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ATP influences the kinetic properties of cytochrome c oxidase. A photoactivatable radioactive ATP analogue was used to localize the nucleotide-binding site on the bovine heart enzyme. Subunits IV and VIII were specifically labelled, suggesting that these two nuclear-coded polypeptides may play a regulatory role on the oxidase functions.
The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.
This study identifies calcium-antagonist-receptor-carrying polypeptides of calcium channels in guinea-pig hippocampus membranes. The arylazide ligands (-)-[3H]azidopine and [N-methyl-3H]LU49888 [(-)-5-[(3-azidophenethyl) [N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl-2- isopropylvaleronitrile] were used to selectively label 1,4-dihydropyridine and phenylalkylamine receptors respectively. In the absence of u.v. light, both ligands reversibly bound to a single class of high-affinity receptors with a calcium-channel-typical pharmacological profile. [N-methyl-3H]LU49888 bound to the extent of 849 +/- 188 fmol/mg of protein (mean +/- S.D., n = 3) with a dissociation constant (Kd) of 1.4 +/- 0.3 nM. Under identical assay conditions (-)-[3H]azidopine labelled to the extent of 562 +/- 132 fmol/mg of protein with a Kd of 0.096 +/- 0.024 nM. After u.v. irradiation of the [N-methyl-3H]LU49888- and (-)-[3H]azidopine-labelled membranes, both photo-affinity probes were found to be incorporated specifically into a 190-195 kDa band as shown by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). Photoincorporation occurred with a protection profile identical with that produced by reversible binding-inhibition. [N-methyl-3H]LU49888, but not (-)-[3H]-azidopine, specifically labelled an additional 265 kDa band. Both photolabelled bands had an identical electrophoretic mobility on SDS/PAGE, irrespective of pretreatment either with 10 mM-N-ethylmaleimide or 10 mM-dithiothreitol. The electrophoretic properties of the 195 kDa polypeptide and the lability of receptor-incorporated (-)-[3H]azidopine to nucleophilic agents resemble those of the previously described drug-receptor-carrying alpha 1 subunit of the purified skeletal-muscle calcium channel. The data suggest that this polypeptide carries both the high-affinity 1,4-dihydropyridine as well as the phenylalkylamine receptor of neuronal calcium channels in guinea-pig hippocampus and is a component of the L-type calcium channel.
The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.
Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.
In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.
Acyl-CoA esters containing the photoreactive acids 12-(4'-azido-2'-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4'-azido-2'-nitro-[3'-5'-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4'-Azido-2'-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4'-azido-2'-nitro[3',5'-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond.
GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.
The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase.
Methylanthraniloyl-8-azido-ATP (Mant-8-N3-ATP), which binds to the 20 kDa C-terminal tryptic fragment of skeletal-muscle myosin subfragment-1 [Maruta, Miyanishi and Matsuda (1989) Eur. J. Biochem. 184, 213-221], was synthesized and used as a probe of the conformational change of smooth-muscle myosin. Mant-8-N3-ATP, like ATP, induced the formation of the 10 S conformation at low ionic strength. In the presence of vanadate, smooth-muscle myosin formed a stable complex with Mant-8-N3-ADP, and this complex showed the 10 S-->6 S transition of myosin. ATP-binding sites for 6 S (extended state) and 10 S (folded state) myosin were studied by photolabelling of myosin with Mant-8-N3-ADP. For both 6 S and 10 S myosin, Mant-8-N3-ATP was incorporated into the 29 kDa N-terminal tryptic fragment of myosin heavy chain. This is unlike the labelling of skeletal-muscle myosin, in which the 20 kDa C-terminal fragment is labelled. The labelling of 29 kDa fragment was diminished significantly by addition of ATP. These results suggest that the conformation of the ATP-binding site of smooth-muscle myosin is different from that of skeletal-muscle myosin. To examine further the possible differences in the labelling site between 6 S and 10 S myosin, the affinity-labelled 29 kDa fragment was subjected to complete proteolysis by lysylendo-peptidase. The fluorescent-labelled-peptide map suggested that the Mant-8-N3-ADP-binding sites for 6 S and 10 S myosin were identical.
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Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.
[(3)H]N(6)-(Ethyl 2-diazomalonyl)-adenosine 3':5'-cyclic monophosphate is incorporated into intact ghosts from human erythrocytes on photolysis at 253.7 nm. Incorporation is blocked in the presence of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and does not occur in the absence of irradiation. Sodium dodecyl sulfate disc gel electrophoresis of solubilized ghosts shows that one protein is labeled. The position of this protein on the gel corresponds exactly to that previously found. [J. Biol. Chem. 247, 8145 (1972)] for the endogeneous protein substrate of the endogenous, cyclic AMP-dependent, protein kinase.
8-Azidoguanosine 3',5'-cyclic monophosphate (8-N3cGMP) has been synthesized for use as a photoactive probe for the labeling of cGMP receptors. The ability of 8-N3cGMP to be bound at specific cGMP binding sites was demonstrated by its ability to activate cGMP-dependent protein kinase isolated from bovine lung (Ka = 1.1 x 10(-7) M) and to inhibit competitively the binding of [3H] cGMP to the enzyme [Kd (8 N3GMP)/Kd (cGMP) = 6]. Photolysis of 8-N3[32P]cGMP in the presence of a crude enzyme preparation resulted in the covalent attachment of analog to cGMP-dependent protein kinase. Half-maximal labeling occurred at 2.2 x 10(-7) M. The incorporation of the analog was completely inhibited by the addition of cGMP.
Lutropin, a pituitary hormone, and human choriogonadotropin bind to the same receptors in the ovary and elicit identical responses. A photoactivable derivative of human choriogonadotropin was used to identify the lutropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycylglycine, and iodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself did but with a slightly lower Ka, 2.98 X 10(9) M-1 compared with 5.1 X 10(9) M-1 for 125I-hormone. The binding could be blocked with untreated hormone or lutropin but not with follitropin, prolactin, insulin, or bovine serum albumin. Its alpha and beta subunits could be crosslinked to produce alpha beta dimer by photolysis, the extent of crosslinking being dependent upon the reagent concentration used for the derivatization: 22.8% at 50 microM, 37.3% at 100 microM, and 67.2% at 150 microM. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels under reducing conditions, three new bands of lower electrophoretic mobility appeared in addition to alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis and the presence of both cells bearing the receptor and the 125I-hormone derivative. It could be blocked by excess untreated hormone. The three bands correspond to molecular weights, 96,000 +/- 6,700, 66,000 +/- 4,600, and 63,000 +/- 4,400. Because the hormone has a high carbohydrate content and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.
An azide derivative of the beta-adrenergic antagonist acebutolol has been synthesized and its effect examined on the isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity of rat reticulocytes. It behaved as an effective competitive antagonist (Kd = 2 X 10(-7) M) prior to photolysis. However, when the reticulocyte preparation pretreated with acebutolol azide was photolyzed, a noncompetitive inhibition of isoproterenol-stimulated adenylate cyclase was obtained. Photolysis of the azide derivative in buffer alone did not convert it to a product of higher affnity. Labeling of the beta-adrenergic receptor appeared to be irreversible; multiple washings could not reverse the inhibition produced during photolysis with the label whereas washing would completely reverse the antagonism produced by the same concentration of label prior to photolysis. The effect appears to be specific for the beta-adrenergic receptor because the inhibition could be blocked stereoselectively by propranolol and there was no inhibition of fluoride- or GMP-P(NH)P-stimulated adenylate cyclase. furthermore, no effect was observed on the glucagon-mediated stimulation of adenylate cyclase of liver membranes, whereas the catecholamine response in the same membranes was inhibited.
Salivary glands of third-instar larvae of Drosophila melanogaster as well as Drosophila K(c) tissue culture cells have been irradiated in the presence of ecdysterone. Irradiation covalently links ecdysterone to a single cellular protein, which is similar, if not identical, in salivary glands and in K(c) cells. This protein has a molecular weight of 130,000 and it has the characteristics of a typical hormone-receptor molecule in terms of hormone-binding properties, translocation into the nucleus, and sedimentation characteristics. The yield of the photoinduced bonding of ecdysterone to receptor protein is around 15%. Ponasterone A competed with ecdysterone for the bonding. Also, ponasterone A itself reacted upon photoactivation with the beta-ecdysterone receptor protein in Drosophila tissue culture cells. We have previously shown that ecdysterone can be bonded upon irradiation to specific hormone-controlled puffs of polytene chromosomes of D. melanogaster third-instar larvae [Gronemeyer, H. & Pongs, O. (1980) Proc. Natl. Acad. Sci. USA 77, 2108-2112]. Because we have now identified the molecular target of the ecdysterone photoreaction, these data show that a hormone-receptor complex translocates to the nucleus and directly binds to the genes, which are under hormonal control. A quantitative assay of hormone-receptor complex in K(c) cells before and after hormone stimulation showed that ecdysterone does not regulate the synthesis and the available amount of its receptor. It was also observed that the translocated hormone-receptor complex resides in the nucleus as long as the hormone is present in the tissue culture medium.