pyrophosphate and diadenosine-tetraphosphate

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.

Topics: Adenosine Triphosphate; Animals; Coleoptera; Dinucleoside Phosphates; Diphosphates; Isotope Labeling; Luciferases; Phosphorus Radioisotopes; Tritium

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Binding Sites; Dinucleoside Phosphates; Diphosphates; Kinetics; Mitochondria; Molecular Structure; Protein Conformation; Proton-Translocating ATPases

The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275-8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959-2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E-aminoacyl-AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E-luciferin-AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl-containing intermediates and releasing PPi may produce dinucleoside polyphosphates.

Topics: Dinucleoside Phosphates; Diphosphates; Luciferases; Pyrophosphatases

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Animals; Dinucleoside Phosphates; Diphosphates; Kinetics; Liver; Lysine; Lysine-tRNA Ligase; Rats

We have identified a system in human lymphocytes which proteolytically cleaves poly(ADPribose) polymerase to specific fragments of molecular weight 96 000, 79 000 and 62 000-60 000. This proteolytic processing is dependent on two different classes of proteinase. One of these proteinases is a serine proteinase, since the processing is inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor and diisopropylfluorophosphate, the other is a cathepsin D-like proteinase, since processing is also inhibited by pepstatin A. The processing that occurs in permeabilized cells can be simulated in vitro by treating purified poly(ADPribose) polymerase with trypsin, but not by treating the polymerase with cathepsin D. Since processing at the cellular level is blocked by inhibitors of either of the two proteinases, but only trypsin could cleave the purified polymerase, this suggests that in the cell the action of the cathepsin D-like proteinase is a prerequisite for cleavage of poly(ADPribose) polymerase by the serine proteinase. Thus, a pathway involving sequential action of these proteinases may exist. Proteolysis in permeabilized human lymphocytes is stimulated by nucleotides containing a pyrophosphate group, such as 5',5'''-P1,P4-tetraphosphate and ATP, or by pyrophosphate itself. In contrast, nucleotides containing only a single phosphate, such as AMP and cyclic AMP, or inorganic sodium phosphate, do not show this stimulation of proteolysis. These results suggest that a pyrophosphate linkage is the minimum molecular requirement for stimulation of proteolytic processing of poly(ADPribose) polymerase. Proteolytic processing of poly(ADPribose) polymerase is independent of ADPribosylation. Following proteolysis, specific fragments of the polymerase, particularly the 62 000-60 000 molecular weight fragment(s), are still capable of being ADPribosylated.

Topics: Adenine Nucleotides; Cathepsin D; Dinucleoside Phosphates; Diphosphates; Enzyme Activation; Humans; Hydrolysis; Molecular Weight; Peptide Hydrolases; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Trypsin

We have identified a proteolytic system that selectively degrades histone H1 in normal human lymphocytes. Treatment of permeabilized human lymphocytes with a series of nucleotides produced a marked decrease in their histone H1 content compared to untreated cells. The nucleotide-stimulated process was selective for histone H1 because gel electrophoresis showed that almost all other lymphocyte protein bands remained constant while histone H1 disappeared. The elimination of histone H1 appears to be the result of proteolysis by a trypsin-like enzyme because it was inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor, and diisopropyl fluorophosphate. Proteolysis was stimulated by P1,P4-di(adenosine-5') tetraphosphate, P1,P3-di(adenosine-5') triphosphate, P1,P5-di(adenosine-5') pentaphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[alpha, beta-methylene]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or sodium phosphate did not show this stimulation of proteolysis. ATP, [alpha, beta-methylene]ATP, [beta, gamma-methylene]ATP, and pyrophosphate all stimulated proteolysis, suggesting that a pyrophosphate linkage was necessary for this process. Thus, resting human lymphocytes contain a trypsin-like protease that is stimulated by nucleotides or pyrophosphate to selectively degrade histone H1.

Topics: Adenine Nucleotides; Dinucleoside Phosphates; Diphosphates; Histones; Humans; In Vitro Techniques; Lymphocytes; Protease Inhibitors