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PubMed1994

Farnesyl-diphosphate synthase is localized in peroxisomes.

In this study, we have investigated the subcellular localization of farnesyl-diphosphate synthase (FPP synthase). FPP synthase produces FPP, which is utilized in the synthesis of squalene, cholesterol, farnesylated and geranylgeranylated proteins, dolichols, coenzyme Q, and the isoprenoid moiety of heme a. This enzyme is found in the 100,000 x g supernatant fraction of cells or tissues and has been considered to be a cytoplasmic protein. In this study, analysis of FPP synthase activity and protein in fractionated rat liver together with immunofluorescent and immunoelectron microscopy studies demonstrated unequivocally that FPP synthase is largely localized in peroxisomes. These data, in combination with the previous observation that mevalonate kinase is predominantly localized in peroxisomes, suggest that peroxisomes are the major site of synthesis of FPP from mevalonate. We also demonstrate that in liver tissue obtained from patients with peroxisomal deficiency diseases (Zellweger syndrome and neonatal adrenoleukodystrophy), the activities of five enzymes involved in isoprenoid synthesis, namely mevalonate kinase, phosphomevalonate kinase, mevalonate-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, and FPP synthase, are significantly reduced, consistent with a peroxisomal localization of these enzymes.

Adrenoleukodystrophy↗
PubMed1993

Distribution of prenyltransferases in rat tissues. Evidence for a cytosolic all-trans-geranylgeranyl diphosphate synthase.

The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

[The effect of fructose-1,6-diphosphate, cytochrome c and their combination on the size of the necrotic area in transient myocardial ischemia].

The effect of fructose-1, 6-diphosphate (200 mg/kg), cytochrome C (20 mg/kg) and their combinations on the size of the zone of necrosis 4 and 72 h after 15-min transitory myocardial ischemia was studied. Combination of the compounds under study inhibited considerably the development of post-occlusion and reperfusion arrhythmias and reduced the size of necrosis 4 h after their administration as prophylactic and arresting measures (to 23.8 +/- 2.9 and 29.3 +/- 3.6% of the ischemic zone, respectively, in 42.8 +/- 3.8% in the control). A combined course of cytochrome C and fructose diphosphate also limited the size of the necrotic zone 72 h after transitory ischemia. Separate single administration of fructose diphosphate and cytochrome C caused no essential changes in the size of myocardial necrosis recorded 72 h after transitory myocardial ischemia.

Animals↗
PubMed1976

[Chromatographic investigation of the substrate properties of 8-bromo-ATP in the nucleoside diphosphate kinase reaction (author's transl)].

8-bromo-ATP is a substrate of nucleoside diphosphate kinase. Its reactivity is similar to that of ATP, and the reaction appears to be unaffected by the different conformations of these two substrates. The nucleoside diphosphate kinase reaction with 8-bromo-ATP cannot be monitored photometrically (optical test) using an NADH/NAD+-dependent enzyme system, because the product 8-bromo-ADP is not a substrate for pyruvate kinase. Measurements of the nucleoside diphosphate kinase reaction with 8-bromo-ATP therefore, were carried out by chromatographic analysis of the nucleoside-triphosphate/diphosphate ratio.

Adenosine Triphosphate↗
PubMed1995

Nm23/nucleoside diphosphate kinase: toward a structural and biochemical understanding of its biological functions.

The nm23 gene, a putative metastasis suppressor gene, was originally identified by its reduced expression in highly metastatic K-1735 murine melanoma cell lines, as compared to related, low metastatic melanoma cell lines. Transfection of nm23 cDNA has been reported to suppress malignant progression in Drosophila and mammalian cells. Highly conserved homologues of nm23 have been found in organisms ranging from the prokaryote Myxococcus xanthus to Drosophila, where the gene is involved in normal development and differentiation. The product of the nm23 gene exhibits a nucleoside diphosphate kinase activity, yet the nucleoside diphosphate kinase activity of Nm23 does not correlate with its apparent biological functions. We review recent cellular, genetic, biochemical and X-ray crystallographic data to formulate and evaluate hypotheses concerning the molecular mechanism of nm23 action.

Animals↗
PubMed1995

Effect of glucose and galactose loading in normal subjects on red and white blood cell uridine diphosphate sugars.

In order to test the assumption that red blood cell (RBC) uridine diphosphate galactose content is regulated in part by dietary galactose and to help comprehend factors influencing RBC and white blood cell (WBC) uridine diphosphate hexose concentrations in vivo, oral loading studies were performed with 50 g of galactose or 75 g of glucose in normal adults. While elevations of blood glucose did not influence RBC or WBC UDPhexose levels, increased blood galactose concentrations caused transient increases in both RBC and WBC UDPgalactose. In both RBC and WBC, the UDPgalactose-4-epimerase was rate limiting. In comparison to RBC, WBC had larger changes in UDPgalactose levels, synthesized additional UDPglucose through the action of UDPglucose pyrophosphorylase and began to restore the equilibrium ratio between UDPglucose and UDPgalactose concentrations while the plasma galactose level was still increasing. Thus, galactose ingestion alters the steady-state levels of UDPhexoses in circulating cells. The modulation of UDPglucose and UDPgalactose concentrations, occurring after the presentation of galactose to cells, as a result of the combined actions of GALT and UDPgalactose-4-epimerase, may be important in determining rates of synthesis of complex glycoconjugates.

Administration, Oral↗
PubMed1995

The 1.9 A crystal structure of a nucleoside diphosphate kinase complex with adenosine 3',5'-cyclic monophosphate: evidence for competitive inhibition.

The X-ray structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase complexed with adenosine 3',5'-cyclic monophosphate (cAMP) has been determined. The structure was solved by difference Fourier analysis. The refined structure has a crystallographic R-factor of 0.17 at 1.9 A resolution. The phosphoryl group and ribose moiety make extensive polar interactions with the protein, whereas the base interacts only with two hydrophobic residues. The comparison with the structure of the enzyme complex with the substrate adenosine diphosphate (ADP) reported earlier shows that cAMP and ADP interact similarly with the enzyme. The base of the cAMP is present in two conformations, syn and anti, with respect to the sugar. The syn conformer is dominant. Based on the effect of cAMP on phosphorylation of the human NDP kinase NM23, it had been proposed that cAMP might interact with NDP kinase in a manner distinct from other nucleotides. However, the structure of the M. xanthus NDP kinase/cAMP complex indicates that the nucleotide is a competitive inhibitor of the enzyme and occupies the usual nucleotide site. Kinetic assays of the NDP kinase activity in the presence of cAMP were done. Their results are consistent with a competitive character of the cAMP inhibition.

Adenosine Diphosphate↗
PubMed1986

Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone.

The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (IC50 values) range from 2 to 13 microM. Four of the five derivatives also were inhibitors of the host cell reductase (IC50 values = 7-34 microM). A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme (IC50 = 340 microM). Four of six compounds were preferential inhibitors of the viral reductase based on a comparison of IC50 values (5- to greater than 85-fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs.

Antiviral Agents↗
PubMed1986

Binding of ATP to nucleoside-diphosphate kinase: a kinetic study.

The binding of nucleotides to pig heart nucleoside-diphosphate kinase was studied using Rose Bengal as an optical probe. ATP, in the absence of Mg2+, binds slowly to the enzyme, with a second order rate constant of about 3000 M-1 . s-1, whereas in its presence the binding is much faster. This finding suggests the regulation of the nucleoside-diphosphate kinase activity by uncomplexed ATP, and that ATP binds normally to the enzyme via a metal ion bridge.

Adenosine Diphosphate↗
PubMed1980

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes.

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.

Adenosine Diphosphate↗
PubMed2003

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases.

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.

Adenosine Diphosphate↗
PubMed2002

Extracellular nucleoside diphosphate kinase NM23/NDPK modulates normal hematopoietic differentiation.

OBJECTIVE: We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibit differentiation of certain hematopoietic cell lines. We further investigated the extracellular effect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3 proteins to in vitro differentiation assays of normal human hematopoietic progenitors. MATERIALS AND METHODS: To study the effect on the earlier stages of hematopoietic maturation, NM23 was added to serum-free pre-colony-forming unit (pre-CFU) assays starting from immature CD34++CD38- bone marrow cells. Serum-free CFU assays starting from CD34+ CD38+ bone marrow cells were used as a model for terminal hematopoietic differentiation. RESULTS: In pre-CFU assays, none of the NM23 isoforms used significantly changed the expansion of CD34++CD38- cells, nor did NM23 alter the CD34++ CD38- cell lineage commitment. In contrast, terminal differentiation of CD34+CD38+ progenitor cells in CFU assays was significantly altered by addition of NM23 protein. More erythroid burst-forming units and fewer macrophage colonies were observed in cultures containing any of the NM23 isoforms examined. Similar effects were observed using the enzymatically inactive H118N mutant of NM23-H1, strongly suggesting that the observed effect is independent of the nucleoside diphosphate kinase activity of NM23. CONCLUSION: We demonstrated a modulating effect of extracellular NM23 proteins on the terminal stages of normal hematopoietic differentiation. Therefore, the fairly high concentrations of NM23 constitutively present in plasma could have a physiologic role in supporting erythropoiesis and inhibiting excessive macrophage formation.

Antigens, CD34↗
PubMed1997

Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis.

Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates. In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes. In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated. Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned. Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes. With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes. The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme. The Xenopus genes share 82-87% identity with their human counterparts. Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart. X1 also binds to a single-stranded (CT)(n) dinucleotide repeat. The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes. We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position. This could provide an autoregulatory translation mechanism. A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

Amino Acid Sequence↗
PubMed2001

Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling.

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.

Alleles↗
PubMed1984

Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin.

We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Cell Fractionation↗
PubMed1986

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis.

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.

Animals↗
PubMed1979

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.

Cell Nucleus↗
PubMed2001

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.

Adenosine Diphosphate↗