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PubMed1989

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells.

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.

Affinity Labels↗
PubMed1991

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.

Animals↗
PubMed1991

Localization of type I iodothyronine 5'-deiodinase to the basolateral plasma membrane in renal cortical epithelial cells.

Type I iodothyronine 5'-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5'-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5'-deiodinase in rat kidney (Köhrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane.

Affinity Labels↗
PubMed1990

Differential interactions of human choriogonadotropin and its antagonistic aglycosylated analog with their receptor.

Human choriogonadotropin (hCG) is a heterodimeric hormone consisting of an alpha subunit and a beta subunit. hCG and aglycosylated hCG (aghCG) have similar receptor binding affinities but differ in their ability to activate hormone-responsive adenylate cyclase. aghCG is an effective antagonist. The mechanisms of this antagonism and interactions of antagonistic aghCG with the receptor are not understood. To address this critical question, we have examined the interaction of this hormone analog with the receptor. The hormone receptor on porcine granulosa cells is a glycoprotein of 86 kDa and thas three domains of 24 kDa, 28 kDa, and 34 kDa, which are disulfide-linked. They undergo proteolysis, particularly when bound to the hormones, to produce three polypeptide components. These three receptor components can readily be identified through the use of affinity labeling with the hormones. Affinity labeling with an amino-specific homobifunctional reagent and subsequent cleavage indicate that hCG is cross-linked directly to the 24-kDa receptor component. In contrast, aghCG is cross-linked directly to the 34-kDa component. The peptide map of the cross-linked aghCG-34-kDa receptor component produced by papain treatment is different from the peptide map of the cross-linked complex of hCG-24-kDa component. This difference in receptor binding may be a factor determining the success or failure of signal transduction from the receptor to the effector system, guanine nucleotide-binding regulatory protein, and adenylate cyclase.

Affinity Labels↗
PubMed1968

Affinity site labeling of a mouse myeloma protein which binds dinitrophenyl ligands.

A mouse myeloma protein of the immunoglobulin A class, which specifically binds dinitrophenyl ligands, has been successfully affinity labeled (site-directed labeling) with several diazonium reagents, leading to inactivation of the combining sites. The labeling reagents reacted only with tyrosine residues of light chain origin. The 7S subunit of the myeloma protein appears to contain only one reactive site.

Animals↗
PubMed2002

Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry.

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

Affinity Labels↗
PubMed1984

Size discrepancy between somatomedin-C and insulin receptors.

Somatomedin-C (Sm-C) and insulin receptors have similar size and structure. Each receptor is a heterotetramer composed two alpha- and two beta-subunits. Sodium dodecyl sulfate (SDS)-polyacrylamide gel autoradiographic studies of affinity labeled receptor preparations demonstrated that each has a whole receptor of greater than 350,000 daltons, a half-receptor of 220,000 daltons, and binding subunit (alpha-subunit) of about 140,000 daltons. Using SDS-polyacrylamide disc gels and double labeling techniques, we demonstrated that the 125I affinity labeled alpha-subunit of the Sm-C receptor from human placenta was 8,000 daltons smaller than the 131I affinity labeled insulin receptor alpha-subunit. Further double label studies demonstrated that [125I]insulin and [131I]insulin cross-linked to placental insulin receptors precisely comigrated on SDS-disc gels, indicating that the difference between insulin and Sm-C alpha-subunits is not an artifact of the system. The difference in ligand size (Sm-C, 7,500 daltons; insulin, 5,700 daltons) would minimize the observed difference and is clearly not the cause of the difference in size observed. These studies provide further evidence in support of two functionally and physically distinct receptor molecules for insulin and Sm-C in human placenta.

Affinity Labels↗
PubMed1992

Alteration of folate analogue transport inward after induced maturation of HL-60 leukemia cells. Molecular properties of the transporter in an overproducing variant and evidence for down-regulation of its synthesis in maturating cells.

Studies are described examining further the decline in folate analogue influx mediated by the one-carbon reduced-folate transport system in HL-60 cells following induction of maturation by cytodifferentiation agents. To facilitate the investigation of the underlying basis of this phenomenon, we derived a variant (HL-60/LCV) with 4-5-fold elevated influx capacity (Vmax) for folate analogues. A commensurate increase in the putative transporter for this system was documented by affinity labeling of these cells with N-hydroxysuccinimide-[3H]aminopterin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity labeled plasma membrane in HL-60/LCV cells delineated a protein peak at Mr = 75,000-80,000. This was substantially greater than the analogous transporter (Mr = 45,000-47,000) we had delineated (Yang, C.-H., Sirotnak, F.M., and Mines, L.S. (1988) J. Biol. Chem. 263, 9703-9709) with the same methodology in the L1210 cell plasma membrane. In addition, the rate of translocation of the Mr = 75,000-80,000 transporter in HL-60 and HL-60/LCV cells was 2-fold lower than the rate of translocation determined for the Mr = 45,000-47,000 transporter in L1210 cells. During induced maturation of HL-60/LCV cells toward the granulocyte pathway, [3H]methotrexate (MTX) influx capacity and the amount of the affinity labeled transporter decreased rapidly in a parallel fashion. The decrease in [3H]MTX influx and in affinity labeling and in the amount of the Mr = 75,000-80,000 transporter was 5-fold following exposure to 210 mM dimethyl sulfoxide (Me2SO) for 5 days during growth in culture. Moreover, during cycloheximide treatment, the decay in [3H]MTX influx at 37 degrees C and in amount of affinity labeled transporter was the same (t1/2 = 144-155 min) for both control and Me2SO-treated HL-60/LCV cells. These results, which reveal no difference in metabolic turnover for control and Me2SO-treated cells, suggest that the decline in folate analogue influx in HL-60/LCV influx cells is a very early event in the program of differentiation and probably occurs by down-regulation of synthesis of the transporter for the one-carbon reduced-folate transport system.

Affinity Labels↗
PubMed1988

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors.

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.

Adenylyl Cyclases↗
PubMed1987

Purification of the 33,000-dalton ligand binding-protein constituent of the lymphoblast substance P receptor.

Substance P (SP) acts as an immunoregulator by binding to specific functional cell surface receptors on a subpopulation of human T-helper lymphocytes. Receptors with similar properties have also been characterized on the human IM-9 B-lymphoblast cell line. Four distinct proteins of molecular weight (MW) 33,000, 58,000, 78,000, and 116,000 can be specifically affinity labeled using [125I]-SP Bolton-Hunter reagent and disuccinymidyl suberate (DSS). Peptide-mapping studies of these individually purified affinity labeled proteins have shown that the 33,000 MW membrane protein is present in the higher molecular weight cross-linked proteins. In the present studies, the 33,000 MW affinity-labeled protein was purified using semipreparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by high-performance hydroxylapatite chromatography. Starting with 800 mg of affinity-labeled protein, the final yield was 39.0 micrograms of 33,000 MW affinity-labeled protein. Based on an estimate of 53 mg receptor per 800 mg membrane protein, this represents an overall yield of greater than 70%. Peptide mapping was done by digesting 20 micrograms of the receptor protein with bovine trypsin. The proteolytic fragments were separated by reverse-phase high-performance liquid chromatography, and the amino acid content of 13 distinct peptides was determined. With this procedure, sufficient receptor can now be purified so that partial amino acid sequences can be obtained for further structural studies.

Amino Acids↗
PubMed1987

Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex.

Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with 125I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azidobenzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (Mr) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate [Gpp-(NH)p]. Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of Mr 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omission of protease inhibitors during the isolation of plasma membranes resulted in the loss of the Mr 85,000 PTH binding species and the appearance of an Mr 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major Mr 85,000 PTH binding species. We conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an Mr 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an Mr 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.

Animals↗
PubMed1982

Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.

A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0--6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). N alpha-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was [125I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.

Affinity Labels↗
PubMed1978

Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein.

Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.

Affinity Labels↗
PubMed1975

Synthesis of a chemically reactive analog of the initiation codon: its reaction with ribosomes of Escherichia coli.

Nitrophenylated 5'-adenylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated pA-U-G. After reduction and subsequent bromoacetylation, an A-U-G analog was obtained, which could be used as an affinity label for the ribosomal A-U-G-binding site(s). After incubating the A-U-G affinity label with 70S ribosomes, 30S subunits programmed for initiation-factor-dependent fMet-tRNAMetf binding were obtained. Hence, the A-U-G analog had irreversibly reacted at the ribosomal decoding site. Initiation complexes which were formed with the labeled 30S subunits were puromycin-resistant. Furthermore, GTP hydrolysis, necessary for proper accommodation of initiator tRNA at the ribosomal donorsite, did not function in these complexes. These data indicate that immobilization of A-U-G at the decoding site of the ribosome allows factor-dependent initiator tRNA binding, but impairs accommodation at the donor site. The ribosomal protein(s) to which A-U-G was covalently bound at the decoding site were identified by polyacrylamide gel electrophoresis in the presence of urea or sarkosyl. The predominant affinity-labeled protein was found to be protein S18. Variation of the incubation conditions of the affinity-labeling reaction leads to attachment of A-U-G label to another ribosomal protein, S4, the ram gene product.

Adenine Nucleotides↗
PubMed1988

A novel approach to the ultrastructural localization of cell surface receptors: affinity-gold labeling of Na, K-ATPase.

We describe here a novel method of affinity-gold labeling for the ultrastructural localization and biochemical characterization of functional cell surface receptors. This approach combines the widely used colloidal gold technique, a previously published method for coating the gold with a matrix of derivatized dextran, small receptor-specific ligands, and a photoactivatable cross-linker. The resulting gold-affinity probe directed to a selected receptor by the ligand, is subsequently attached to the receptor by light activation of the cross-linker. As a specific example, a gold affinity probe prepared with ouabain, a selective inhibitor of Na,K-ATPase, as the directing ligand was used to investigate the ultrastructural localization of this enzyme complex in cell membranes. The biological activity of ouabain covalently linked to derivatized dextran containing the photoactivatable cross-linker was examined by its action on ion transport across dog trachea epithelium and on the enzyme activity of Na,K-ATPase preparations obtained from the rectal gland of the elasmobranch, Mustelus californicus. By these tests the probe mimics the effects of free ouabain. Electron micrographs of labeled human erythrocytes and cultured human foreskin fibroblasts showed an apparent random distribution of Na,K-ATPase on the plasma membranes of these cells. Binding of the probe was blocked in the presence of excess ouabain, a result demonstrating that the affinity probe binds at the same sites as free ouabain. Covalent attachment of probe by light activation of the nitroarylazido groups greatly enhanced retention during washing and standard procedures of fixation and dehydration. The high density of the gold probe was utilized to isolate the covalently attached membrane components from labeled human foreskin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1992

Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol.

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.

Affinity Labels↗
PubMed1975

Identification of the 30 S protein adjacent to peptidyl transferase catalytic center of Escherichia coli ribosomes.

Iodoacetylphenylalanyl-tRNAPhe was used as an affinity label to localize the ribosomal components involved in the peptidyl transferase catalytic center of Escherichia coli ribosomes. When labeling was carried out at pH 5.0, the affinity label could specifically label the ribosomal components which comprise the catalytic center. Analysis of ribosomal proteins which had reacted with the affinity label revealed that a 30 S subunit protein, S 20, was located at or near to the ribosomal binding site of the 3'-terminus of aminoacyl- or peptidyl-tRNA.

Acyltransferases↗
PubMed1995

A novel endoprotease responsible for the specific cleavage of transducin gamma subunit.

Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.

Amino Acid Sequence↗