Further studies of urea-catalyzed phosphorylation reactions

Summary We have analyzed the products formed when mixtures of a nucleoside and ammonium dihydrogen phosphate are heated with an excess of urea. If there is more phosphate than nucleoside in the mixture, compounds containing pyrophosphate bonds are obtained. If uridine, as nucleoside, is in excess over phosphate, di- and oligonucleotides are formed.On leave from Department of Medical Biochemistry, University of Göteborg, Sweden.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Radioactive phosphorylation of alcohols to monitor biocatalytic Diels-Alder reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope (32)P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme-an RNA sequence that catalyzes the eponymous reaction. We used the (32)P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the (32)P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Modulation of natural killer cell activity by surface phosphorylation reactions

To determine whether phosphorylation of cell surface proteins is involved in NK cell activity, the phosphorylation patterns of a rat NK cell line (RNK-16) incubated with 12.5 microM [gamma-32P]ATP were characterized before and after exposure to YAC-1 cells, which serve as targets for killing, and K562 cells, which are not killed by RNK-16 cells. By 51Cr release assays, the inhibitory effect of ATP on RNK-16 killing activity previously reported was corroborated. RNK-16 cells prelabeled with 12.5 microM ATP show enhanced labeling of a 70- to 72,000-Da protein after exposure to unlabeled target YAC-1 cells but not after exposure to K562 cells. A protein of similar apparent molecular size is also labeled upon exposure of RNK-16 cells to OX-34, an antibody which binds and inhibits killing, as well as upon exposure to OX-18, which also binds but does not inhibit NK activity. These findings are indicative of the activation of a kinase with high affinity for [gamma-32P]ATP, which phosphorylates an endogenous surface substrate of 70-72,000 Da upon binding of macromolecules to the RNK-16 cells. RNK-16 cells, previously labeled with micromolars [gamma-32P]ATP and subsequently treated with millimolars unlabeled ATP, showed loss of label from a 110,000-Da protein component, indicative of the rapid turnover of a phosphate group on a surface protein. Thus, extracellular ATP enhances the phosphorylation of a 70- to 72,000-Da component upon binding of RNK-16 cells to target cells or upon binding of antibodies at micromolar concentrations of ATP and catalyzes the loss of phosphate from a 110,000-Da component at millimolar concentrations of ATP. These findings reflect a complex repertoire of surface phosphorylation changes which occur in RNK-16 cells.     

In vitro phosphorylation of serum albumin by two protein kinases: a potential pitfall in protein phosphorylation reactions

Bovine albumin was phosphorylated by both cAMP-dependent protein kinase and casein kinase I to a significant extent. Other albumins were also tested and it was found that the extent of phosphorylation varied with the species of origin of the albumin, but was between 1 and 3 mol phosphate per mole albumin for the cAMP-dependent protein kinase-catalyzed reactions. The phosphorylation occurred at and above pH 7.5 and required the presence of thiol reagents. Phosphoamino acid analyses of bovine albumin showed that it was phosphorylated on at least two serine residues. The phosphorylation could not be demonstrated in vivo.     

Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.