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.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

A simple enzymic method for the synthesis of adenosine 5''-[alpha-32P]triphosphate on a preparative scale.

A simple, rapid and inexpensive method is described for the enzymic synthesis of [alpha-32P]ATP from [32P]Pi on a preparative scale with an overall yield of 53%. The final product contained all of the detectable radioactivity (less than 99.9%) in the alpha position and has been shown to behave identically with commerically availabe [alpha-32P]ATP during the synthesis of 3':5'-cyclic AMP in the reaction catalysed by adenylate cyclase.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.     

Functionally similar vanadate-induced 8-azidoadenosine 5'-[alpha-(32)P]Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.     

Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP

It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.     

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.     

A 3-aminobenzamide-resistant labeled protein in [32P]NAD+-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [32P]NAD+. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [32P]NAD+ was shown to be Mg2+-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [alpha-32P]ATP, [gamma-32P]ATP and [32P]orthophosphate, but not from [3H]NAD+ or [14C]NAD+. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [32P]NAD+. This phosphorylation appears to occur via an Mg2+-dependent conversion of NAD+ to AMP with the eventual utilization of the alpha-phosphate for phosphorylation of the 72 000 molecular weight protein.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.     

Covalent modification of the non-catalytic sites of the H(+)-ATPase from chloroplasts with 2-azido-[alpha-(32)P]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (beta-Tyr362) and non-catalytic (beta-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 microM 2-azido-[alpha-(32)P]ATP, that with the intermediate dissociation constant (site 5) at 2 microM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[alpha-(32)P]ATP was possible. CF(0)F(1) with non-covalently bound 2-azido-[alpha-(32)P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by Delta pH/Delta phi. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF(0)F(1). Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between beta- and alpha-subunits which is abolished by covalent derivatization of beta-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F(0)-part and the F(1)-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three beta-Tyr385.     

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.     

Adenylate cyclase assay with [alpha-32P]ATP as substrate. A simple modification for lowering blanks

In the assay of adenylate cyclase using [α-³²P]ATP as the substrate and alumina chromatography as the separating procedure for labeled nucleotides, blank levels are dependent on the quality of the labeled ATP and also on that of the alumina. In order to lower the blanks by eliminating the radioactive material contaminating the commercial [α-³²P]ATP preparations, the following treatment is proposed: The reaction mixture resulting from the incubation is heated for 4 min at 95°C in 0.165 n HCl, then it is chromatographed on a selected alumina (Woelm) column. In the conditions used, cyclic AMP was unaffected, while blank values were low. The detection limit of [³²P]cyclic AMP was thus higher and the precision of enzyme activity determination was improved, while the advantages of one-step chromatography were retained.     

Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses

Incubation of crude extracts from cells lytically infected with polyoma virus in the presence of periodate-oxidized [alpha-32P]ATP led to the radioactive labeling of one main polypeptide immunoprecipitated by anti-T antigen antibodies. It was absent from extracts of mock-infected cells and exhibited an apparent Mr value of 105,000, identical with that of the large-T viral protein. This polypeptide was unambiguously identified as large-T on the basis of the heat sensitivity of the in vitro labeling in extracts from cells infected with a tsa mutant. The amount of incorporated radioactivity was found to increase linearly with that of infected cell extract and with the incubation time. Labeling resulted from the specific binding of an oxidized ATP molecule to a nucleotide binding site of the large-T protein, since it was efficiently completed by unlabeled ATP. Study of the dependence on the concentration of oxidized ATP evidenced a first order kinetic for the labeling reaction. Similar results were obtained for the large-T protein of SV40 virus.     

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.     

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.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.     

Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.     

Enzymatic synthesis of uniformly 32P-labeled polyribonucleotides and high-specific-activity ribonucleoside 5'-[alpha-32P]diphosphates

Uniformly 32P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5'-[alpha-32P]triphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition.