Autosomal recessive hypotrichosis with loose anagen hairs associated with TKFC mutations.

BACKGROUND: Loose anagen hair is a rare form of impaired hair anchorage in which anagen hairs that lack inner and outer root sheaths can be gently and painlessly plucked from the scalp. This condition usually occurs in children and is often self-limiting. A genetic basis for the disorder has been suggested but not proven. A better understanding the aetiology of loose anagen hair may improve prevention and treatment strategies. OBJECTIVES: To identify a possible genetic basis of loose anagen hair using next-generation DNA sequencing and functional analysis of variants identified. METHODS: In this case study, whole-exome sequencing analysis of a pedigree with one affected individual with features of loose anagen hair was performed. RESULTS: The patient was found to be compound heterozygous for two single-nucleotide substitutions in TKFC resulting in the following missense mutations: c.574G> C (p.Gly192Arg) and c.682C> T (p.Arg228Trp). Structural analysis of human TKFC showed that both mutations are located near the active site cavity. Kinetic assays of recombinant proteins bearing either of these amino acid substitutions showed almost no dihydroxyacetone kinase or D-glyceraldehyde kinase activity, and FMN cyclase activity reduced to just 10% of wildtype catalytic activity. CONCLUSIONS: TKFC missense mutations may predispose to the development of loose anagen hairs. Identification of this new biochemical pathobiology expands the metabolic and genetic basis of hypotrichosis.

Purification, characterization, and substrate and inhibitor structure-activity studies of rat liver FAD-AMP lyase (cyclizing): preference for FAD and specificity for splitting ribonucleoside diphosphate-X into ribonucleotide and a five-atom cyclic phosphodiester of X, either a monocyclic compound or a cis-bicyclic phosphodiester-pyranose fusion.

An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4',5'-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881-888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes affinity for ADP-agarose (adsorption required the activating cation Mn2+ and desorption required its removal), to a final activity of 2.2 units/mg after a 240-fold purification with a 15% yield. By SDS-PAGE, only one protein band was observed (Mr = 59 000). The correspondence between protein and enzyme activity was demonstrated by renaturation after SDS-PAGE, by gradient ultracentrifugation followed by analytical SDS-PAGE, and by native PAGE with visualization of enzyme activity by fluorescence. A native Mr of 100 000 (ultracentrifugation) or 140 000 (gel filtration) indicated that FAD-AMP lyase could be a dimer. The enzyme required millimolar concentrations of Mn2+ or Co2+, exhibited different optimum pH values with these cations (pH 8.5 or 7.3, respectively), and was strongly inhibited by ADP or ATP, but not by dADP, dATP, or the reaction products AMP and cFMN. A specificity study was conducted with 35 compounds related to FAD, mostly nucleoside diphosphate-X (NDP-X) derivatives. Besides FAD, the enzyme split 11 of these compounds with the pattern NDP-X --> NMP + P=X. Structure-activity correlations of substrates, nonsubstrates, and inhibitors, and the comparison of the enzymic reactivities of NDP-X compounds with their susceptibilities to metal-dependent chemical degradation, pinpointed the following specificity pattern. FAD-AMP lyase splits ribonucleoside diphosphate-X compounds in which X is an acyclic or cyclic monosaccharide or derivative bearing an X-OH group that is able to attack internally the proximal phosphorus with the geometry necessary to form a P=X product, either a five-atom monocyclic phosphodiester or a cis-bicyclic phosphodiester-pyranose fusion. For instance, NDP-glucose and GDP-alpha-L-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP-beta-L-fucose were not. Judging from kcat/Km ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (kcat/Km only 2-fold lower, but not a physiological compound in mammals), whereas other substrates exhibited 50-500-fold lower kcat/Km values. However, there was no evidence for specific flavin recognition. Instead, what seems to be recognized is the NDP moiety of NDP-X, with a strong preference for ADP-X. Splitting would then depend on the presence of an adequate X-OH group. The possibility that, besides FAD, there could be in mammals other ADP-X substrates of FAD-AMP lyase is discussed, with emphasis placed on some ADP-ribose derivatives.

Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase.

Free ADP-ribose has a reducing ribose moiety and it is hazardous due to its nonenzymic reactivity toward protein side chains. ADP-ribose hydrolases are putative protective agents to avoid the intracellular accumulation of ADP-ribose. In mammalian sources, two types of enzymes with ADP-ribose hydrolase activity are known: (i) highly specific ADP-ribose pyrophosphatases, which in a Mg(2+)-dependent fashion hydrolyse only ADP-ribose and the nonphysiological analogue IDP-ribose, and (ii) less specific nucleoside diphosphosugar or diphosphoalcohol (NDP-X) pyrophosphatases, which besides A(I)DP-ribose hydrolyse also some nonreducing NDP-X substrates. So far, of these two enzyme types only the less specific one has been reported in human sources: an ADP-sugar pyrophosphatase purified from erythrocytes or expressed from cDNA clones. Here we report that human placenta extracts contain two ADP-ribose hydrolases, which were characterised after a near 1000-fold purification. One is an ADP-sugar pyrophosphatase: it hydrolysed ADP-ribose, ADP-glucose and ADP-mannose, but not e.g. UDP-glucose, at similar rates. It resembles the erythrocyte and recombinant enzyme(s), but showed a 5-20-fold lower K(m) for ADP-ribose (7 microM). The other enzyme is a highly specific ADP-ribose pyrophosphatase (the first of this kind to be reported in humans): it hydrolysed only ADP-ribose and IDP-ribose at similar rates, with a very low, 0.4 microM K(m) for the former. This is a major candidate to control the accumulation of free ADP-ribose in humans. It remains to be seen whether it belongs to the 'nudix' protein family, which includes several ADP-ribose hydrolases and other 'housecleaning' enzymes (M.J. Bessman, D.N. Frick, S.F. O'Handley, J. Biol. Chem. 271 (1996) 25059-25062).

Rat liver nucleotide pyrophosphatase/phosphodiesterase is an efficient adenylyl transferase.

Rat liver nucleotide pyrophosphatase/phosphodiesterase I (NPP/PDE) catalysed efficiently the transfer of adenylate from ATP to alcohols (methanol, ethanol, propanol, ethylene glycol, glycerol, 2, 2-dichloroethanol and glycerol 2-phosphate), which acted as adenylate acceptors competing with water with different efficiencies. NPP/PDE kinetics in alcohol/water mixtures were accounted for by rate equations for competitive substrates, modified to include alcohol negative co-operativity and, depending on the nature of the alcohol, enzyme denaturation by high alcohol concentrations or activation by low alcohol concentrations. The correlation of alcohol efficiencies with alcohol acidities, the comparison of rat liver with snake venom NPP/PDE, and the different effects of ionic additives on the efficiencies of glycerol 2-phosphate and glycerol provided evidence for interaction of the alcohols with a base catalyst, a non-polar and a cationic subsite in the active centre of rat liver NPP/PDE. The enzyme thus appears to be well suited to act as transferase, and we propose that NPP/PDE could be an adenylylating agent in the membrane.

ADP-ribose pyrophosphatase-I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol.

Free ADP-ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low-Km ADP-ribose pyrophosphatase-I (ADPRibase-I) is well suited to control free ADP-ribose and nonenzymatic ADP-ribosylation. In vitro, the acetaminophen metabolite N-acetyl-p-benzoquinoneimine (NAPQI) decreases ADPRibase-I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase-I in vivo. Rats pretreated with 3-methylcholanthrene and L-buthionine-[S,R]-sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/ kg; n = 5) or vehicle (n = 3). ADPRibase-I partially purified from acetaminophen-overdosed rats showed a decreased Vmax (0.32+/-0.09 versus 0.60+/-0.03 mU/mg of liver protein; p<0.01) not reverted by DTT and an increased Km for ADP-ribose (1.39+/-0.31 versus 0.67+/-0.05 microM; p<0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase-I from normal rat liver with oxidized glutathione elicited a time- and dose-dependent, DTT-reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase-I can be regulated by thiol exchange and that the increase of Km, elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP-ribose concomitant to ADPRibase-I inhibition could contribute to the hepatotoxicity of acetaminophen.

Enzymic formation of riboflavin 4',5'-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver1.

An enzyme activity splitting FAD to AMP and riboflavin 4',5'-cyclic phosphate (4',5'-cFMN), with a Km of 6-8 microM, was partially purified from the cytosolic fraction of rat liver homogenates. 4', 5'-cFMN was characterized by enzyme, HPLC, UV-visible and NMR spectroscopic analyses. The data suggest that a novel enzyme, tentatively named FAD-AMP lyase (cyclizing) or FMN cyclase, is involved. Also, 4',5'-cFMN was hydrolysed to 5'-FMN by a rat liver cyclic phosphodiesterase. The results indicate a novel enzymic pathway for flavins in mammals, and support the biological relevance of 4',5'-cFMN, perhaps as a flavocoenzyme or a regulatory signal.

Glycine-enhanced inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase-I by EDTA: a full account of the reported inhibition by commercial preparations of acidic fibroblast growth factor (FGF-1).

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture-grade acidic fibroblast growth factor (FGF-1) correlates with a low-Mr contaminant. 1H-NMR analyses revealed EDTA in the total-volume fractions of a gel-filtration experiment, where all the inhibitory activity of the FGF-1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF-1 or the EDTA-containing fractions) was time-dependent, blocked by the substrate p-nitrophenyl-dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF-1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Use of potato tuber nucleotide pyrophosphatase to synthesize adenosine 5'-monophosphate methyl ester: evidence that the solvolytic preferences of the enzyme are regulated by pH and temperature.

Nucleotide alkyl esters are pharmacologically important as potential (ant)agonists of purinoceptors and inhibitors of enzymes. Potato nucleotide pyrophosphatase (PNP) was compared with snake venom phosphodiesterase (SVP) as a catalyst to synthesize nucleotide alkyl esters. In methanol-water mixtures, the methanolysis/hydrolysis ratio of PNP, but not SVP, changed with pH and temperature, being optimal at high pH and low temperature. In a semi-preparative experiment, a crude PNP preparation produced 0.17 mM AMP-O-methyl ester (AMP-OMe) from 1 mM diadenosine 5',5"'-P1,P2-diphosphate (AppA) and 5M methanol, at pH 9 and 0 degrees C. Drawbacks to large-scale use are: low rates inherent to low temperatures, ATP unsuitability as a substrate for alcoholysis, and high cost of AppA. Advantages of PNP vs. SVP are cheapness, non-toxicity, and availability of the enzyme source.

Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine.

N-acetyl-p-benzoquinoneimine (NAPQI) is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-dependent alterations of ADPRibase-I leading to strong inhibition: a fast Km increase produced by low concentrations, and a time-dependent Vmax decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to acetaminophen toxicity.

Identification of rat liver glucose-3-phosphatase as an inositol monophosphatase inhibited by lithium.

Glucose-3-phosphatase (Glc3Pase) from rat liver has been purified 780-fold with a 4% recovery. The substrate specificity of the purified enzyme agreed with that of inositol monophosphatase (EC 3.1.3.25). D-Glucose 3-phosphate (D-Glc(3)P; K(m) = 200 microM) was hydrolyzed with an efficiency similar to DL-myo-inositol 1-monophosphate (DL-Ins(1)P; K(m) = 80 microM), since the ratio V(max)/K(m) was similar for both substrates. Purification data, coelution of activities, thermal inactivation curves, optimal pH, bivalent cation requirements, inhibition by Li+, molecular weight, and isoelectric pH comparisons supported that the hydrolysis of D-Glc(3)P and DL-Ins(1)P was catalyzed by a unique phosphohydrolase identified as a hepatic form of the lithium-sensitive inositol monophosphatase. That the hydrolysis of D-Glc(3)P is a genuine feature of inositol monophosphatases was confirmed because the enzyme purified from bovine brain showed also Glc3Pase activity, and inspection of published 3D models of inositol monophosphatase complexes with D(L)-Ins(1)P or D(L)-Ins(4)P indicated that beta(alpha)-D-Glc(3)P in a pyranose conformation with all (but one) the hydroxy groups in equatorial orientation would fit in the active site as other good substrates do. The results of this work are suggestive of possible relationships between inositol and sugar 3-phosphate metabolism.

Specific ADP-ribose pyrophosphatase from Artemia cysts and rat liver: effects of nitroprusside, fluoride and ionic strength.

One specific ADP-ribose pyrophosphatase (ADPRibase) has been identified in Artemia cysts, following a protocol that in rat liver allows the identification of three ADPRibases. Artemia ADPRibase resulted similar, but not identical, to rat liver ADPRibase-I with respect to known and novel properties disclosed in this work. In the presence of Mg2+, Artemia ADPRibase was highly specific for ADP-ribose and showed a low, 0.7 microM Km. Preincubation with the nitric oxide donor nitroprusside and dithiothreitol, elicited dose- and time-dependent, severalfold increase of Km and decrease of Vmax. At saturating ADP-ribose concentrations, fluoride was a strong inhibitor (IC50 approximately equal to 10-20 microM), whereas bringing ionic strength to 0.3-1.3 mol/l doubled the activity measured at lower or higher strengths. The novel fluoride and ionic strength effects were studied also with rat liver ADPRibase-I. Differences between the Artemia enzyme and ADPRibase-I concerned molecular weight (31,000 versus 38,500, respectively), Mn2+ ability to substitute for Mg2+ as the activating cation (better for the rat enzyme), and Vmax decrease by nitroprusside (not seen with the rat enzyme). The results are discussed in relation with the role of specific ADPRibases as protective factors limiting free ADP-ribose accumulation and protein glycation, and as targets for cytotoxic agents.

High efficiency of glycerol 2-phosphate and sn-glycerol 3-phosphate as nucleotidyl acceptors in snake venom phosphodiesterase esterifications. Formation of primary and secondary AMP-O-glyceryl and AMP-O-glycerophosphoryl esters and evidence for an acceptor-binding enzyme site.

Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP by primary R-CH2OH alcohols with uncharged R residues, yielding AMP-O-CH2R esterification products. The alcohols compete with water for an SVP-bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2-phosphate and sn-glycerol 3-phosphate with ATP to yield AMP-O-glycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP-O-glyceryl esters by alkaline phosphatase. The results demonstrated that R-CH2OH alcohols with negatively charged R residues, as well as secondary alcohols, act as adenylyl acceptors in SVP reactions, thus extending the usefulness of SVP as a tool to produce 5'-nucleotide derivatives. The efficiencies (EA) of glycerol phosphates as adenylyl acceptors were very high at low, millimolar concentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2-phosphate, when Pi or NaCl was present. In contrast, glycerol EA was independent of its own concentration, Pi, and NaCl. The responses of glycerol phosphates indicate that they act as adenylyl acceptors via a mechanism different from uncharged R-CH2OH alcohols. The occurrence of an acceptor-binding enzyme site, specific for negatively charged R residues, and its potential relevance to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucleotidyl transferases are discussed.

Inhibition and ADP-ribose pyrophosphatase-I by nitric-oxide-generating systems: a mechanism linking nitric oxide to processes dependent on free ADP-ribose.

Rat liver ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes ADP-ribose with high specificity and a low Km. Thus it can participate in the control of free ADP-ribose and nonenzymatic ADP-ribosylation of proteins. Here we show that ADPRibase-I was inactivated by acidified nitrite, whereas sodium nitroprusside (SNP) or 3-morpholinosydnonimine (SIN-1) at pH 7.5 produced a dose- and time-dependent Km increase from 0.5 microM to 2 microM. The effects of SNP and SIN-1 depended on the presence and concentration of dithiothreitol, pointing to S-nitrosylation of enzyme thiols. It is suggested that, by inhibiting ADPRibase-I, NO can stimulate nonenzymatic ADP-ribosylation of targets susceptible to micromolar free ADP-ribose. This is discussed in relation to apparently contradictory earlier reports on the role of NO in the ADP-ribosylation of actin.

Rat liver nucleoside diphosphosugar or diphosphoalcohol pyrophosphatases different from nucleotide pyrophosphatase or phosphodiesterase I: substrate specificities of Mg(2+)-and/or Mn(2+)-dependent hydrolases acting on ADP-ribose.

Three rat liver nucleotides(5') diphosphosugar (NDP-sugar) or nucleoside(5') diphosphoalcohol pyrophosphatases are described: two were previously identified in experiments measuring Mg(2+)-dependent ADP-ribose pyrophosphatase activity (Miró et al. (1989) FEBS Lett. 244, 123-126), and the other is a new, Mn(2+)-dependent ADP-ribose pyrophosphatase. They are resolved by ion-exchange chromatography, and differ by their substrate and cation specificities, KM values for ADP-ribose, pH-activity profiles, molecular weights and isoelectric points. The enzymes were tested for activity towards: reducing (ADP-ribose, IDP-ribose) and non-reducing NDP-sugars (ADP-glucose, ADP-mannose, GDP-mannose, UDP-mannose, UDP-glucose, UDP-xylose, CDP-glucose), CDP-alcohols (CDP-glycerol, CDP-ethanolamine, CDP-choline), dinucleotides (diadenosine pyrophosphate, NADH, NAD+, FAD), nucleoside(5') mono- and diphosphates (AMP, CMP, GMP, ADP, CDP) and dTMP p-nitrophenyl ester. Since the enzymes have not been purified to homogeneity, more than three pyrophosphatases may be present, but the co-purification of activities, thermal co-inactivation, and inhibition experiments give support to: (i) and ADP-ribose pyrophosphatase highly specific for ADP(IDP)-ribose in the presence of Mg2+, but active also on non-reducing ADP-hexoses and dinucleotides (not on NAD+) when Mg2+ was replaced with Mn2+; (ii) a Mn(2+)-dependent pyrophosphatase active on ADP(IDP)-ribose, dinucleotides and CDP-alcohols; (iii) a rather unspecific pyrophosphatase that, with Mg2+, was active on AMP(IMP)-containing NDP-sugars and dinucleotides (not on NAD+), and with Mn2+, was also active on non-adenine NDP-sugars and CDP-alcohols. The enzymes differ from nucleotide pyrophosphatase/phosphodiesterase-I (NPPase/PDEaseI) by their substrate specificities and by their cytosolic location and solubility in the absence of detergents. Although NPPase/PDEaseI is much more active in rat liver, its known location in the non-cytoplasmic sides of plasma and endoplasmic reticulum membranes, together with the known cytoplasmic synthesis of NDP-sugars and CDP-alcohols, permit the speculation that the pyrophosphatases studied in this work may have a cellular role.

Rat liver mitochondrial ADP-ribose pyrophosphatase in the matrix space with low Km for free ADP-ribose.

A study involving markers of subcellular and submitochondrial fractions, gradient centrifugation, latency measurements and extraction with digitonin, demonstrates the association of a specific ADP-ribose pyrophosphatase with rat liver mitochondria and its localization in the matrix space. The enzyme hydrolyses ADP-ribose to AMP, with a Km of 2-3 microM. The results support the occurrence of a specific turnover pathway for free ADP-ribose and its relevance in mitochondria.

Detection of specific glucose-3-phosphatase activity in rat liver.

Sugar-3-phosphates are related to aspects of diabetes which depend on protein glycosylation events. Sorbitol-3-phosphate and fructose-3-phosphate occur in normal and diabetic individuals, and glucose-3-phosphate is a potential intermediate in their biosynthesis. Almost nothing is known about enzyme pathways for their metabolic turnover. We have found that part of the phosphohydrolytic activity on glucose-3-phosphate in rat liver supernatants corresponds to a specific, Mg(2+)-dependent, glucose-3-phosphatase much less or not active on other phosphate esters, including glucose-1-phosphate, glucose-6-phosphate, fructose-1-phosphate, fructose-6-phosphate and p-nitrophenyl-phosphate. This finding opens a route to a better understanding of the metabolism and role of sugar-3-phosphates.

Alcohol esterification reactions and mechanisms of snake venom 5'-nucleotide phosphodiesterase.

In a previous study we have shown that snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes methanol-esterification reactions [García-Díaz, M., Avalos, M. & Cameselle, J. C. (1991) Eur. J. Biochem. 196, 451-457]. Now we have demonstrated that SVP catalyzes AMP transfer from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2-chloroethanol or 2,2-dichloroethanol. The AMP-O-alkyl ester products were identified by HPLC, enzyme analysis, ultraviolet and NMR spectroscopy. Those results show the potential of SVP as a tool to prepare 5'-nucleotide esters and agree with the formation of a covalent 5'-nucleotidyl-SVP intermediate susceptible to nucleophilic attack by short-chain (poly)alcohols as acceptors alternative to water. To test the kinetic influence of the solvent nucleophile in SVP mechanisms, initial rates of ATP solvolysis were assayed in different water/alcohol mixtures. Relatively high alcohol concentrations inactivated SVP but lower concentrations gave proportional rates of alcoholysis. An efficiency parameter (EA), defined as the ratio of the mole fraction of AMP-O-alkyl ester as a product to that of alcohol as an acceptor in water/alcohol mixtures, made possible the comparison of alcohols and water as AMP acceptors at low concentrations, as it could be reasoned that EA = 1 for water. Rates of hydrolysis (VH) of substrates yielding AMP and different leaving groups were also assayed. The higher EA and VH values corresponded, respectively, to those acceptors and leaving-group conjugate acids with lower pKa and higher polar-substituent constants (sigma*). The results support the occurrence of general acid-base catalysis in the active center of SVP and the identification of rate-limiting steps. A model is proposed for the mechanisms of SVP-catalyzed hydrolysis and alcoholysis which accounts for the influence of the acid-base properties of alcohols on the kinetic profile of SVP reaction sequences.

Dinucleoside tetraphosphatase from human blood cells. Purification and characterization as a high specific activity enzyme recognized by an anti-rat tetraphosphatase antibody.

Dinucleoside tetraphosphatase (Np4Nase; EC 3.6.1.17) has been purified 170,000-fold from a 30-60% ammonium sulfate fraction of a human blood cell extract. Purification included a dye-ligand affinity elution step using the inhibitor adenosine 5'-tetraphosphate. Human blood Np4Nase resembled rat liver Np4Nase, including recognition by anti-rat Np4Nase, but differed from homogeneous human leukemia Np4Nase in the 1000-fold lower specific activity of the latter. The results are discussed in relation to the potential role of diadenosine tetraphosphate (Ap4A) in the control of cell division and the turnover of Ap4A in blood.

Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria.

The submitochondrial location of dinucleoside triphosphatase (EC 3.6.1.29), previously shown to be in part associated with mitochondria, has been studied in rat liver. The precipitability and latency of activity in organelle suspensions, and the profile of solubilization by digitonin, were like those of the matrix space marker glutamate dehydrogenase, and differed from those of other submitochondrial fractions. This, and the synthesis of diadenosine polyphosphates by mitochondrial aminoacyl-tRNA synthetases, suggest the occurrence of a pathway for the intramitochondrial turnover of diadenosine 5',5'''-P1,P3-triphosphate (Ap3A).