability to shift [3H]triamcinolone acetonide-labeled GR (4S protein) from two human leukemic cell lines to a higher density in sucrose gradients; Western and autoradiographic analysis of affinity-labeled ([3H]dexamethasone 21-mesylate) receptor revealed an immunoreactive and competitively labeled band of 94 kDa. CCRF-CEM cell membrane GR (mGR) resolved as a > 7S protein on density gradients and immunoselected cell surface protein labeled by whole cell biotinylation or affinity-labeling with [3H]dexamethasone 21-mesylate was approximately 145 kDa, demonstrating that mGR was larger in size than iGR, as has been shown previously for the mGR of mouse lymphoma cells. Analysis of mGR in lymphocytes of leukemic patients and the CCRF-CEM cell line indicated differences in levels of expression as shown by FACS and immunocytochemical analyses. We are currently using this system to study the correlation between the quantity of membrane-resident GRs and the glucocorticoid-induced lytic response, a relationship previously shown in the murine (S-49 cell) system.

Amino Acid Sequence↗
PubMed2000

Site-directed mutagenesis and biochemical analysis of the endogenous ligands in the ferrous active site of clavaminate synthase. The His-3 variant of the 2-His-1-carboxylate model.

The facial 2-His-1-carboxylate (Asp/Glu) motif has emerged as the structural paradigm for metal binding in the alpha-ketoglutarate (alpha-KG)-dependent nonheme iron oxygenases. Clavaminate synthase (CS2) is an unusual member of this enzyme family that mediates three different, nonsequential reactions during the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In this study, covalent modification of CS2 by the affinity label N-bromoacetyl-L-arginine near His297, which is within the HRV signature of a His-2 motif, suggested this histidine could play a role in metal coordination. However, site-specific mutagenesis of eight His residues to Gln identified His145 and His280, but not His297, as involved in iron binding. Weak homology of His145 and its flanking sequence and the presence of Glu147 fitting the canonical acidic residue of the His-Xaa-Asp/Glu signature are consistent with His145 being a coordinating ligand (His-1). His280 and its flanking sequence, which give poor alignments to most other members of this enzyme family, are similar among a subset of these enzymes and notably to CarC, an apparent oxygenase involved in carbapenem biosynthesis. The separation of His145 and His280 is more than twice that seen in the current 2-His-1-carboxylate model and may define an alternative iron binding motif, which we propose as His-3. These ligand assignments, based on kinetic measurements of both oxidative cyclization/desaturation and hydroxylation assays, establish that no histidine ligand switching occurs during the catalytic cycle. These results are confirmed in a recent X-ray crystal structure of CS1, a highly similar isozyme of CS2 (81% identical). Tyr299, Tyr300 in CS2 modified by N-bromoacetyl-L-arginine, is hydrogen bonded to Glu146 (Glu147 in CS2) in this structure and well-positioned for reaction with the affinity label.

Affinity Labels↗
PubMed1990

Two dissociable phases in the contractile response of the guinea pig isolated vas deferens to adenosine triphosphate.

The effects of the ATP affinity label periodate-oxidized ATP (ATP-2',3'-dialdehyde; P-ATP) on contractile responses of the guinea pig vas deferens to ATP was characterized and compared to the effects of the specific P2x-purinoceptor photoaffinity label antagonist, arylazido aminopropionyl ATP (ANAPP3). After incubation of vas deferens with 10(-2) M P-ATP for 5 min, the second phase of biphasic contractions to ATP was inhibited selectively. The inhibitory effect of P-ATP was specific for ATP, and resulted from affinity labeling in that it was long-lasting, was not reversed by washing, was related in magnitude to exposure period and was attenuated by ATP present during the incubation. In contrast to P-ATP, ANAPP3 inhibited selectively the first phase of ATP-induced responses. P-ATP and ANAPP3 together inhibited both phases of response. P-ATP inhibited selectively the second phase of responses to 5'-substituted ATP analogs which develop a prolonged second phase [e.g., adenosine 5'-O-(3-thiotriphosphate) and adenosine tetraphosphate] or an abbreviated second phase (e.g., beta,gamma-methylene ATP, beta,gamma-imido ATP and alpha,beta-methylene ATP). The greater the duration of the second phase, the more pronounced was the inhibitory effect. Two distinct and dissociable mechanisms mediate the biphasic response to ATP: the initial phase involves ANAPP3-sensitive P2x-purinoceptors, whereas the second is blocked by P-ATP and appears to involve cleavage of the phosphate chain. An additional effect observed as a result of P-ATP-treatment was potentiation of the first phase of contraction to ATP. This novel and irreversible effect may arise from the inhibition of degradative ecto-phosphohydrolase activity.

Adenosine Triphosphate↗
PubMed1980

Evidence for participation of transglutaminase in receptor-mediated endocytosis.

We report evidence that the enzyme transglutaminase (glutaminyl-peptide gamma-glutamyltransferase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC2.3.2.13) participates in receptor-mediated endocytosis. Clustering and internalization of rhodamine-labeled alpha 2-macroglobulin (R alpha 2 M) in normal rat kidney (NRK) cells is inhibited by a wide spectrum of compounds that inhibit transglutaminases, including that from NRK cells. The pattern of clustering inhibition resembles the pattern of transglutaminase inhibition as follows: (i) The most potent transglutaminase inhibitors are dansylcadaverine and the transglutaminase-directed affinity label N-benzyloxy-carbonyl-5-diazo-4-oxonorvaline p-nitrophenyl ester; these were also the most potent inhibitors of clustering and internalization of R alpha 2M. (ii) The inhibition of clustering of R alpha 2M occurs in the same concentration range as that required for transglutaminase inhibition. (iii) Linear primary amines are more effective blockers than the iso-chain primary amines. (iv) The transglutaminase affinity label N-benzyloxycarbonyl-5-diazo-4-oxonorvaline p-nitrophenyl ester irreversibly inhibits a significant fraction of the NRK transglutaminase and the clustering and internalization of R alpha 2M. A closely related compound, N-trifluoroacetyl-6-diazo-5-oxonorleucine ethyl ester, does not significantly inhibit transglutaminase or clustering and internalization. (v) Clustering and internalization is inhibited 10-fold more effectively by the heptapeptide Ac-Gly2-LLeu-LLys-Gly3 than by the heptapeptides Ac-Gly2-LLeu-DLys-Gly3 or AcGly3-DLys-DLeu-Gly2. This is the pattern of stereospecificity for the inhibition of purified transglutaminases.

Affinity Labels↗
PubMed1993

Selenium-dependent regulation of type I 5'-deiodinase expression.

Selenite concentration regulates activity and expression of the p27 substrate-binding subunit of type I 5'deiodinase (5'-D) and of a protein labeled with bromoacetylthyroxin (BrAcT4), or p30, with yet unknown function in a porcine-kidney epithelial cell line (LLC-PK1) cultured in serum-free medium. p27 is metabolically labeled by 75-selenite and affinity labeled by BrAc[125I]T4. Compared with glutathione peroxidase, expression of the p27 5'D subunit (5'-DI) is observed at 10-fold-lower concentrations of selenium in the growth medium, suggesting an intracellular hierarchy of selenite utilization. Selenium deficiency retards cell growth and prevents 5'-DI expression and may thus impair thyroid hormone action in vivo.

Affinity Labels↗
PubMed1997

Roles of the structure and orientation of ligands and ligand mimics inside the ligand-binding pocket of the vitamin D-binding protein.

1alpha,25-Dihydroxyvitamin D3, the vitamin D hormone, manifests its diverse biological properties by specifically binding to the vitamin D sterol-binding pockets of vitamin D-binding protein (DBP) and vitamin D receptor. In the past, several affinity, photoaffinity, and chemical modification studies have been carried out to probe the vitamin D sterol-binding pocket of DBP and to evaluate the relationship between the structure of this pocket and the functions of the protein. In the present study, we examined the steric requirements inside this pocket by considering conformational structures of various bromoacetate derivatives of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 and their abilities to covalently and specifically modify this pocket. We observed that, although 25-hydroxyvitamin D3 3beta-bromoacetate (25-OH-D3-3-BE), 1alpha,25-dihydroxyvitamin D3 3beta-bromoacetate [1alpha,25(OH)2D3-3-BE], 1alpha,25-dihydroxyvitamin D3 1alpha-bromoacetate [1alpha,25(OH)2D3-1-BE], and 1alpha,25-dihydroxyvitamin D3 1alpha,3beta-dibromoacetate [1alpha,25(OH)2D3-1,3-di-BE] bound DBP in a specific manner, only [3H]-25-OH-D3-3-BE and [3H]-1alpha,25(OH)2D3-3-BE affinity labeled the protein. BNPS-skatole cleavages of [3H]-25-OH-D3-3-BE- and 3H-1alpha,25(OH)2D3-3-BE-labeled DBP samples produced the same labeled peptide (N-terminal), demonstrating the specificity of labeling by these analogs. Energy-minimized conformational structures of these bromoacetate derivatives indicated significant changes in the A-ring conformations of these analogs. These structural changes were invoked to explain the inability of [3H]-1alpha,25(OH)2D3-1-BE and [3H]-1alpha,25(OH)2D3-1,3-di-BE to affinity label DBP. Overall, these studies suggested that the vitamin D sterol-binding pocket in DBP is sterically quite restrictive. This information could be potentially important in designing future vitamin D-based drugs for several diseases.

Acetates↗
PubMed1979

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.

Animals↗
PubMed1992

Preparation of a chemiluminescent imidoester for the non-radioactive labelling of proteins.

A chemiluminescent aryl acridinium ester was synthesized which possesses an imidate ester group capable of reacting with proteins under mild conditions. The compound can be detected at levels as low as 5.2 x 10(-19) mol using commercially available luminometers and can therefore be used to produce high specific activity labelled antibodies for use in immunochemiluminometric assays. The imidate ester compares favourably with a previously reported N-succinimidyl ester in terms of its labelling properties but is easier to synthesize, requiring one less step. The compound was used to label affinity purified to synthesize, requiring one less step. The compound was used to label affinity purified sheep antibodies to human parathyroid hormone to demonstrate its utility in a two-site immunochemiluminometric assay for the measurement of intact parathyroid hormone.

Acridines↗
PubMedDate not supplied

Uptake of oligonucleotides by keratinocytes.

Oligonucleotides (ODNs) conjugated to rhodamin (Rh) and 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODNs transport into keratinocytes. Affinity labeling of two proteins, 63 and 35 kDa, and the inhibition of the affinity labeling and ODNs uptake by the cells in the presence of nucleic acids, polyanions and trypsin suggest, that the proteins are involved in transport of nucleic acids in keratinocytes.

Autoradiography↗
PubMed1979

Binding specificity of rat alpha-fetoprotein for a series of estrogen derivatives: studies using equilibrium and nonequilibrium binding techniques.

The binding specificity of rat alpha-fetoprotein (AFP) for a series of estrogen derivatives has been investigated and compared to that of the uterine cytosol estrogen receptor. In addition, several equilibrium and nonequilibrium binding techniques have been examined for their appropriateness in studying AFP-estrogen interactions under different experimental conditions. The ratio of association constants (RAC) of various estrogen derivatives (compared to 17 beta-estradiol; RAC = 100%) were determined using a rapid and convenient miniaturized equilibrium assay that utilizes the partitioning properties of Sephadex G-25. AFP has the highest affinity for estrone derivatives and is especially tolerant of small, nonpolar substituents in the D ring; it binds nonsteroidal estrogens and antiestrogens weakly or not at all. Thus, it has a binding specificity quite unlike that of the estrogen receptor. Several potential affinity labels were studied. Of these, 16-diazoestrone, a photoreactive compound, has the highest affinity for AFP (RAC = 121%). Another equilibrium technique, steady state polyacrylamide electrophoresis, effects a clear separation between AFP and albumin. It is superior to conventional polyacrylamide electrophoresis under conditions where dissociation is rapid. Charcoaldextran adsorption also rapidly removes estradiol that is bound to AFP (t1/2 = approximately 10 min at 0 C). The AFP-estradiol complex appears to be stabilized by adsorption to hydroxylapatite, however, so that hydroxylapatite can be employed in a batchwise manner to assay binding to AFP. This is the most appropriate technique when high concentrations of ligand are used. These studies highlight the marked differences in binding specificity between rat AFP and the uterine cytosol estrogen receptor and indicate the techniques most appropriate for binding measurements under different conditions. This work should facilitate the design of affinity labeling reagents for rat AFP as well.

Animals↗
PubMed1987

Characterization of a monoclonal antibody against the lymphoblast substance P receptor.

A murine monoclonal antibody to the IM-9 lymphoblast substance P (SP) receptor has been produced which recognizes the membrane-associated proteins of the SP receptor as demonstrated by immunoprecipitation of [125I]SP affinity-labeled and [35S]methionine biosynthetically labeled IM-9 soluble membranes. SP and anti-SP receptor binding to [35S]methionine-labeled IM-9 cell proteins were directly compared by attachment of each to affinity supports. Eluants from these affinity columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed an equivalent 33-kDa protein in both cases. This protein corresponds to one of the previously described [125I]SP specifically affinity-labeled membrane-associated proteins. In addition, two-color fluorescence-activated cell sorter analysis with human peripheral blood T lymphocytes with fluorescein-SP and rhodamine-labeled antireceptor antibody revealed a distinct population of cells (20 to 30%) that were equally labeled by both the fluorescent peptide and antibody. This result indicates that the anti-SP receptor antibody recognizes an epitope of the receptor that is common to both human peripheral blood T lymphocytes and IM-9 lymphoblast cells.

Animals↗
PubMed1991

Labeling of integrin alpha v beta 3 with 58Co(III). Evidence of metal ion coordination sphere involvement in ligand binding.

Integrin-mediated cell adhesion to the extracellular matrix is divalent metal ion-dependent; however, a demonstration of the interaction between native integrins and divalent metal ions is lacking. Here we provide direct evidence that the vitronectin receptor (VNR) is a metalloprotein. The unique electron shell of Co(II), an ion which we show supports ligand recognition by VNR, enables its oxidative conversion to inert Co(III). This property facilitated "affinity labeling" of VNR with 58Co(III) by oxidation of the metal ion in situ (i.e. in position). An average of 3.5 +/- 0.5 mol of cobalt were incorporated per mol of VNR. The ability of VNR to bind metal ions was independently confirmed by examining the interaction between VNR and Mn2+ under native conditions. The apparent high affinity between VNR and Mn2+ allowed us to observe the specific binding between 54Mn2+ and VNR by equilibrium gel filtration studies. Interestingly, the oxidative incorporation of Co(III) into VNR specifically blocked ligand binding, suggesting that the coordination sphere of metal ion bound to VNR is a critical determinant in integrin-ligand recognition. Furthermore, Mn2+ abolished the oxidative affinity labeling of VNR with Co(III) and consequently blocked the inactivation of VNR by in situ incorporation of Co(III). Thus, Mn2+ and Co2+ bind to the same or mutually exclusive sites on VNR. These observations provide the first demonstration that an integrin, specifically VNR, is a metalloprotein and demonstrate a functional link between the coordination sphere of the bound metal ion and ligand recognition by this receptor.

Affinity Labels↗
PubMed1996

A mass spectrometry-based approach for probing enzyme active sites: identification of Glu 127 in Cellulomonas fimi exoglycanase as the residue modified by N-bromoacetyl cellobiosylamine.

We have identified the residue in Cellulomonas fimi exoglycanase modified by N-bromoacetyl cellobiosylamine as Glu 127 using a new combination of experimental approaches. The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved with pepsin. The N-acetyl cellobiosylamine-modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chromatography connected online to an electrospray ionization mass spectrometer. The modified residue in the labeled peptide was determined by using a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry. Tandem mass spectrometry was used for further structural characterization of the cleaved residue. We show that the residue modified by N-bromoacetyl cellobiosylamine is Glu 127. This residue has been identified previously as the acid-base catalyst by using a combination of mutagenic and kinetic analyses. Our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residues in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current methods used to identify labeled residues within proteins.

Actinomycetales↗
PubMed1994

12-O-tetradecanoylphorbol 13-acetate and fibroblast growth factor increase the 30-kDa substrate binding subunit of type II deiodinase in astrocytes.

Type II 5'-deiodinase (D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) in the brain. The D-II activity in astroglial cell cultures is induced by several pathways including cyclic AMP (cAMP), 12-O-tetradecanoylphorbol 13-acetate (TPA), and fibroblast growth factors (FGFs). We have examined the effect of TPA and FGFs on the 30-kDa substrate binding subunit of D-II, by affinity labeling with N-bromoacetyl-[125I]T4 in astroglial cells. TPA (0.1 microM), 20 ng/ml acidic FGF (aFGF), and 1 mM 8-bromo cyclic AMP all caused an increase in the 30-kDa protein. cAMP induced the greatest increase (fivefold) followed by TPA (3.2-fold) and FGF (2.8-fold). Glucocorticoids acted synergistically with cAMP and aFGF and promoted the effect of TPA. Affinity labeling was competitively inhibited by bromoacetyl-T4 > bromoacetyl-T3 > T4 > reverse T3 > iopanoic acid > T3 > 3,5,3'-triiodothyroacetic acid. The effect of TPA (0.1 microM) was maximum at 8 h and then gradually decreased. aFGF (20 ng/ml) plus heparin (17 micrograms/ml) induced a maximal 30-kDa increase at 8 h, which stayed stable for up to 24 h. The effect of aFGF was concentration dependent. Of the other growth factors studied, only basic FGF and platelet-derived growth factor induced small increases in the 30-kDa protein. Epidermal growth factor had little effect. In vitro labeling of cAMP, TPA, and aFGF-stimulated cell sonicates resulted in an increase in the 30-kDa protein that paralleled the increase in D-II activity.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗