Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.     

The influence of diphosphonic analogues of inorganic pyrophosphate on activity of RNA-polymerases from the calf thymus

Diphosphonic analogues of inorganic pyrophosphate (PPi): methylene-, oxyethylidene-, aminomethylenediphosphonic acids as well as phosphonacetic, imidodiphosphoric bis- (phosphonomethyl)-phosphonic acids and methylenediphosphonic and phosphonic acid monoanhydrides were studied for their effect on the RNA-synthesizing activity of thymocytes. DNA-dependent RNA-polymerases I and II from the calf thymus nuclei were used for these studies. The analogues and PPi under study are shown to be inhibitors of both RNA-polymerases in nuclei from calf thymus and of purified RNA-polymerase II, which is more sensitive to the effect of diphosphonates. Methylenediphosphonic acid is the strongest inhibitor among the studied analogues, and imidodiphosphoric and phosphonacetic acids are the weakest inhibitors. Inhibition of purified RNA-polymerase II by diphosphonates has a complex character and includes both interaction of the PPi analogues with enzymes and chelating by them of Mn ions which are cofactors for RNA polymerase.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.     

Hydrolytic activity of bovine tryptophanyl-tRNA-synthetase cause by removal of Zn2+

Bovine tryptophanyl-tRNA synthetase (E.C.6.1.1.2) lacking Zn2+ ions removed by chelation with phosphonate analog of P1,P4-bis-(5'-adenosyl)tetraphosphate (Ap4A) was obtained (E-Zn). E-Zn lost the ability to form tryptophanyl adenylate, however it hydrolyses ATP to ADP and further on to AMP and Pi. GTP serves as a substrate with Km approximately 0.6 mM. It is proposed that the hydrolysable nucleotides bind to a nucleotide binding site(s) distinguishable from the substrate (catalytic) ones. After incubation of E-Zn with Zn2+ and Mg2+ the initial catalytic activity (ATP-PPi exchange and amino-acylation reactions) is restored whereas the hydrolytic activity becomes fully suppressed.     

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Modification of human DNA polymerase alpha by N-acetylimidazole

The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.     

Bis(adenosyl-5′)tetraphosphate as a partial agonist of ATP receptors in rat sensory neurons

The effects of bis(adenosyl-5)oligophosphates on cation conductance-promoting ATP receptors at the somatic membrane of rat sensory ganglia neurons were investigated. Both bis(adenosyl-5)tetraphosphate and bis(adenosyl-5)pentaphosphate act as partial agonists of ATP receptors at saturating concentration and activate ionic currents of almost one order of magnitude below the amplitude of peak ATP-activated currents. Combined application of these two substances to the neuron produces a considerable decline in the amplitude of ATP-activated ionic currents, competing with ATP for common binding sites.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 4, pp. 427–431, July–August, 1988.