Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Photoaffinity Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,711 records · Page 95Linked to original sources
PubMed1997

Human K562 transfectants expressing high levels of reduced folate carrier but exhibiting low transport activity.

A human reduced folate carrier (hRFC) cDNA was transfected into transport-deficient K562 cells to circumvent complications that may result from carrier expression in a heterologous mammalian species. Relative to wild-type cells, hRFC transcript levels were increased 11- and 19-fold, respectively, in the K43-6 and K43-1 transfectants. Although photoaffinity labeling of hRFC protein revealed similar increases of 15- and 19-fold, respectively, only a 2-fold enhancement in methotrexate (Mtx) transport was observed. This suggests that only a small portion of the cDNA-encoded hRFC protein is actively engaged in membrane transport. Kinetic analysis of [3H]Mtx transport indicated that K43-6 cells exhibited a similar affinity (Kt) but an increased Vmax (1.7-fold) when compared with K562 cells. The restored transport was similar to that of wild-type cells in its capacity to be trans-stimulated by intracellular folates and in its sensitivity to competitive transport inhibitors (1843U89, bromosulfophthalein, folic acid, leucovorin, and ZD1694) and to irreversible inhibition by N-hydroxysuccinimide-methotrexate. Further, deglycosylated photoaffinity-labeled hRFC protein in both K562 and K43-6 cells migrated at approximately 65-70 kDa on SDS-gels, consistent with the molecular mass from the predicted amino acid sequence. These data further establish that the expression of hRFC, alone, is sufficient to confer transport properties typical of the "classical" hRFC. However, the discrepancy between the stoichiometry of carrier expression and transport activity implies that membrane translocation of bound substrate may be regulated by additional undefined mechanisms.

Biological Transport↗
PubMed1975

Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation.

Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex. It catalyzes ATP-Pi exchange and ATP hydrolysis. The exchange reaction is sensitive to uncouplers, rutamycin, valinomycin plus K-+, dicyclorexylcarboditmide, arsenate, azide, and adenylyl imidodiphosphate. It is also specific for ATP; ITP, GTP and UTP are essentially ineffective. Studies with the photoaffinity labeling uncoupler, 2-azido-4-nitrophenol (NPA), have shown that the mitochondrial uncoupler-binding sites are located exclusively in complex V. Complexes I, III and IV, which carry the three coupling sites of the respiratory chain, had negligible capacity for the binding of NPA, whereas the uncoupler-binding capacity of complex V appeared to be increased two- to threefold as compared to mitochondria. Complexes I, II, III, IV and V are obtained from the same batch of mitochondria by a simple fractionation procedure, which employs cholate, deoxycholate, ammonium acetate and ammonium sulfate. Studies with NPA have shown that mitochondria contain per milligram protein about 0.6 nmole of uniformly reacting uncoupler binding site. All of the uncouplers tested appeared to interact competitively with this site. Photoaffinity labeling with tritiated NPA has shown that a major portion of NPA binds to a polypeptide of molecular weight between 26,000 and 30,000. Other studies on the mechanism of uncoupling have shown that picrate is a membrane-impermeable uncoupler. It cannot uncouple mitochondria. However, it is an effective uncoupler of ATP synthesis and ATP-induced transhydrogenation or reverse electron transfer when used in conjunction with sonicated submitochondrial particles, which have an inside-out orientation of the inner membrane with respect to the medium. In these particles, picrate binds to the same uncoupler-binding site as NPA and other uncouplers. However, unlike the membrane-permeable uncouplers, picrate is a poor protonophore. It has a very small effect on the proton permeability of phosphorylating submitochondrial vesicles, even at two to three times the concentration needed for complete uncoupling. The increase in the proton permeability of submitochondrial vesicles caused by such high concentrations of picrate (500 mum) can be achieved with approximately 5 mum 2,4-dinitrophenol. At this concentration, dinitrophenol results in only about 20% uncoupling.

Adenosine Triphosphate↗
PubMed1981

Somatomedin receptor of human placenta: solubilization, photolabeling, partial purification, and comparison with insulin receptor.

Using a recently isolated human basic somatomedin (basic SM) similar to insulin-like growth factor I (IGF-I), we studied both the photoaffinity-labeled and unlabeled basic-SM receptor solubilized from human placental cell membranes. Unlike the result with the insulin receptor, high yields of soluble basic-SM-binding activity are obtained with Triton X-100. The soluble basic-SM receptor retains high-affinity (Kd approximately 0.3 nM) peptide-specific binding of basic SM, similar to the binding present in particulate placenta membranes; the receptor exhibits a comparatively low affinity for insulin (Kd approximately 3 microM). On Sepharose 6B, like the crude soluble insulin receptor, the basic-SM receptor migrates as a species with an apparent Stokes radius of 7.2 nm; unlike the insulin receptor, the basic-SM receptor does not, under similar conditions, yield a smaller binding species (apparent Stokes radius 3.8 nm). Upon photoaffinity labeling with 125I-labeled basic SM, one principal specifically labeled constituent is detected. Upon gel electrophoresis in the presence of 2-mercaptoethanol, the photolabeled constituent, like the insulin receptor, migrates as a species with an apparent molecular weight of about 140,000; in the absence of reducing agent, a molecular weight greater than 240,000 is observed. Lectin-agarose affinity chromatography yields a 30-fold purification both of the basic-SM-binding activity and the photolabeled constituent. Anti-insulin receptor antibody does not appear to precipitate the basic-SM receptor. We conclude that the basic-SM receptor of human placenta is a glycoprotein, remarkably similar to (an isoreceptor) but distinct from the insulin receptor previously characterized in this tissue.

Female↗
PubMed1988

Identification of 5-hydroxytryptamine1A receptor proteins in bovine frontal cortex.

5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.

Affinity Labels↗
PubMed1983

Cyclic AMP-dependent protein kinases in semen from men with subnormal sperm motility or morphology.

Photoaffinity labelling by 8-N3-/32P/cAMP of human sperm homogenates with normal pathology and normal progressive motility of the gametes (N) revealed the presence of 2 specific cAMP-binding activities with MW 47 000 (R-I) and 52 000 (R-II). Similar amounts of R-I and R-II were found in sperm homogenates obtained from semen samples with either reduced progressive motility (A50: 30-50% progressive motility and A30: less than 30% progressive motility) or abnormal morphology of the gametes (T: 5-30% normal heads). In contrast, the seminal plasma of the respective semen samples of N, A50, A30 and T incorporated the 8-azido cAMP photolabel in similar quantities into 2 additional cAMP-binding proteins with MW 42 000 and 37 000. Quantitative analysis of the photoaffinity labelling of the seminal plasma obtained from normal (N) or subnormal semen (A50, A30, T) showed that cAMP was predominantly bound to the 37 000 protein. Furthermore, the cAMP-dependent protein kinase and cAMP-binding activities of sperm homogenates were quantitatively comparable within the 4 groups. These results provide the first evidence that neither quantitative nor qualitative differences in the molecular and/or enzymatic properties of cAMP-dependent protein kinases can be detected between normal human sperm (N) and those with either reduced progressive motility (A50, A30) or abnormal morphology (T).

Adolescent↗
PubMed2000

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin.

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.

Antineoplastic Agents↗
PubMed1998

[Estimation of the binding site of drugs by means of new types of photoactive ligands].

Photoaffinity labeling is a powerful technique to identify ligand-binding proteins in a crude mixture and localize their binding-site. Benzophenone photophore has several favorable features compared with the classical photoreactive unit, aromatic azides, that is why its application is expanding. Benzophenone can easily be attached to biologically active ligands by using a tritiated heterobifunctional crosslinker reagent: [3H]-BZDC-NHS or a photoreactive amino acid; [3H]-4-benzoyl-L-phenylalanine. Tethered inositol polyphosphates and antimitotic agents were prepared first and reacted with the high specific activity photocrosslinker reagent in microscale. The photoactivatable ligands obtained were used for studying IP3 receptor, alpha-Trinozitol receptor, PLC delta enzime, beta-tubullin and glycoprotein P. Thrombin receptor was investigated by a short peptide antagonist containing the photoactivatable amino acid. The results demonstrate the versatility of photoaffinity labeling and prove that this technique has a potential providing much more information about the receptor and mechanism than simply identifying a single polypeptide: it contributed to solve the 3D structure of the IP3 binding domain and to reveal a unique activation of the PLC delta. The results confirmed the superiority of benzophenone. Using that photophore glycoprotein P, which is responsible for the MDR, and thrombin receptor were labelled at the first time.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Multiple distinct subunits of the gamma-aminobutyric acid-A receptor protein show different ligand-binding affinities.

The purified gamma-aminobutyric acid/benzodiazepine receptor protein from mammalian brain contains at least four discrete polypeptides (Mr 51,000, 53,000, 55,000 and 58,000) by a variety of visualization techniques and in three species (rat, cow, and human). These polypeptide bands vary in their affinity for gamma-aminobutyric acid analogs as shown by inhibition of [3H]muscimol binding, demonstrated by photoaffinity labeling and gel electrophoresis in sodium dodecyl sulfate. One-dimensional peptide maps of proteolytic digests revealed that distinct fragments were produced, indicating that the four polypeptides represent discrete sequences. The four bands were identified by Western blotting with subunit-specific monoclonal antibodies as two species each of previously identified alpha and beta subunits. [3H]Muscimol photolabeled all four bands (beta and alpha) to varying degrees not proportional to the extent of protein staining. The Mr 58,000 beta subunit subtype showed a higher affinity for 4,5,6,7-tetrahydro-isoxazolo-[5,4-c]pyridin-3-ol than the Mr 56,000 beta subtype, whereas the Mr 56,000 beta and Mr 51,000 alpha bands were more enhanced by pentobarbital than the Mr 58,000 band. Furthermore, the alpha subunit pattern revealed by photoaffinity labeling with [3H]flunitrazepam was significantly different for three regions of bovine brain, showing only one major band in cerebellum at Mr 51,000, two major bands in cortex at Mr 53,000 and 51,000, and three bands in hippocampus at Mr 55,000 as well as Mr 53,000 and 51,000. Because the ratio of the amounts of the various polypeptides varies with brain region and the pharmacological properties of the peptides vary, it is likely that a family of oligomeric gamma-aminobutyric acid/benzodiazepine receptors exists in the brain. This is consistent with the reported variable expression of different subunit subtype mRNAs and with brain region-dependent variation in pharmacology and binding behavior.

Affinity Labels↗
PubMed1984

Proteolytic and chemical dissection of the human erythrocyte glucose transporter.

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.

Affinity Labels↗
PubMed1988

Solubilization of the omega-conotoxin receptor associated with voltage-sensitive calcium channels from bovine brain.

The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes.

Animals↗
PubMed1994

Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and other pyridines. Human liver NNMT activity has a bimodal frequency distribution, an observation which raises the possibility that this enzyme activity might be regulated by a genetic polymorphism, a polymorphism that could have functional implications for individual differences in drug and xenobiotic toxicity. As a first step toward testing that hypothesis, we set out to clone and express a cDNA for human liver NNMT. Human liver NNMT was partially purified, photoaffinity-labeled, subjected to limited proteolysis, and partial amino acid sequence information was obtained. The polymerase chain reaction was then used to amplify a 550-nucleotide sequence with human liver cDNA as template and primers designed on the basis of the NNMT amino acid sequence. The 5'- and 3'-ends of a human liver NNMT cDNA were obtained by use of the rapid amplification of cDNA ends. The combined use of these approaches resulted in the isolation of a human liver NNMT cDNA that was 969 nucleotides in length, with a 792-nucleotide open reading frame that encoded a 264-amino acid protein with a calculated molecular mass of 29.6 kDa. The human liver NNMT cDNA was transcribed in vitro and translated with a reticulocyte lysate system to yield a protein with a molecular mass of approximately 29 kDa that comigrated during SDS-polyacrylamide gel electrophoresis with photoaffinity-labeled human liver NNMT. The NNMT cDNA was also subcloned into the eukaryotic expression vector p91023(B). COS-1 cells transfected with this construct expressed a high level of NNMT enzymatic activity, and the biochemical properties of this activity were similar to those of human liver NNMT. Human liver NNMT and transfected COS-1 cell NNMT had apparent Km values for the two cosubstrates for the reaction, nicotinamide and S-adenosyl-L-methionine, of 0.43 and 0.38 mM and of 1.8 and 2.2 microM, respectively. IC50 values for the inhibition of NNMT by N1-methylnicotinamide were 60 and 30 microns for human liver and COS-1 cell-expressed NNMT, respectively. Cloning of a cDNA for human liver NNMT will help make it possible to test the hypothesis that inheritance may play a role in the regulation of individual differences in human liver NNMT activity.

Amino Acid Sequence↗
PubMed1984

Heterogeneous deoxyribonucleic acid-binding forms of rabbit uterine progesterone receptor.

This study investigated the subunit structure of the rabbit uterine progesterone receptor (PR) using ion-exchange and DNA-cellulose chromatography. The mol wts of receptor were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling the receptors with the progestin [3H]R5020. Cytosols were labeled with [3H]progesterone or 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione [( 3H]R5020) and chromatographed on DE-52 cellulose. Greater than 80% of the PR bound to DE-52 and elution with a KCl gradient gave two peaks of activity at 50-75 mM (peak I) and at 125-200 mM KCl (peak II). Chromatography on QAE-Sephadex also separated two peaks. Peak I was abolished by addition of molybdate, or by passage over phosphocellulose. All (greater than 80%) of the PR from peaks I and II bound to DNA-cellulose and eluted as a single, symmetrical peak at 0.29 M KCl in both cases. Peak I could not be directly generated from peak II by exposure to salt or by ammonium sulfate precipitation. After peak II receptor had been bound and eluted from a DNA-cellulose column, however, it eluted subsequently from QAE as peak I. Photoaffinity labeling of peak I and peak II with [3H] R5020 in the presence of 10 microM cortisol revealed two proteins in each peak with mol wt of 102,000 and 78,000 (n = 13). Both mol wt forms were present in the DNA-eluates both of peak I and peak II. The [3H]R5020 binding to these mol wt forms could be completely displaced under exchange conditions with 10 nM progesterone or R5020. Deoxycorticosterone, dihydrotestosterone, and estradiol were nearly ineffective competitors at 10 nM. Without phosphocellulose chromatography before chromatography on DE-52 and DNA-cellulose, numerous receptor fragments were found. Fragments containing both steroid- and DNA-binding domains were found at 102,000, 78,000, 54-60,000, 43,000, 33-34,000, and 21,000, suggesting proteolysis. This proteolytic activity was removed by passage over phosphocellulose. The rabbit uterine PR contains at least two major proteins of 78,000 and 102,000 mol wt which cannot be distinguished on the basis of their ionic or DNA-binding characteristics or steroid-binding specificity.

Animals↗
PubMed1993

Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

Acetylcholine↗
PubMed1985

Reduced levels of cardiac cAMP-dependent protein kinase in spontaneously hypertensive rat.

Cardiac cAMP-dependent protein kinases were compared between the spontaneously hypertensive rat and the age-matched normotensive Wistar-Kyoto rat by DEAE-cellulose chromatography, photoaffinity labeling with 8-N3[32P]cAMP, and Western blots using the antiregulatory and 125I-anticatalytic subunit antibodies. DEAE-cellulose chromatography revealed that the ratio of type I to type II cAMP-dependent protein kinase was 3:1 in the cytoplasmic soluble proteins from the heart of normotensive rat. In contrast, the ratio of type I to type II was 1:1 in the heart of hypertensive rat. Type I protein kinase was reduced by 3-fold in hypertensive rat compared to normotensive rat. The levels of type II protein kinase were similar in both normotensive and hypertensive rats. The ratio of regulatory subunits of type I (RI) to type II (RII) cAMP-dependent protein kinase was 2.5 in the soluble proteins from the heart of normotensive rat compared to a ratio of 0.62 for hypertensive rat. RI was reduced by 4-fold in hypertensive rat compared to normotensive rat. The decrease in RI from hypertensive rat was also demonstrated by photoaffinity labeling with 8-N3[32P] cAMP. Western blot analysis of the catalytic subunit revealed a 2-fold decrease in catalytic subunit (C) in the soluble proteins from the hypertensive rat compared to normotensive rat. These results show that the reduced level of activity of cardiac type I protein kinase in hypertensive rat was the result of a decrease in both the RI and C subunits, thus reducing the number of type I cAMP-dependent protein kinase holoenzyme molecules. Comparison of type I protein kinase from "prehypertensive" and "hypertensive" stages of hypertensive rat indicated that the type I protein kinase was reduced by 3-fold before an increase in the blood pressure was detectable. Cardiac type I protein kinase is predominantly associated with the cytoplasmic proteins in both the normotensive and hypertensive rats. The levels of RI, RII, and C associated with the membrane-solubilized proteins were not affected in the hypertensive rat. The levels of RII were similar in the brain tissue of normotensive and hypertensive rats, suggesting that the decrease in type I protein kinase is specific in hypertensive rat. In conclusion, a decrease in cardiac type I cAMP-dependent protein kinase may affect the degree of phosphorylation of cardiac regulatory proteins, thus impairing normal cardiac physiology in hypertensive rat.

Animals↗
PubMed1979

Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases.

Cyclic AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified, and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit molecular weight of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These data suggest an asymmetric tetrameric (R2C2) structure (Mr approximately equal to 160,000) for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit molecular weight, pI values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 microgram/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but are clearly dissimilar from those of the soluble type II enzyme. Moreover, regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit molecular weight, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the present results suggest that the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.

Cyclic AMP↗
PubMed1994

Inhibition and inactivation of horse liver alcohol dehydrogenase with the imidazobenzodiazepine Ro 15-4513.

The imidazobenzodiazepine ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine -3-carboxylate (Ro 15-4513) is important as a potential "drink and drive" drug due to effects on receptors in brain neurones, resulting in alcohol intoxication-antagonistic properties. Because of the molecule's importance its effect on alcohol metabolism in liver has been investigated. Ro 15-4513 was found to be, like its parent compound the 8-fluoro analogue flumazenil, a reversible alcohol competitive inhibitor of horse liver alcohol dehydrogenase (EC 1.1.1.1) with a dissociation constant of 345 microM at pH 7.0. Due to its azido group Ro 15-4513 was developed as a potential photoaffinity-labeling reagent for benzodiazepine receptors. Used with horse liver alcohol dehydrogenase, the enzyme is chemically modified and inactivated in a Michaelis-Menten type reaction via a reversible enzyme-Ro 15-4513 complex with a dissociation constant of 8.6 mM at pH 7.0. The inactivation reaction has been studied over the pH 6.0-10.0 range. The dissociation constants for the binding of Ro 15-4513 to the enzyme and the first-order rate constants for inactivation have been determined as a function of pH. These give pKa values of 7.2 and 8.8 for the free enzyme, the latter being assigned to the zinc-water ionization. The enzyme is protected from inactivation in a competitive manner by flumazenil and by many heterocyclic and thiol compounds which combine with the active-site zinc. Flumazenil has a similar binding affinity as Ro 15-4513 with an enzyme-flumazenil dissociation constant of 6.0 mM at pH 7.0. Ro 15-4513 may also have potential as a photoaffinity-labeling reagent for other metallo enzymes. Whether the effects of Ro 15-4513 on alcohol-metabolizing enzymes are also of clinical significance remains to be determined.

Affinity Labels↗
PubMed1992

Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity.

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.

Adrenal Cortex↗
PubMed1986

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia.

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.

Animals↗