Synthesis of [gamma-32P]thiamine triphosphate

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%.     

Enzymatic synthesis of [beta-32P]ADP using adenylate kinase and [gamma-32P]ATP

An enzymatic method for the synthesis of [beta-32P]ADP from [gamma-32P]ATP is described. This substrate is required for the assay of ADPase and is not commercially available. The method described results in a preparation of [beta-32P]ADP of high purity with a yield of approximately 40% the theoretical obtainable.     

Carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate

We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.     

Activation of Go-proteins by membrane depolarization traced by in situ photoaffinity labeling of galphao-proteins with [alpha32P]GTP-azidoanilide

Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838). In the present work we identify the activated G-proteins as Go-proteins by tracing their depolarization-induced in situ photoaffinity labeling with [alpha32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was introduced into transiently permeabilized rat brain-stem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [alpha32P]GTPAA to Galphao1- and Galphao3-proteins, but not to Galphao2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha32P]GTPAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of Go-protein-coupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The alpha subunit of VGSC was cross-linked and co-immunoprecipitated with Galphao-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha subunit most efficiently cross-linked with guanosine 5'-O-2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound Galphao-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.     

Direct photoaffinity labeling of junctional sarcoplasmic reticulum with [14C]doxorubicin

Doxorubicin, an anticancer drug, induces Ca2+ release from the terminal cisternae (TC) of skeletal muscle (Zorzato, F., Salviati, G., Facchinetti, T., and Volpe, P. (1985) J. Biol. Chem. 260, 7349-7355). Long wave ultraviolet irradiation of a TC fraction with morphologically intact feet structures (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) in the presence of [14C]doxorubicin, led to covalent photolabeling of two proteins that exhibited apparent Mr values of 350,000 and 170,000. Such proteins were found to be absent in a fraction of longitudinal sarcoplasmic reticulum but enriched in junctional face membranes obtained by Triton X-100 treatment of the TC fraction. Three additional proteins with Mr values of 80,000, 60,000, and 30,000 were also faintly labeled in the junctional face membrane fraction. On a molar basis the highest level of incorporation was found in the 170,000-Da protein, probably a Ca2+-binding protein (Campbell, K. P., MacLennan, D. H., and Jorgensen, A. O. (1983) J. Biol. Chem. 258, 11267-11273). A lower level of labeling was observed in the 350,000-Da protein, tentatively identified as a component of the feet structures (Cadwell, J. J. S., and Caswell, A. H. (1982) J. Cell Biol. 93, 543-550). Photolabeling of junctional TC proteins did not occur if a 10-50-fold excess cold doxorubicin was included in the assay medium, indicating that it was displaceable and specific, and if ultraviolet irradiation was omitted. Photolabeling was inhibited by caffeine or ruthenium red, i.e. by an activator and an inhibitor of Ca2+ release from TC, respectively. Furthermore, photolabeling was prevented by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid suggesting that doxorubicin binding is Ca2+-dependent. Doxorubicin-binding proteins are constituents of the junctional sarcoplasmic reticulum and might be involved in modulating Ca2+ release from TC.     

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.     

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.     

Decreased incorporation of the photoaffinity probe 8N3-[gamma-32P]-GTP into a 45kD protein in lung tumors

Photoincorporation of 8N3-[gamma-32P]-GTP into tissue and cell extracts was examined using gel electrophoresis and autoradiography. Decreased photoincorporation into a 45kD band was observed in extracts from mouse lung tumors as compared to normal mouse lung, and in extracts from lung tumor-derived cell lines when compared to isolated bronchiolar epithelial cells. Decreased 45kD photolabelling was also observed in extracts of S49 lymphoma cyc- cells (deficient in Gs alpha, a 45kD GTP binding protein of receptor-coupled adenylate cyclase) when compared to wild type S49 cells. This, and the observation that there was no cholera toxin-catalyzed ADP-ribosylation in the 45kD band of lung tumor extracts, suggests that the 45kD band contains Gs alpha.     

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

[gamma-32P]ATP as a tracer of the fragmentation of Ca-F-actin.

[gamma-32P]ATP-G-actin was polymerized in 4 mM-CaCl2, and the distribution of the radioactive nucleotide among the oligomeric and the polymeric species was studied. The results obtained are best explained by assuming spontaneous fragmentation.     

Direct photoaffinity labeling of the putative sulfonylurea receptor in rat beta-cell tumor membranes by [3H]glibenclamide

The oral antidiabetic sulfonylurea [3H]glibenclamide specifically binds to plasma membranes from a rat beta-cell tumor indicating a receptor for sulfonylureas in these membranes. Irradiation of [3H]glibenclamide at 254 or 300 nm in the presence of albumin resulted in covalent labeling of the albumin molecule. Direct photoaffinity labeling of beta-cell membranes with [3H]glibenclamide resulted in the covalent modification of two membrane polypeptides with apparent molecular masses 140 and 33 kDa. The extent of labeling of the 140 kDa polypeptide was specifically decreased by sulfonylureas. This suggests that a membrane polypeptide of 140 kDa is a component of the sulfonylurea receptor in the beta-cell membrane.     

Covalent labeling of adenylyl cyclase cytosolic domains with gamma-methylimidazole-2',5'-dideoxy-[gamma-(32)P]3'-ATP and the mechanism for P-site-mediated inhibition

A truncated first cytosolic domain of type V adenylyl cyclase (VC(1)) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC(2)) were used alone and in the readily reversible complex (VC(1).IIC(2)) to evaluate interactions with each other and with reversible and irreversible P-site ligands. Enzyme activity was used to assess formation and dissolution of VC(1).IIC(2). The data suggest that binding of 2',5'-dideoxy-3'-ATP to VC(1) and IIC(2) prevented formation of VC(1).IIC(2) and that 2',5'-dideoxy-3'-ATP dissociation occurred slowly. To enable configuration specific cross-linking to the catalytic site, 2',5'-dideoxyadenosine 3'-[gamma-(1-methylimidazole)-triphosphate] (gamma-MetIm-2', 5'-dd-3'-ATP) and 2',5'-dd-adenosine 3'-(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2', 5'-dd-3'-ATP) were synthesized, the former also as its gamma-(32)P-labeled analog. gamma-Azidoanilido-2',5'-dd-3'-ATP exhibited an inhibitory potency comparable with that of 2', 5'-dd-3'-ATP. gamma-MetIm-2',5'-dd-[gamma-(32)P]3'-ATP labeled the individual VC(1) and IIC(2) domains comparably and covalently to approximately 20% within 1 h. Formation of VC(1).IIC(2) resulted in reduced labeling of VC(1) but enhanced labeling of IIC(2). The data imply that formation of the catalytically active VC(1).IIC(2) complex affects the interaction of each domain with the 2', 5'-dd-3'-ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo-irreversible inhibition.     

Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling

A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.     

Analogues of [3H] chloramphenicol for photoaffinity labeling.

The synthesis of [3H]chloramphenicol and its erythro-diastereoisomer with specific activities of 1.25 Ci/mmol, and the further transformation of the [3H]chloramphenicol to a series of azido and diazo-substituted derivatives are described. The antibiotic activity of the compounds was considered insufficient for their use as photoaffinity labels.     

Preparation of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine by using soya beans.

This method describes a procedure that can be carried out easily to obtain large amounts of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine. The method involves germinating soya beans in the presence of [32P]Pi. The yield was 0.58% for [P]phosphatidylcholine and 0.52% for [32P]lysophosphatidylcholine, and the specific radioactivity of both was 10(7) d.p.m./mumol.