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PubMed1988

Characterization of human poly(ADP-ribose) polymerase with autoantibodies.

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.

Animals↗
PubMed1999

Hydrolysis of NADP+ by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2'-phosphate.

CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD+ and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP+ by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP+ resulted in the rapid and time-dependent accumulation of ADP-ribose 2'-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2'-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP+ glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2'-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2'-phosphate did not result in the formation of ADP-ribose 2'-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2'-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD+, displays exclusively a NADP+ glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2'-phosphate.

ADP-ribosyl Cyclase↗
PubMed1981

Extinction and expression of the ribose-positive phenotype in hybrid Novikoff hepatoma cells.

Expression of the ribose-positive phenotype was examined in hybrids obtained from the fusion of parental pentose-negative Novikoff hepatoma cells and ribose-positive variants. The two ribose-positive variants used differed phenotypically in their ability to use pentoses other than ribose for growth. One variant used D-ribose, D-xylose, and L-arabinose for growth, while the other variant used only D-ribose. Each variant was fused to pentose-negative parental hepatoma cells, and resultant hybrids were tested for the ability to use ribose. In both instances extinction of ribose utilization was the primary event, suggesting the existence of a trans-acting negative control element in the parental cells. In addition, hybrids from both fusion experiments eventually reexpressed the ribose phenotype. The rate of reexpression, however, was different for the two fusion experiments. Reexpression of ribose utilization in hybrids derived from the nonspecific variant occurred at approximately 10(-3) segregants/cell/day. Reexpressing segregants arose from the specific-derived hybrids at a rate of 0.5 segregants/cell/day. Possible reasons for this difference include a differential rate in chromosomal segregation or a difference in the regulation of ribose metabolism.

Animals↗
PubMed2003

Formation of aroma compounds from ribose and cysteine during the Maillard reaction.

The headspace volatiles produced from a phosphate-buffered solution (pH 5) of cysteine and a 1 + 1 mixture of ribose and [(13)C(5)]ribose, heated at 95 degrees C for 4 h, were examined by headspace SPME in combination with GC-MS. MS data indicated that fragmentation of ribose did not play a significant role in the formation of the sulfur aroma compounds 2-methyl-3-furanthiol, 2-furfurylthiol, and 3-mercapto-2-pentanone in which the carbon skeleton of ribose remained intact. The methylfuran moiety of 2-methyl-3-(methylthio)furan originated from ribose, whereas the methylthio carbon atoms came partly from ribose and partly from cysteine. In 3-mercapto-2-butanone one carbon unit was split from the ribose chain. On the other hand, all carbon atoms in 3-thiophenethiol stemmed from cysteine. In another trial cysteine, 4-hydroxy-5-methyl-3(2H)-furanone and [(13)C(5)]ribose were reacted under the same conditions. The resulting 2-methyl-3-furanthiol was mainly (13)C(5)-labeled, suggesting that it stems from ribose and that 4-hydroxy-5-methyl-3(2H)-furanone is unimportant as an intermediate. Whereas 2-mercapto-3-pentanone was found unlabeled and hence originated from 4-hydroxy-5-methyl-3(2H)-furanone, its isomer 3-mercapto-2-pentanone was formed from both 4-hydroxy-5-methyl-3(2H)-furanone and ribose. A new reaction pathway from ribose via its 1,4-dideoxyosone is proposed, which explains both the formation of 2-methyl-3-furanthiol without 4-hydroxy-5-methyl-3(2H)-furanone as an intermediate and a new way to form 3-mercapto-2-pentanone.

Carbon Isotopes↗
PubMed2004

NAD glycohydrolase activities and ADP-ribose uptake in erythrocytes from normal subjects and cancer patients.

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.

ADP-ribosyl Cyclase↗
PubMed2000

Functional competition between poly(ADP-ribose) polymerase and its 24-kDa apoptotic fragment in DNA repair and transcription.

Poly(ADP-ribose) polymerase is a 113-kDa nuclear enzyme that binds to both damaged DNA and to RNA associated with actively transcribed regions of chromatin. Binding of poly(ADP-ribose) polymerase to DNA lesions activates it, catalyzing the covalent addition of multiple ADP-ribose polymers to the enzyme (automodification). During apoptosis, poly(ADP-ribose) polymerase is cleaved by caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic fragment. The functional relevance of this cleavage is not well understood. We therefore prepared a recombinant 24-kDa poly(ADP-ribose) polymerase fragment and investigated the role of this fragment in DNA repair and transcription. The 24-kDa fragment retained its binding affinity for both DNA breaks and RNA. In an in vitro cell-free DNA repair assay, this fragment inhibited rejoining of DNA breaks and suppressed ADP-ribose polymer formation by competing with poly(ADP-ribose) polymerase in binding to DNA breaks. With regard to transcription, it has recently been demonstrated that binding of poly(ADP-ribose) polymerase to transcribed RNA reduces the rate of transcript elongation and that automodification of poly(ADP-ribose) polymerase bound to DNA breaks results in up-regulation of transcription. We tested the 24-kDa fragment for its ability to suppress transcript elongation, and we found that it competed against the up-regulation of transcription mediated by full-length poly(ADP-ribose) polymerase. The ability of the 24-kDa fragment to inhibit DNA repair, ADP-ribose polymer formation, and damage-dependent up-regulation of transcription may contribute to the apoptotic shift from cell survival to cell death mode.

Apoptosis↗
PubMed2002

Ribose supplementation in maximally exercising Thoroughbreds.

A diverse group of studies, which are equine exclusive, indicate that ribose administered to myocardial and skeletal muscle tissue stimulates ATP production and recovery. This study investigated the effects of ribose supplementation on blood and muscle metabolites and performance in Thoroughbred geldings performing a maximal treadmill standardised exercise test (SET). In Experiment 1, 6 conditioned Thoroughbred geldings performed a baseline SET and horses were assigned to one of 2 experimental treatment groups, placebo or ribose, based on VO2max. The placebo treatment group received 0.07 g glucose/kg bodyweight (bwt) and ribose treatment group received 0.07 g ribose/kg bwt top dressed on the feed twice daily. Following a 2 week treatment period, a second SET was performed. After a one-week washout period, the horses switched treatment groups. Following another 2 week treatment period, a third SET was performed. Blood ammonia-N was lower in the ribose treatment group at 15 min (P = 0.06) and 30 min (P = 0.02) postexercise. Plasma lactic acid was lower in the ribose treatment group at 30 min postexercise (P = 0.07). In Experiment 2, 1 h before a SET, 2 horses received 3 l water (control) and 3 horses 250 g of ribose dissolved in 3 l water (single ribose dose) via a nasogastric tube. Following a 2 week washout period, the horses switched treatment groups and another SET was performed. There were no differences in blood ammonia-N, plasma lactic acid or glucose between treatment groups. No differences in performance were detected between treatment groups in either experiment. In conclusion, the results from Experiment 1 show a trend that daily ribose supplementation may be beneficial during recovery from exercise. However, a single dose of ribose 1 h before exercise revealed no effect on the variables measured. Because moderate to intense daily exercise can cause a decrease in total adenine nucleotide (TAN) pool with no meaningful recovery even after 72 h rest, future experiments should be designed to futher elucidate the effects of ribose supplementation on TAN metabolism in horses exercising at high intensity.

Adenosine Triphosphate↗
PubMed1985

Naturally occurring antibodies to poly(ADP-ribose) in autoimmune MRL/Mp-lpr/lpr mice.

The presence of antibodies to poly(ADP-ribose) was demonstrated in the sera of MRL/Mp-lpr/lpr (MRL/l) mice by an enzyme linked immunosorbent assay (ELISA). Antibody to poly(ADP-ribose) was strongly inhibited by single stranded DNA (ssDNA) and poly(ADP-ribose), and less by double stranded DNA (dsDNA). Affinity purified anti-poly(ADP-ribose) antibodies bound more with immobilized ssDNA than with poly(ADP-ribose), and were significantly inhibited by soluble ssDNA, although poly(ADP-ribose) was the best soluble inhibitor. On the contrary, affinity purified anti-ssDNA antibodies bound best with ssDNA and significantly but less with poly(ADP-ribose); however, they were scarcely inhibited by poly(ADP-ribose). These results suggest that similar antigenic determinants exist in poly(ADP-ribose) and ssDNA. It is conceivable, however, at the present moment that 'naturally occurring antibodies to poly(ADP-ribose)' in MRL/l mice are subpopulations of anti-ssDNA antibodies that react equally well with poly(ADP-ribose) and ssDNA.

Animals↗
PubMed1987

Direction of elongation of poly(ADP-ribose) chains. Addition of residues at the polymerase-proximal terminus.

The mechanism of elongation of poly(ADP-ribose) on poly(ADP-ribose) polymerase was examined in two ways. The first technique involved a pulse-chase protocol. Poly(ADP-ribose) polymerase was labeled with radioactive NAD, excess precursor was removed by rapid gel filtration chromatography, and nonradioactive NAD was supplied for a second incubation. The products were released with alkali and digested with venom phosphodiesterase which generates AMP uniquely from the distal terminus. The distal residue that was labeled during the pulse remained at the distal terminus and was not converted to an internal residue during the chase. The second technique employed the NAD analog, 2'-deoxyNAD (dNAD), which can engage in mono-ADP-ribose addition reactions but lacks the 2'-OH that is required for polymer formation. dNAD inhibits ADP-ribose incorporation competitively but is not incorporated at the enzyme-distal chain terminus. These findings are inconsistent with a model of poly(ADP-ribose) synthesis in which new residues are added to the 2'-OH terminus of the growing chain, distal to the polymerase attachment. They are consistent with the alternative possibility that new residues are added at the 1" terminus, adjacent to the polymerase. Any such "proximal addition" model requires that there be at least two active center sites (akin to the ribosomal A and P sites), which at a certain stage of each elongation cycle will be occupied by ADP-ribose monomers and ADP-ribose polymers, respectively. Although dNAD does not enter poly(ADP-ribose), it does engage in a slow side reaction whereby a single dADP-ribose residue is added covalently to the polymerase itself, thereby inactivating the enzyme.

ADP Ribose Transferases↗
PubMed1983

Properties of poly(ADP-ribose) synthetase and ADP-ribosyl histone splitting enzyme.

Poly(ADP-ribose) synthetase catalyzes initiation, elongation, and branching of poly(ADP-ribose) on various acceptor proteins including itself. The automodification took place at about 15 sites on the synthetase molecule of rat liver; at each site was bound poly(ADP-ribose) as large as greater than 80 ADP-ribose units long with branched structures. Such extensive automodification accompanied a marked increase in the apparent Mr (to greater than 500,000) of the synthetase. Based on this unique property, the synthetase was identified as the main acceptor of poly(ADP-ribose) in isolated nuclei and permeabilized cells. The enzyme-bound poly(ADP-ribose) was not transferred to histone under the various conditions tested. These results suggested that the automodified enzyme does not serve as an intermediate in the modification of other proteins, but plays some biological role(s) as a structural element. ADP-ribosyl histone splitting enzyme, an enzyme responsible for the cleavage of ADP-ribose-protein bonds, was purified 5,000-fold to apparent homogeneity from rat liver. The purified enzyme was composed of a single polypeptide of Mr approximately equal to 80,000. The substrate specificity was broad for the protein portion, but strict for the ADP-ribose portion; only mono(ADP-ribosyl) proteins served as the substrates. The splitting reaction appeared to proceed by a non-hydrolytic mechanism producing a derivative of ADP-ribose with an altered terminal ribose.

ADP Ribose Transferases↗
PubMed1976

The effect of ribose 5-phosphate and 5-phosphoribosyl-1-pyrophosphate availability on de novo synthesis of purine nucleotides in rat liver slices.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.

Animals↗
PubMed1978

Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.

Bdellovibrio↗
PubMed1988

Poly(ADP-ribose) degradation by glycohydrolase starts with an endonucleolytic incision.

We have recently shown that poly(ADP-ribose) polymerase forms poly(ADP-ribose) by adding ADP-ribose residues to the polymerase-proximal end of an enzyme-bound nascent chain. In this light we have reexamined the mode of hydrolysis of enzyme-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. When the substrate has been labeled by a pulse-chase protocol, soluble glycohydrolase releases a significant amount of labeled oligomer which can only come from the enzyme-distal (2') end of the polymer. This constitutes additional evidence for the proximal growth of chains. Oligomer is infrequently released from the proximal (1") end of enzyme-bound chains. Rather, the bulk of the poly(ADP-ribose) is digested directly to ADP-ribose monomers. We conclude that poly(ADP-ribose) glycohydrolase starts digestion with an endonucleolytic incision and then removes ADP-ribose residues processively in the 2'----1" direction. Therefore, in contrast to earlier models of polymer growth and hydrolysis, a single poly(ADP-ribose) chain may be extended at one end and simultaneously degraded at the other end. The balance between synthesis and degradation may control the quantity and distribution of polymer around the DNA break which occasions its synthesis.

Glycoside Hydrolases↗
PubMed1983

Aberration of poly(adenosine diphosphate-ribose) metabolism in human colon adenomatous polyps and cancers.

The activities of three principal enzymes engaged in the biosynthesis and degradation of poly(adenosine diphosphate-ribose) [poly(ADP-ribose)] were examined in cell nuclei isolated from adenomatous polyps (tubular adenomas of familial polyposis coli, villous adenoma, and tubulovillous adenoma), cancers, and normal mucosa of human colon. The activities of poly(ADP-ribose) synthetase in adenomatous polyps [161 +/- 46 (S.E.) pmol/min/mg DNA] and cancers (114 +/- 32 pmol/min/mg DNA) were, on an average, about 3 and 2 times, respectively, higher than those in normal mucosa (52 +/- 24 pmol/min/mg DNA); the difference was statistically significant (p less than 0.001). The activity of poly(ADP-ribose) glycohydrolase was also significantly high in adenomatous polyps (13.0 +/- 3.4 nmol/min/mg DNA), but not in cancers (10.1 +/- 2.5 nmol/min/mg DNA), compared with normal mucosa (5.2 +/- 1.4 nmol/min/mg DNA) (p less than 0.001). The activity of ADP-ribosyl protein lyase, in contrast, was lower in adenomatous polyps (152 +/- 40 pmol/min/mg DNA) than in normal mucosa (345 +/- 111 pmol/min/mg DNA) and cancers (288 +/- 80 pmol/min/mg DNA) (p less than 0.001). Analyses of reaction products with snake venom phosphodiesterase digestion revealed that poly(ADP-ribose) synthesized in nuclei of normal mucosa, adenomatous polyps, and cancers had the average chain lengths of 2.9, 1.7, and 9.7 ADP-ribose units, respectively. Based upon these values and total amounts of ADP-ribose incorporated, the amount of poly(ADP-ribose) synthesized per mg DNA in 30 min was calculated as 308, 1510, and 106 pmol in the above three types of colon tissues, respectively. These results suggested that a larger amount of monomers and short oligomers of ADP-ribose was synthesized in adenomatous polyps, while a smaller number of longer polymers was produced in cancers as compared with normal mucosa. Immunohistochemical analysis of these tissues using anti-poly(ADP-ribose) antibody supported this view.

Adenoma↗
PubMed2002

Formation of sulfur aroma compounds in reaction mixtures containing cysteine and three different forms of ribose.

The headspace volatiles produced from buffered and unbuffered cysteine model systems, containing inosine 5'-monophosphate, ribose 5-phosphate, or ribose, were examined by GC-MS. Sulfur compounds dominated the volatiles of all systems and included mercaptoketones, furanthiols, and disulfides. The inosine monophosphate systems produced much lower quantities of volatiles than ribose phosphate or ribose systems. In the systems buffered with phosphate or phthalate buffers, both ribose and ribose phosphate systems gave similar quantities of sulfur volatiles. However, in the absence of buffer, the ribose system was relatively unreactive, especially for volatiles formed via the 2,3-enolization route in the Maillard reaction, where 4-hydroxy-5-methyl-3(2H)-furanone is a key intermediate. A number of keto-enol tautomerisms, which are known to be acid-base-catalyzed, occur in the 2,3-enolization route. This may explain the catalysis of the ribose systems by the buffers. In the ribose phosphate systems, however, Maillard mechanisms probably played a less important role, because ribose 5-phosphate readily dephosphorylated to give 4-hydroxy-5-methyl-3(2H)-furanone on heating and thus provided an easier route to aroma compounds than the Maillard reaction.

Cysteine↗
PubMed1999

Ribose utilization in Lactobacillus sakei: analysis of the regulation of the rbs operon and putative involvement of a new transporter.

A 7-kb DNA fragment of Lactobacillus sakei, containing the rbsD, rbsK and rbsR genes was sequenced. The genes responsible for ribose utilization are organized differently from what was previously described for model organisms such as Escherichia coli and Bacillus subtilis. No gene encoding RbsA, RbsB and RbsC, the subunits of the ribose ABC-transporter, were present in the rbs gene cluster. Instead, we found an open reading frame coding for RbsU, a protein similar to GltA, the glucose transporter of Staphylococcus xylosus. The disruption of rbsK, encoding the ribokinase, impaired growth on ribose. The disruption of rbsR, encoding the repressor, had no effect on the ability to grow on ribose, but led to overexpression of a large transcript corresponding to rbsU, rbsD and rbsK, suggesting that RbsU might be involved in ribose utilization. Ribose uptake and phosphorylation assays on the wild type strain and various mutants showed that, in ptsI mutants, both ribose uptake and phosphorylation are increased. These increased activities can explain the faster growth rate on ribose that was observed in ptsI mutants. The phosphotransferase system is thus involved in the negative regulation of ribose utilization. This regulation might not act at the transcriptional level since the overexpression of the rbs genes in the rbsR mutant did not lead to the same phenotype. A gene sharing high similarity scores with ackA genes, encoding the acetate kinase, was found upstream from the rbs gene cluster. The unusual location of this gene is maybe not fortuitous since acetate kinase is involved in ribose catabolism.

ATP-Binding Cassette Transporters↗
PubMed1993

Poly(ADP-ribose) polymerase stimulates DNA polymerase alpha by physical association.

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.

Animals↗
PubMed1978

Poly(adenosine dephosphate ribose) metabolism and regulation of myocardial cell growth by oxygen.

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.

Animals↗