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PubMed2001

The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling.

Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.

Adenosine Triphosphate↗
PubMed1989

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the porcine anterior pituitary.

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr approximately equal to 150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr approximately equal to 44,000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44,000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.

Animals↗
PubMed1996

Identification of the biosynthetic leukotriene C4 export pump in murine mastocytoma cells as a homolog of the multidrug-resistance protein.

A membrane glycoprotein of 190 kDa has been identified previously by photoaffinity labeling as a candidate for the ATP-dependent export pump for leukotriene C4 in mastocytoma cells [Leier, I., Jedlitschky, G., Buchholz, U. & Keppler, D. (1994) Eur. J. Biochem. 220, 599-606]. The present study indicates that this protein represents the murine homolog of the human multidrug resistance protein (MRP). In immunoblot analyses several polyclonal anti-MRP antibodies and one monoclonal antibody recognized the protein of 190-kDa in plasma membranes of mastocytoma cells. Immunoprecipitation after photoaffinity labeling with [3H]leukotriene C4 precipitated the labeled 190-kDa glycoprotein. Deglycosylation by glycopeptide N-glycosidase F of mastocytoma membrane proteins was performed in comparison with membranes from MRP-overexpressing cells and resulted in a reduction of the molecular mass of 190 kDa by about 20 kDa in all membrane preparations. The expression of the murine mrp gene in the mastocytoma cells was analyzed by amplification and sequencing of two mrp cDNA fragments in the first nucleotide binding domain (182 bp) and in a domain proximal to the 3'-end (291 bp). The deduced amino acid sequences of these fragments were identical with murine Mrp and 86.7% and 89.7% identical with the corresponding sequences of human MRP. These results indicate that the ATP-dependent release of leukotriene C4 by murine mastocytoma cells is mediated by murine Mrp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps.

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.

Affinity Labels↗
PubMed1988

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries.

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.

Animals↗
PubMed1987

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes.

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.

Animals↗
PubMed1989

Identification of an intracellular pool of gamma-aminobutyric acidA/benzodiazepine receptors en route to the cell surface of brain neurons in culture.

We have previously shown that approximately 20% of the gamma-aminobutyric acid (GABA)A/benzodiazepine receptors in intact neurons are intracellular [J. Neurosci. 6:2857-2863 (1986); Mol. Pharmacol., 35: 75-84], but the nature of this pool remained unknown. In this report, we describe the synthesis, appearance in the plasma membrane, and degradation of the GABAA/benzodiazepine receptor complex in nerve cells derived from embryonic chick brain and grown in primary monolayer cell culture. Irreversible photoaffinity labeling of the benzodiazepine-sensitive modulator site on the GABAA/benzodiazepine receptor using [3H]flunitrazepam as a permeable probe was used in conjunction with exhaustive trypsinization of intact cells and competition binding using Ro7-0213 (a benzodiazepine bearing a charged quarternary ammonium moiety). Newly synthesized intracellular receptors that are transported to the surface membrane comprise at most only 4% of total receptors and up to one fifth of the steady state intracellular pool. The kinetics of receptor recovery after photoaffinity blockade are consistent with a model in which newly synthesized receptors first appear within the cell and then undergo intracellular transit before appearing on the cell surface. This suggests a "precursor-product" relationship between newly synthesized intracellular receptors and surface receptors. However, the majority of intracellular receptors are not transported to the cell surface and may represent a "nonprecursor" pool. The kinetics of degradation for intracellular receptors are consistent with the overall kinetics for total (surface plus intracellular) receptors. Intracellular and surface receptors are pharmacologically similar with regard to their sensitivity to methyl-beta-carboline-3-carboxylate binding and their affinity for flunitrazepam. Degradation of the individual receptor subunits (with apparent molecular weights of 51,000 and 48,000) was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both photoaffinity-labeled subunits degrade with indistinguishable biphasic kinetics. From these results, and the results of our previous studies, we propose a minimal model describing the dynamic cellular pathway for GABAA/benzodiazepine receptor metabolism in central nervous system neurons.

Animals↗
PubMed1989

A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.

Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.

Affinity Labels↗
PubMed2000

Recent bioorganic studies on rhodopsin and visual transduction.

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.

Amino Acid Sequence↗
PubMed1994

Purification and amino-terminal sequencing of the high affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig liver endoplasmic reticulum.

A high affinity phenylalkylamine Ca2+ antagonist binding polypeptide (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann, H. (1993) Mol. Pharmacol. 43, 139-148) was purified to homogeneity from the endoplasmic reticulum of guinea pig liver with the aid of [3H]emopamil, an antiischemic agent, and [3H]azidopamil, a photoaffinity label. The purified protein retained its high affinity for the antiischemic drugs emopamil (Kd = 4 nM), opipramol (IC50 = 15 nM), trifluoperazine (IC50 = 2 nM), and for Zn2+ (IC50 = 2 microM). Ferguson plots revealed a molecular mass of 27.2 kDa. Partial amino acid sequence information was obtained by Edman degradation and revealed no homology to known protein sequences. Antibodies raised against a synthetic peptide corresponding to the first 25 NH2-terminal amino acid residues specifically immunoprecipitated the [3H]azidopamil photoaffinity-labeled polypeptide and recognized the protein in Western blots. Cross-linking with a variety of homo- and heterobifunctional agents lead to the formation of dimers. Since in the purified preparation no other subunit could be identified with different protein stains, our results indicate that the [3H]emopamil binding site is formed by the homodimer of a novel membrane protein.

Amino Acid Sequence↗
PubMed1987

Prostaglandin-E2-induced activation of adenosine 3'-5' cyclic monophosphate-dependent protein kinases of a murine macrophage-like cell line (P388D1).

Changes in the activities of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinases in response to prostaglandin (PG)E2-induced elevation of intracellular cAMP level were investigated with a murine macrophage-like cell line, P388D1. Photoaffinity labeling with 8-azido-[32P]cAMP showed that untreated P388D1 cells possess two types of cAMP-binding proteins of m.w. 49,000 and 52,000, respectively, in the cytosol fraction in a ration of 1:8. They must represent regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinases, because affinity chromatography on a column of omega-aminohexyl-agarose of the cytosol fraction clearly separated two fractions that exhibited the enzymatic activities and cAMP-binding activities. Photoaffinity labeling of these fractions with 8-azido-[32P]cAMP confirmed the separation of two types of isoenzymes, because each cAMP-dependent protein kinase active fraction was associated with only one type of regulatory subunit. The exposure of P388D1 cells to exogenously added PGE2 (1 microM) caused about 7.5-fold increase in the intracellular cAMP level within 30 sec. The cAMP level then sharply dropped to about 100 pmol/10(7) cells, remained at this level for about 20 min, and then gradually increased to 200 pmol/10(7) (about fivefold over the control). The enzyme assay of the cytosol demonstrated that the activation of cAMP-dependent protein kinases closely follows the kinetics of the intracellular cAMP level. The activation of the enzyme was specific for PGE2 and was not triggered by 1 microM PGF2 alpha or PGD2 which have been shown to be unable to activate adenylate cyclase of P388D1 cells. The PGE2-induced increase in the intracellular cAMP level appeared to activate preferentially the type I isoenzyme, inasmuch as the enzymatic activity of this type separated by the affinity chromatography of the cytosol of PGE2-exposed cells was lower in the presence than in the absence of cAMP, whereas the type II enzyme activity remained responsive to exogenously added cAMP.

Animals↗
PubMed1992

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.

Affinity Labels↗
PubMed1986

Structural homologies in the lutropin/human choriogonadotropin receptor and the follitropin receptor on porcine granulosa cells.

In order to examine the structure of the human choriogonadotropin (hCG) receptor and the follitropin (FSH) receptor on porcine granulosa cells, the hormone receptors were photoaffinity-labeled or affinity-cross-linked. The resulting hormone-receptor complexes were analyzed by alkaline cleavage of cross-links, reduction of disulfides, and peptide maps. The results revealed striking similarities in the structure of the hormone receptors. Both appear to be oligomeric; the hCG receptor has at least four components of 18, 24, 28, and 34 kDa, whereas the FSH receptor shows three distinct components of 18, 22, and 34 kDa. The 24- and the 22-kDa components are the sites for the primary photoaffinity labeling or affinity cross-linking by hCG and FSH, respectively. These components were linked by intercomponent disulfides. Reduction of cross-linked complexes revealed that in the hCG receptor the 24-, the 28-, and the 34-kDa components were disulfide-linked sequentially in a linear form as were the 22-, the 18-, and the 34-kDa components in the FSH receptor. The peptide maps of cross-linked hCG-receptor and FSH-receptor complexes, however, were distinct, indicating that the hCG receptor and the FSH receptor were not identical.

Affinity Labels↗
PubMed1997

Hyper- and hypocardiodynamic states are associated with externalization and internalization, respectively, of alpha-adrenergic receptors in rat heart during sepsis.

Alterations in the distribution of alpha-adrenergic receptors (alpha ARs) in two subcellular organelles, the sarcolemmal membrane and the light vesicle, of rat heart during the progression of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). alpha ARs were assayed by using [3H]prazosin binding and photoaffinity labeling with [125I]arylazidoprazosin in combination with polyacrylamide gel electrophoresis. Septic rat hearts exhibit two distinct phases: an initial hypercardiodynamic (9 h after CLP; early sepsis) followed by a hypocardiodynamic (18 h after CLP; late sepsis) phase. [3H]prazosin binding studies show that during early sepsis, the Bmax (maximal binding capacity) was increased by 21.4% in sarcolemma but was decreased by 22.5% in light vesicles, while during late sepsis, the Bmax was decreased by 25.4% in sarcolemma but was increased by 60.8% in light vesicles. The photoaffinity labeling studies revealed three binding peptides with M(r) of 77, 68, and 39 kDa. The total binding for the three label peptides during early sepsis was increased by 25.5% in sarcolemma but was decreased by 40% in light vesicles, while during late sepsis, the total binding was decreased by 32.1% in sarcolemma but was increased by 35.8% in light vesicles. These data indicate that alpha ARs in the rat heart were externalized from light vesicles to sarcolemma during early hypercardiodynamic phase while they were internalized from surface membranes to intracellular compartment during late hypocardiodynamic phase of sepsis. Because alpha ARs play an important role in regulating myocardial contractility, an initial externalization followed by internalization of alpha ARs may contribute to the development of the initial hypercardiodynamic and the subsequent hypocardiodynamic states during sepsis.

Animals↗
PubMedDate not supplied

A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins.

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase beta in the reaction of primer elongation displaying substrate properties as an analogue of dCTP and dTTP. FAP-dCTP was used for the photoaffinity modification of mammalian DNA polymerase beta. Two approaches to photoaffinity labeling were utilized. In one approach, photoreactive FAP-dCTP was first incorporated into radiolabeled primer-template, and photoreactive DNA was UV-irradiated in the presence of DNA polymerase beta, which resulted in the polymerase labeling by photoreactive primer. In an alternate approach, FAP-dCTP was first UV-cross-linked to the enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked FAP-dCTP was incorporated into the 3'-end of radioactive primer. This "catalytic" modification pathway was shown to be less specific in recognition of FAP-dCTP as an analogue of dCTP than dTTP. FAP-dCTP was used as substrate of endogenous DNA polymerases of HeLa cell extract to synthesize photoreactive DNAs for photoaffinity modification of cell proteins. UV irradiation results in modification of DNA binding proteins of cell extract. The level of photoaffinity labeling of protein targets in the cell extract was strongly dependent on the efficiency of synthesis of photoreactive DNA.

Base Sequence↗
PubMed1982

Internalization and molecular processing of insulin receptors in isolated rat adipocytes.

The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with 125I-labeled NAPA-DP-insulin were identified by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands of Mr 330,000, 295,000, and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16 degrees C were immediately trypsinized, all of the receptor bands were degraded into lower molecular weight fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 37 degrees C for 1 hr prior to trypsin exposure, approximately equal to 30% of the receptors were found to be trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37 degrees C (but not at 16 degrees C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16 degrees C and during the subsequent incubation at 37 degrees C showed that this agent (i) increased the trypsin-insensitive (intracellular) receptor pool, (ii) blocked conversion of the Mr 125,000 subunit into the Mr 115,000 component, and (iii) prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulin-receptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).

Adipose Tissue↗
PubMed1996

Initial externalization followed by internalization of beta-adrenergic receptors in rat heart during sepsis.

Changes in the distribution of beta-adrenergic receptors in two subcellular fractions, the sarcolemma and the light vesicle, of rat heart during sepsis were studied, using [3H]dihydroalprenolol ([3H]DHA) binding and photoaffinity labeling with [125I]iodocyanopindolol ([125I]ICYP). Sepsis was induced by cecal ligation and puncture (CLP). Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP; early sepsis) and a subsequent hypocardiodynamic (18 h after CLP; late sepsis) state. [3H]DHA-binding studies show that, during early sepsis, the maximum binding capacity (Bmax) was increased by 35% in sarcolemma but was decreased by 25% in light vesicles, whereas during late sepsis, the Bmax was decreased by 39% in sarcolemma but was increased by 30% in light vesicles. Photoaffinity labeling studies show that the incorporation of [125I]ICYP into 64,000-Da peptide during early sepsis was increased by 32% in sarcolemma but was decreased by 27% in light vesicles, whereas during late sepsis, the incorporation was decreased by 30% in sarcolemma but was increased by 35% in light vesicles. These data indicate that beta-adrenergic receptors in the rat heart were externalized from light vesicles to sarcolemma during the hyperdynamic phase but were internalized from surface membranes to intracellular sites during the hypodynamic phase of sepsis. Because beta-adrenergic receptors mediate adrenergic control of cardiac muscle contraction, a biphasic intracellular redistribution of beta-adrenergic receptors in the heart may contribute to the development of the initial hypercardiodynamic and subsequent hypocardiodynamic states during sepsis.

Animals↗
PubMed1983

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.

Preincubation of turkey erythrocytes with catecholamines desensitizes the beta-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the beta-adrenergic receptors with 125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J.M., Nambi, P., Lavin, T.N., Heald, S.L., Caron, M.G. & Lefkowitz, R.J. (1982) J. Biol. Chem. 257, 9242-9245]. The time course of alteration in beta-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are first purified and then photoaffinity labeled with 125I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3',5'-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the beta-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [32P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/polyacrylamide gel electrophoresis. Desensitization is associated with a 2- to 3-fold increase in 32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the beta-adrenergic antagonist propranolol. Purified turkey erythrocyte beta-adrenergic receptors could be phosphorylated by incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the beta-adrenergic receptor and is associated with agonist-promoted phosphorylation of beta-receptor peptides.

8-Bromo Cyclic Adenosine Monophosphate↗