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PubMed2000

Enzymatic synthesis of methylbutenol from dimethylallyl diphosphate in needles of Pinus sabiniana.

Methylbutenol (2-methyl-3-buten-2-ol) is an abundant volatile organic compound released from Western U.S. pines. To understand the mechanism of methylbutenol formation, we developed a sensitive gas chromatographic assay for its detection and determined that needles of gray pine (Pinus sabiniana) contain an enzyme that catalyzes the synthesis of methylbutenol from dimethylallyl diphosphate (DMAPP). The methylbutenol synthase activity was partially purified; its pH optimum was 7-8, and, like other prenyl diphosphate utilizing enzymes, it was dependent on the presence of a divalent cation, preferably Mn2+. The enzyme also required K+ or NH4+ for activity. The Km values for DMAPP and Mn2+ were about 4.8 and 6 mM, respectively. Geranyl diphosphate was not a substrate for the enzyme, so it is distinct from linalool synthase, a plant enzyme that catalyzes an analogous reaction. The methylbutenol synthase reaction may be responsible for the majority of light-dependent methylbutenol production by many pine species in the Western United States.

Butanols↗

PubMed2001

Reduction of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal Down syndrome brain.

Information on the various factors leading to impairments in the developing brain of fetal Down Syndrome patients is limited to few histological reports. We therefore attempted to describe expression levels of proteins in brain using the proteomic technique of two-dimensional electrophoresis with subsequent mass spectroscopical identification of protein spots and quantification with specific software. Cortical tissue was obtained from autopsy of human fetal abortus. Protein levels of GTP-binding nuclear protein ran, guanine nucleotide-binding protein g(o), alpha subunit 2, guanine nucleotide-binding protein g(i)/g(s)/g(t) beta subunit 1, -beta subunit 2, guanine nucleotide-binding protein beta subunit 5, nucleoside diphosphate kinase A, nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta, Rho GDP-dissociation inhibitor 1, biphosphate 3'-nucleotidase, small glutamine-rich tetra-tricopeptide repeat-containing protein and histidine triad nucleotide-binding protein were studied. Quantification revealed statistically significant reduced levels of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal DS brain as compared to controls. We conclude that in early prenatal life proteins involved in neural differentiation, migration and synaptic transmission are impaired in DS cortex. These results may help to understand the abundant mechanisms leading to abnormalities in the wiring, structure and function of DS brain.

Brain↗

PubMed1983

Further observations in a case of uridine diphosphate galactose-4-epimerase deficiency with a severe clinical presentation.

The red-cell concentrations of galactose-1-phosphate and uridine diphosphate galactose have been studied in relation to dietary galactose in a case of uridine diphosphate galactose-4-epimerase deficiency (McKusick 23035). Uridine diphosphate galactose accumulates rapidly in response to very small amounts of galactose but the concentration of galactose-1-phosphate increases proportionately to galactose intake. The significance of the observation is discussed with respect to the pathogenesis and treatment of the disease.

Biotransformation↗

PubMed2004

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides.

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.

Alkyl and Aryl Transferases↗

PubMed1998

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization.

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer gamma-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.

Amino Acid Sequence↗

PubMed1978

IV. Synthesis and assay of analogs of adenosine 3',5'-diphosphate as inhibitors of bovine adrenal estrogen sulfotranferase.

Analogs of adenosine 3',5'-diphosphate are described which aid in the characterization of the inhibition of estrone sulfurylation by 3'-phosphoadenosine 5'-phosphosulfate as mediated by bovine adrenal estrogen sulfotransferase (3'-5'-phosphosulfate:estrone 3-sulphotransferase, EC 2.8.2.4). The facile conversion of ribonucleosides to 2',3'-cyclic phosphate 5'-phosphate in neat pyrophosphoryl chloride is utilized to provide a reliable route to the requisite intermediates for enzymatic regiospecific conversion to ribonucleoside 3',5'- and 2',5'-diphosphates. The importance of the 3'-phosphate ester to inhibition of estrone sulfurylation is confirmed by Ki measurements. Replacement of the 6-amino group by hydrogen or oxygen leads to considerable loss in affinity for the enzyme as does also dimethylation of the exocylic amino group. Alterations in the pyrimidine ring are not well tolerated by the sulfotransferase but modifications in the imidazole ring as in tubercidin (7 -deazaadenosine) and 8-bromoadenosine 3',5'-diphosphate lead to an enhanced affinity. The latter findings are discussed in terms of an hypothesis of stacking of the aromatic ring of the estrogen substrate and the purine moiety and its analogs.

Adenine Nucleotides↗

PubMed1989

Formation of a stable and catalytically active complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26.

Formation of a stable complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26, which represents the catalytically active state of this enzyme, is observed in the presence of a relatively high concentrations of inorganic pyrophosphate or one of the substrates, isopentenyl diphosphate or farnesyl diphosphate. The apparent molecular mass of the complex is estimated to be about 50 kDa by gel filtration with Superose 12.

Alkyl and Aryl Transferases↗

PubMed1990

Nucleoside diphosphate kinase from Xenopus oocytes; partial purification and characterization.

We have identified and partially purified a soluble nucleoside diphosphate kinase (NDP kinase) from Xenopus laevis oocytes. The enzyme preparation can catalyze the transfer of phosphate from ATP to all of the major oxy- and deoxynucleotides. It can also catalyze the transfer of a phosphorothioate group from gamma-S-ATP to an acceptor GDP forming gamma-S-GTP. Like NDP kinases from other sources, the catalytic mechanism appears to involve a phosphoenzyme intermediate which can be isolated. Transfer of phosphate from nucleoside triphosphates to protein is rapid, reaching saturation within 1 min following the addition of nucleoside triphosphates. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. While nucleoside diphosphate kinases are generally thought to require magnesium for activity, both the oocyte enzyme preparation and a commercial bovine liver enzyme preparation are only partially inhibited by short (10 min) exposures to 25 mM EDTA. Both enzyme preparations are, however, further inhibited by long incubations with this metal chelator (2 h, 70% inhibition). Zinc enhances the inhibition of NDP kinase by EDTA, but is ineffective on its own. Rapid phosphorylation in the presence of [gamma-32P]ATP and EDTA could be used to identify the phosphoenzyme intermediate in homogenates of Xenopus oocytes and facilitated its isolation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with autoradiography indicated the presence of only a single phosphorylated species of Mr 21,500 in supernatants of fresh oocyte homogenates. Partial purification of this protein utilizing salt precipitation, hydrophobic-interaction chromatography and an affinity step with Affi-Gel Blue Sepharose resulted in a 100-fold purification and a 29% overall yield of NDP-kinase activity. Size-exclusion chromatography of the purified preparation yielded two peaks containing enzyme activity. They eluted with apparent molecular weights of 45,000 and 70,000, suggesting a native enzyme that is multimeric or associated with other proteins.

Adenosine Triphosphate↗

PubMed1978

Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Saccharomyces cerevisiae.

Yeast membranes incorporate radioactivity from GDP[14C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components. The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditions. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol. When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharides is formed besides the previously-described Dol-PP-(GlcNAc)2 mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [14C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.

Cell Membrane↗

PubMed1979

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars. I. Determination of the degradation rate during the glycosyltransferase assays.

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phosphatases; 3, chemical decomposition of UDP-D-[14C]Gal; UDP-D-[14C]Glc and UDP-D-[14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn2+, Mg2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. These results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.

Adult↗

PubMed2005

A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release.

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PPi) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PPi group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (Pi), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PPi release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.

Cloning, Molecular↗

PubMed2006

Juvenile hormone biosynthesis in M. sexta: substrate specificity of insect prenyltransferase utilizing homologous diphosphate analogs.

Analogs of dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) were prepared and tested as potential substrates of prenyltransferase of the tobacco hornworm, Manduca sexta, and of a sesquiterpene synthase derived from pig liver. Enzyme derived from corpora allata homogenates of both the larval and adult stage of M. sexta coupled each of the DMAPP analogs to produce homologous geranyl and farnesyl diphosphate products in the order (Z)-3-ethyl>(Z)-3-n-propyl>(Z)-3-methyl (DMAPP)>(Z)-3-i-propyl(Z)-3-n-butyl. In competition studies, the ethyl and n-propyl analogs either enhanced or had no effect on DMAPP coupling, whereas the larger analogs were inhibitors. (Z)-7-ethyl and (2Z,6Z)-3,7-diethyl analogs of GPP were as good, if not better substrates of larval prenyltransferase, while the C-3 ethyl analog of GPP, which is precursor to an isomeric form of juvenile hormone (JH) that is not typically found in insects, was poorly coupled by the enzyme. While similarities were seen for whole-cell extracts derived from adult and larval M. sexta, adult prenyltransferase derived from cytosolic and 16,000xg pellet fractions displayed distinct competitive coupling of GPP and its homologs, suggesting differences in substrate specificity as a result of enzyme localization. In contrast to M. sexta, the pig liver enzyme poorly coupled each of the homologous DMAPP derivatives, and the homologous derivatives of GPP were less efficiently coupled than GPP. These results indicate that prenyltransferase in M. sexta possesses high steric latitude at the (Z)-C-3 and C-7 alkyl positions of DMAPP and GPP, respectively, in contrast to other animal prenyltransferases but in keeping with the enzyme's presumptive role in homologous JH metabolism.

Animals↗

PubMed2003

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein.

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Diphosphates↗

PubMed1998

Adenosine diphosphate-induced platelet activation inhibited by magnesium in a dose-dependent manner.

OBJECTIVE: To examine the hypothesis that magnesium inhibits platelet activation at concentrations equivalent to therapeutic levels. METHODS: Fifteen subjects were enrolled: five healthy, female donors with regular, spontaneous menstrual cycles; five women with uncomplicated third-trimester pregnancies; and five preeclamptic subjects before magnesium therapy. Anticoagulated whole blood was added to tubes containing 0.5 micromol/L adenosine diphosphate (to activate platelets), HP1-1D (activation-independent platelet antibody), CD62 antibody and CD63 antibody (activation-dependent platelet antibodies), and magnesium sulfate in increasing concentrations (2-100 mg/dL). The percentage of activated platelets (CD62 or CD63 positive) was determined using three-color flow cytometric analysis. Data were analyzed using the Student t test, repeated measures analysis of variance, two-way analysis of variance, and Student-Newman-Keuls for pairwise comparison in appropriate cases. P < .05 was considered significant. RESULTS: Adenosine diphosphate-induced platelet activation was inhibited with increasing magnesium concentration in all subjects (P < .001). Significant inhibition of CD62 and CD63 expression first occurred at a magnesium concentration of 4 mg/dL in the normal pregnant group (P < .05), at 6 mg/dL in the preeclamptic group (P < .05), and at 8 mg/dL in the nonpregnant group (P < .05). CONCLUSION: Magnesium inhibits adenosine diphosphate-induced platelet activation in a dose-dependent manner. This effect initially attains statistical significance at concentrations equivalent to therapeutic levels.

Adenosine Diphosphate↗

PubMed1984

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1990

Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator.

UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5'-diphosphate chloroacetol (UDC) and uridine 5'-diphosphate bromoacetol (UDB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a KD of 0.110 mM and kinact of 0.84 min-1 at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD+. The inactivation of epimerase by uridine 5'-diphosphate [2H2]chloroacetol proceeds with a primary kinetic isotope effect (kH/kD) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD+ at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD+ is proposed to be the chromophore with lambda max at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

Binding Sites↗

PubMed2004

Positively charged side chains in protein farnesyltransferase enhance catalysis by stabilizing the formation of the diphosphate leaving group.

Protein farnesyltransferase (FTase) requires both Zn(2+) and Mg(2+) for efficient catalysis of the formation of a thioether bond between carbon-1 of farnesyldiphosphate (FPP) and the cysteine thiolate contained in the carboxy-terminal CaaX sequence of target proteins. Millimolar concentrations of Mg(2+) accelerate catalysis by as much as 700-fold in FTase. Although FTase lacks a typical DDXXD Mg(2+) binding site found in other enzymes that use Mg(2+) for diphosphate stabilization, D352beta in FTase has been implicated in binding Mg(2+) (Pickett et al. (2003) J. Biol. Chem. 278, 51243). Structural studies demonstrate that the diphosphate (PPi) group of FPP resides in a binding pocket made up of highly positively charged side chains, including residues R291beta and K294beta, prior to formation of an active conformation. Analysis of the Mg(2+) dependence of FTase mutants demonstrates that these positively charged residues decrease the Mg(2+) affinity up to 40-fold. In addition, these residues enhance the farnesylation rate constant by almost 80-fold in the presence of Mg(2+), indicating that these residues are not simply displaced by Mg(2+) during the reaction. Mutations at R291beta increase the pK(a) observed in the magnesium affinity, suggesting that this arginine stabilizes the deprotonated form of the PPi leaving group. Furthermore, binding and catalysis data using farnesylmonophosphate (FMP) as a substrate indicate that the side chains of R291beta and K294beta interact mainly with the beta-phosphate of FPP during the chemical reaction. These results allow refinement of the model of the Mg(2+) binding site and demonstrate that positive charge stabilizes the developing charge on the diphosphate leaving group.

Alanine↗

PubMed1998

Syntheses of photoactive analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol and photoaffinity labeling of poly(ADP-ribose) glycohydrolase.

Two isomeric azidoadenosyl analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J. Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(ADP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 microM, a concentration 80-fold lower than that where the isomeric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [alpha-32P]-8-N3-ADP-HPD was therefore synthesized and used to photoderivatize poly(ADP-ribose) glycohydrolase. Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-32P]-8-N3-ADP-HPD with short-wave UV light resulted in the covalent incorporation of the photoprobe into the protein, as demonstrated by gel electrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occurred in the absence of UV light. The photoincorporation saturated at low concentrations of the photoprobe and photoprotection was observed in the presence of low concentrations of ADP-HPD, an indication of the specificity of the photoinsertion reaction. These results demonstrate that [alpha-32P]-8-N3-ADP-HPD can be used to specifically covalently photoderivatize the enzyme to characterize the polypetides that constitute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. The photoincorporation reaction was further used to determine the ability of ADP-ribose polymers of varying size to compete with [alpha-32P]-8-N3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha-32P]-8-N3-ADP-HPD was inhibited by 80% in the presence of low concentrations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose units in length). No similar photoprotection was afforded by the addition of a high-molecular weight highly branched polymer. These results indicate that the photolabel shares a binding site with the short, linear polymer, but not with the long, highly branched polymer.

Adenosine Diphosphate↗