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PubMed1989

Synthesis, in vitro acetylcholine-storage-blocking activities, and biological properties of derivatives and analogues of trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol).

Eighty-four analogues and derivatives of the acetylcholine-storage-blocking drug trans-2-(4-phenylpiperidino)-cyclohexanol (vesamicol) were synthesized, and their potencies were evaluated with the acetylcholine active-transport assay utilizing purified synaptic vesicles from Torpedo electric organ. The parent drug exhibits enantioselectivity, with (-)-vesamicol being 25-fold more potent than (+)-vesamicol. The atomic structure and absolute configuration of (+)-vesamicol were determined by X-ray crystallography. The absolute configuration of (-)-vesamicol is 1R,2R. Structure-activity evidence indicates that (-)-vesamicol does not act as an acetylcholine analogue. Alterations to all three rings can have large effects on potency. Unexpectedly, analogues locking the alcohol and ammonium groups trans-diequatorial or trans-diaxial both exhibit good potency. A potent benzovesamicol family has been discovered that is suitable for facile elaboration of the sort useful in affinity labeling and affinity chromatography applications. A good correlation was found between potencies as assessed by the acetylcholine transport assay and LD50 values in mouse.

Acetylcholine↗
PubMed1993

Tissue-related and species-specific differences in the 2-5A oligomer size requirement for activation of 2-5A-dependent RNase.

2',5'-Oligoadenylate (2-5A)-dependent RNase (L or F) is the final enzyme in the 2-5A pathway and a key component in the molecular mechanism of interferon (IFN) action. Here we demonstrate differences in the 2-5A oligomer size requirement between rabbit 2-5A-dependent RNase from reticulocytes and from cultured kidney cells. The rabbit reticulocyte enzyme was activated by tetramer 2-5A, whereas the ribonuclease from rabbit kidney cells required only trimer 2-5A. Interestingly, in contrast to the 2-5A-dependent RNase from rabbit reticulocytes, that from murine reticulocytes could be activated by trimer 2-5A. Partial proteolysis of affinity-labeled, 80-kD 2-5A-dependent RNase from rabbit reticulocytes and rabbit kidney cells resulted in the same pattern of labeled peptides. However, the affinity labeling reaction with a 32P-labeled 2-5A analog did produce some different labeled polypeptides in rabbit kidney cell extract and rabbit reticulocyte lysate. These results could indicate specialized functions for the 2-5A system in different organ systems.

Adenine Nucleotides↗
PubMed1990

Mapping the active site of yeast RNA polymerase B (II).

Yeast RNA polymerase B (II) was incubated with a collection of 13 different nucleotide derivatives and affinity labeled by allowing DNA-directed phosphodiester bond formation. The 32P-labeled site was localized in the C-terminal part of the B150 subunit by microsequencing a proteolytic fragment, then further mapped by a combination of extensive or single-hit chemical cleavage reactions and analysis of the labeled peptide patterns. The affinity label was mapped to between Asn946 and Met999, within one of the nine regions that are conserved between B150 and the bacterial beta subunit. The results underscore the conservative evolution of the catalytic center of eukaryotic and bacterial RNA polymerases.

Amino Acid Sequence↗
PubMed1989

p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue.

The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.

Adipose Tissue↗
PubMed1994

Fatty acid ethyl ester-synthesizing activity of lipoprotein lipase from rat postheparin plasma.

Lipoprotein lipase (LPL) was obtained from rat postheparin plasma by chromatographies on heparin-Sepharose and hydroxyapatite. The enzyme was associated with fatty acid ethyl ester synthase (FAEE synthase) as judged by their co-elution profiles and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular weight 57,000 in purified LPL fraction was labeled by affinity labeling with [3H]-diisopropyl fluorophosphate. The FAEE synthase activity of LPL was not affected by addition of apolipoprotein C-II. Digestion of the enzyme with trypsin resulted in almost complete loss of the triolein-hydrolyzing activity without change in FAEE synthase activity. The tributyrin-hydrolyzing activity of LPL was also not affected by addition of apolipoprotein C-II or trypsin digestion. On addition at progressively higher concentrations, bovine serum albumin increased FAEE synthesis to a maximum at 2 mg/ml and at higher concentrations inhibited its activity. On incubation of purified LPL with chylomicrons in an ethanol/water mixture, FAEE was formed in the presence of a high concentration of bovine serum albumin. The specific activity of FAEE synthesis from chylomicrons was about 65 times that from oleic acid. Triolein/gum arabic emulsion was used for identification of reaction products. We propose the following mechanism of FAEE formation from chylomicrons by LPL. The enzyme attacks chylomicrons forming an acyl-enzyme intermediate, and during the deacylation process, ethanol binds to fatty acids as an acceptor. These results suggest that LPL contributes to nonoxidative ethanol metabolism (FAEE formation) through degradation of triglyceride-rich lipoproteins such as chylomicrons.

Acyltransferases↗
PubMed1985

Asparagine-linked oligosaccharides on formyl peptide chemotactic receptors of human phagocytic cells.

Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.

Acetylglucosaminidase↗
PubMed1978

Insulin and epidermal growth factor-urogastrone: affinity crosslinking to specific binding sites in rat liver membranes.

Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with (125)I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with (125)I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.

Affinity Labels↗
PubMed2003

Superselective labelling of proteins: approaches and techniques.

Affinity labelling is a popular method used for the study of macromolecules and their interactions with ligands. The method is based on the targeted delivery of a chemically cross-linkable group, attached to a reactive molecule with affinity for a particular site in the biopolymer of interest. In complex multicomponent systems, the applications of affinity labelling are restricted by the tendency of the reagents to randomly label nontargetted molecules. This review highlights techniques developed to minimize non-specific cross-linking and to achieve high selectivity for the labelling of target protein. Such techniques might be termed 'superselective labelling', as opposed to traditional, less selective approaches.

Animals↗
PubMed1992

Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines.

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.

Blotting, Northern↗
PubMed1987

Biochemistry of protein-isocyanate interactions: a comparison of the effects of aryl vs. alkyl isocyanates.

In addition to their use in the polyurethane and pesticide industries, isocyanates have proven to be useful probes for the exploration of protein structure. This paper focuses on three aspects of isocyanates: their broad reactivity, their reversible interaction with cholinesterases, and the relative hydrolysis rates of alkyl and aryl isocyanates. The broad reactivity of isocyanates as well as the demonstrated affinity labeling of serine and sulfhydryl esterases are discussed. Extension of the affinity labeling studies to include the analysis of the inhibition of cholinesterases by methyl isocyanate shows that methyl isocyanate is not an effective inhibitor of any of the cholinesterases. The inhibition of cholinesterases by alkyl isocyanates shows a pattern of decreased specificity with decreased alkyl chain length. The inhibition of cholinesterases by isocyanates is shown to be reversible, with a maximum rate of reversal seen at physiological pH. This reversal is characteristic of the reaction of an isocyanate with a sulfhydryl group. Finally, the affinity labeling of proteins must compete successfully with the hydrolysis of isocyanates in aqueous solution. The hydrolysis of alkyl isocyanates is shown to be significantly slower than that of the aryl isocyanates.

Affinity Labels↗
PubMed1989

Chemical characterization of ligand binding site fragments from turkey beta-adrenergic receptor.

Affinity-labeled beta-adrenergic receptor from turkey erythrocyte membranes was specifically cleaved near cysteine residues after S-cyanylation. Analysis of the labeled polypeptide fragments suggests that iodocyanopindolol diazirine reacted with an amino acid residue which is located in the non-glycosylated region containing the sixth and seventh transmembrane domains of the receptor. However, the possibility cannot be excluded that a second residue, located between the third and fifth transmembrane domains, was also labeled. Since treatment with either hydroxylamine or triethylamine resulted in removal of the affinity label from the protein, the present study suggests that aspartic or glutamic acid residues are present in the adrenergic-binding site which is located in the above-mentioned domains. The procedure for specific chemical cleavage of the affinity-labeled adrenergic receptor should also be useful for future structural and comparative studies of other adrenergic receptors.

Affinity Labels↗
PubMed1994

Plasmin cleaves betaglycan and releases a 60 kDa transforming growth factor-beta complex from the cell surface.

Plasmin regulates the activity and distribution of transforming growth factor beta (TGF-beta) and other growth factors. The purpose of the present investigation was to determine the effects of plasmin on cellular receptors for TGF-beta. AKR-2B fibroblasts were affinity-labelled with 125I-TGF-beta 1 and 125I-TGF-beta 2, demonstrating betaglycan, the type-I TGF-beta receptor and the type-II TGF-beta receptor. Treatment of TGF-beta-affinity-labelled cells with plasmin (10-100 nM) for 1 h profoundly and selectively decreased recovery of TGF-beta-betaglycan complex. The type-I and type-II receptors were not plasmin substrates. A radiolabelled complex with an apparent mass of 60 kDa was detected by SDS/PAGE in both the medium and cell extracts of plasmin-treated affinity-labelled cells. In order to demonstrate that plasmin cleavage of betaglycan did not require prior exposure of the betaglycan to cross-linking agent, AKR-2B cells were treated with plasmin first and then affinity-labelled. Markedly decreased TGF-beta binding to cellular betaglycan was observed. Although plasmin treatment of AKR-2B cells decreased overall binding of 125I-TGF-beta 1 and 125I-TGF-beta 2, the rate at which the cells degraded bound 125I-TGF-beta at 37 degrees C was not changed. AKR-2B cells treated with plasmin demonstrated slightly increased [3H]thymidine incorporation; the plasmin-treated cells retained their ability to respond to TGF-beta. Conditioned medium from plasmin-treated AKR-2B cells contained increased amounts of active TGF-beta as determined in Mv 1 Lu epithelial-cell-proliferation assays. Specific cleavage of betaglycan represents a novel mechanism whereby plasmin may regulate the assortment of receptors available for TGF-beta. In addition, plasmin may facilitate transfer of active TGF-beta between neighbouring cells by releasing the active growth factor from the cell surface.

Affinity Labels↗
PubMed1983

Chemical modification of the nicotinic cholinergic receptor of PC-12 nerve cell.

The identity of the protein that mediates the nicotinic acetylcholine sensitivity in neuronal cells has been investigated by chemical modification and affinity labeling. When an ion flux assay is used, it is possible to measure specifically the activity of the ionophore associated with the nicotinic acetylcholine receptor in cultured nerve cells (PC-12 pheochromocytoma). This activity is modulated by modification of the redox state of at least one disulfide bridge located at the vicinity of the agonist binding site. The oxidizing agent 5,5'-dithiobis(nitrobenzoic acid) restores the complete receptor response which had been inhibited by reduction with dithiothreitol. N-Ethylmaleimide and the nicotinic affinity labels [4-(N-maleimido)benzyl]-alpha-trimethylammonium iodide and bromoacetylcholine react also with the reduced receptor and irreversibly block the agonist-dependent response of the ionophore. The two affinity labels show strong affinities for the receptor, and apparent IC50 values of 20 and 560 nM can be respectively evaluated. Bromoacetylcholine, being an acetylcholine analogue, blocks the receptor function by desensitization, a process in which the constant interaction with the activator causes a shift into an inactive form of the receptor. Bromoacetylcholine can also be shown to activate untreated as well as reduced cells. In this case, the bound label induces a lasting response which is terminated by the irreversible desensitization of the modified receptor. These experiments thus show that the PC-12 nicotinic ionophore shares functional and structural similarities with peripheral receptors. They suggest that nicotinic affinity labels developed for the muscle receptor can also be used as specific markers of the nicotinic neural ionophore.

Acetylcholine↗
PubMed2006

Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha.

A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile covalent attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The covalent attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the covalent attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).

Affinity Labels↗
PubMed1985

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

Dexamethasone 21-methanesulfonate, an affinity label for glucocorticoid-binding proteins, was incubated with rat liver cytosol preparations. The predominant covalently labeled component was identified as Yb-glutathione-S-transferase on the basis of chromatographic properties, electrophoretic mobility, and specific retention by an anti-Yb-immunoadsorbent. Affinity labeling of this protein was blocked by excess dexamethasone. Preferential reactivity of dexamethasone 21-methanesulfonate with the Yb subclass of glutathione-S-transferase (glutathione transferase, EC 2.5.1.18) was also evident with mixtures containing the multiple forms of the enzyme. Yb-glutathione-S-transferase, the nonsaturable glucocorticoid-binding component of rat liver cytosol should, therefore, be reclassified; because of its high concentration and selective interaction with steroids, this enzyme may be an intracellular glucocorticoid-binding protein and, thereby, influence transport, metabolism, and action of the steroids.

Affinity Labels↗
PubMed1986

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate.

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1988

Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking.

We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

Affinity Labels↗
PubMed1989

Expression of hepatic transforming growth factor receptors during late gestation in the fetal rat.

Transforming growth factor-alpha (TGF alpha) promotes DNA synthesis in adult rat hepatocytes, an effect opposed by picomolar concentrations of TGF beta. Recently, the presence of these growth factors in fetal rat liver has been demonstrated. Since a regulatory role for TGF alpha and TGF beta in fetal hepatic growth requires the presence of high affinity receptors, the receptors for these hormones were studied in membranes from normal fetuses at 17-21 days gestational age and growth-retarded fetuses of mothers fasted for 48 h. Fetal liver membranes bound [125I]epidermal growth factor ([125I]EGF) with high affinity (Kd = 1-2 nM). TGF alpha could compete with EGF for the same binding site, albeit at 4-fold lower affinity. EGF receptor number increased from nearly undetectable levels at 17 days to adult levels (0.15-0.3 nmol/mg membrane protein) by 21 days. Affinity labeling of fetal liver membranes with [125I]TGF alpha identified the 170,000 mol wt (Mr) EGF receptor. The intensity of labeling correlated with EGF receptor number based on binding analyses. TGF beta bound to fetal liver membranes with high affinity (Kd = 30 pM) and at a level (20-30 pmol/mg throughout late gestation) that was 3-fold higher than in adult liver. Affinity labeling of fetal hepatic membranes with [125I]TGF demonstrated high affinity 85,000 Mr TGF beta receptors and lower affinity 66,000 and 130,000 Mr receptors. Although TGF beta binding did not change with advancing gestation, affinity labeling of the 85,000 Mr protein doubled from day 18 to day 21 and was decreased by 50% in fetuses from fasted mothers. These data, demonstrating the presence and regulation of the receptors for TGF alpha and TGF beta, support roles for these hormones in the regulation of fetal hepatic growth.

Affinity Labels↗