Hyperoxaluria in L-glyceric aciduria: possible nonenzymic mechanism

Hydroxypyruvate inhibited the oxidation of [1-14C]glyoxylate to [14C] oxalate whether catalyzed by a purified preparation of glycolic acid oxidase from human liver, lactate dehydrogenase, a human liver extract, or a lobe of rat liver. It also brought about the nonenzymic decarboxylation of [1-14C]glyoxylate when it was present in the above assay systems. Radioactive isotope dilution and high-performance liquid chromatography analysis revealed the autooxidation of hydroxypyruvate to oxalate on standing in buffered solution at pH 7.4. In view of these observations, the current hypothesis of the role of lactate dehydrogenase in inducing hyperoxaluria in L-glyceric aciduria has been reexamined, and a possible nonenzymic mechanism by which oxalate may originate from hydroxypyruvate under such conditions has been proposed.     

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of (14)CO(2) from [1-(14)C]glycollate and [1-(14)C]glyoxylate were investigated. 2. At pH8, (14)CO(2) production from [1-(14)C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H(2)O(2), which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. (14)CO(2) is shown to be generated from [1-(14)C]glycollate at pH8 by a similar reaction, but the H(2)O(2) is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

Oxalate synthesis from [14C1]glycollate and [14C1]glycoxylate in the hepatectomized rat

Hepatectomy significantly altered the metabolism of [1-¹⁴C]glyoxylate and [1-¹⁴C]glycollate in the rat. The production of ¹⁴CO₂ was reduced by 47% and 77%–86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primary glycine, serine and ethanolamine, were also reduced by over 50%, was would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [¹⁴C]oxalate from [1-¹⁴C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [¹⁴C]oxalate from [¹⁴₁]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [¹⁴C]oxalate from [¹⁴₁]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.     

The synthesis of oxalate from hydroxypyruvate by isolated perfused rat liver the mechanism of hyperoxaluria in L-lyceric aciduria

Hydroxypyruvate and glycolate inhibited the oxidation of [U-¹⁴C]glyoxylate to [¹⁴C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [¹⁴C]Oxalate synthesis from [¹⁴C]glycine similarly inhibited by hydroxypyruvate, but conversion of [¹⁴C₁]glycolate to [⁴C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [¹⁴C]oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [¹⁴C₃]Hydroxypyruvate, but not [¹⁴C₁]hydroxypyruvate, was oxidized to [¹⁴C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.     

Absorption of glyoxylate and oxalate in thiamine and pyridoxine deficient rat intestine

Dietary deficiency of thiamine or pyridoxine has been shown to produce hyperoxaluria and renal stone formation in man and experimental animals. To determine the possible contribution of exogenous glyoxylate and oxalate, the intestinal transport of [14C] - oxalate and [14C] - glyoxylate was measured in vitamin B1 and B6 deficient rats and their respective pair-fed controls. Results indicate that glyoxylate and oxalate are passively diffused from lumen to lamina propria in thiamine deficient and their pair-fed controls with no significant change in the rate of uptake of both the substrates. However B6 deficient rats showed a significant enhancement in the rate of oxalate uptake due to development of a new biphasic transport system. The rate of glyoxylate uptake by simple passive diffusion remained unaltered in pyridoxine deficiency.     

Hepatic oxalate production: the role of hydroxypyruvate

The metabolism of hydroxypyruvate to oxalate was studied in isolated rat hepatocytes. [14C]Oxalate was produced from [2-14C]- and [3-14C]- but not [1-14C]hydroxypyruvate. No oxalate was produced from similarly labeled pyruvate. The mechanism by which hydroxypyruvate is metabolized to oxalate involves decarboxylation at the carbon 1 position as the initial step. This activity was distinct from that which produced CO2 from the carbon 1 position of pyruvate. Hydroxypyruvate decarboxylase activity was found mainly in the mitochondria, with the remainder (25%) in the cytosol. No activity was present in the peroxisomes, the probable site of oxalate production from glycolate and glyoxylate. Hydroxypyruvate, but not pyruvate stimulated [14C]oxalate production from [U-14C]fructose, suggesting that hydroxypyruvate is either an intermediate in the fructose-oxalate pathway, or that it prevents carbon from leaving that pathway. The lack of effect of pyruvate in this regard is evidence against redox being the primary effect of hydroxypyruvate and focuses attention on hydroxypyruvate and its precursors as important sources of carbon for oxalate synthesis from both carbohydrate and protein.     

Oxalate accumulation from citrate by Aspergillus niger

Carbon-14 was incorporated from citrate-1,5-¹⁴C, glyoxylate-¹⁴C(U), or glyoxylate-1-¹⁴C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-¹⁴C(U) was superior to glyoxylate-1-¹⁴C and citrate-1,5-¹⁴C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC 3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC 1.1.3.1; glyoxylate oxidase, EC 1.2.3.5; NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC 1.2.1.17) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.Abbreviations Used DCPIP 2,6-dichlorophenolindophenol     

The uptake of oxalate by rat liver and kidney mitochondria

Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxalate metabolism by tobacco leaf discs

The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [¹⁴C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as ¹⁴CO₂, and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O₂ concentration from 1% to 21% to 60%, nor was the formation of [¹⁴C]oxalate from [2-¹⁴C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO₂.     

Dependence of ADP-ribosylation of histones of chicken liver nuclei on the intranuclear content of NAD

The dependence of ADP-ribosylation of chicken liver nuclear histones on NAD concentration in the nuclei was studied under conditions of stimulation of coenzyme synthesis by the nicotinamide and nicotinic acid as well as upon addition of various concentrations of the [Ado-U-14C]NAD nuclei to the incubation mixture. In the first case, the rate of [Ado-U-14C]NAD incorporation into the histones was decreased due to the dilution of the label by the de novo synthesized NAD. The amount of the latter formed under effects of nicotinic acid and nicotinamide increased, correspondingly, from 2,2 X 10(-5) mmol up to 4.1 X 10(-5) and 7.0 X 10(-5) mmol per mg of nuclear protein. The incorporation of [Ado-U-14C]NAD into the histones decreased from 12.0 X 10(-8) mmol after incubation of liver slides with nicotinic acid and nicotinamide down to 8.0 X 10(-8) and 7.0 X 10(-8) mmol, respectively. With a rise in the concentration of exogenous [Ado-U-14C]NAD, the level of ADP-ribosylation of nuclear histones increased, the plot [14C]NAD incorporation at the labeled coenzyme concentration of 25 X 10(-7) mM/mg of histone had a plateau. Changes in the labeled substrate concentration brought about corresponding changes in the average length of the histone-linked poly-(ADP-ribose) chain.     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme     

Modes of action of a shortening system for erucic acid at the mitochondrial level of rat liver

In this work, were studied the conditions of erucic acid (cis-docosenoic, n-9) shortening by using Rat liver mitochondrial preparations which were incubated in vitro with [14-14C] erucic acid (22:1), with inhibitors of the respiratory chain (rotenone, cyanide) or not, with activators of either the shortening reaction (NAD+, NADP+), or beta-oxidation (malate, carnitine, cytochrome c) or not. The shortening activity was measured by the amount of 14C radioactivity recovered in the shorter fatty acids formed (20:1, 18:1, 16:1) when beta-oxidation was inhibited. The beta-oxidation activity was measured by the amount of 14C recovered in the acid-soluble products (P A S). The incubations were performed under conditions which were the least favourable to peroxysomal activity. Data showed that, with increasing amounts of albumin, which inhibits peroxysomal activity, increasing amounts of shorter fatty acids (20:1, 18:1, 16:1) were formed from erucic acid. This shortening reaction was strongly stimulated by NAD+, more than by NADP+; it was also stimulated by cytochrome c and much more when both NAD+ and cytochrome c were added. Similar data were observed in beta-oxidation, except that practically NADP+ did not exhibit any stimulating effect. Oxidation of NADH by mitochondria only occurred when cytochrome c was added to the medium and was not modified by the addition of ADP or rotenone. These data show that liver mitochondria are capable of shortening erucic acid, as are peroxysomes. This shortening reaction is highly NAD+-dependent and seems to be localized outside the matrix. This system could constitute a second route for utilization of fatty acids in mitochondria, besides the well-known path of beta-oxidation.     

The formation of oxalate from glycolate in rat and human liver

In this study, we attempted to elucidate the metabolic pathway and enzymes actually involved in oxalate formation from glycolate in rat and human liver. In rat liver, the formation of oxalate from glycolate appeared to take place predominantly via glyoxylate. The oxalate formation from glycolate observed with crude enzyme preparations was almost entirely accounted for by the sequential actions of glycolate oxidase and xanthine oxidase (XOD) or lactate dehydrogenase (LDH). Under the conditions used, no significant activity was attributable to glycolate dehydrogenase, an enzyme reported to catalyze the direct oxidation of glycolate to oxalate. Among the three enzymes known to catalyze the oxidation of glyoxylate to oxalate, glycolate oxidase and XOD showed much lower activities (a higher Km and lower Vmax) toward glyoxylate than those with the respective primary substrates. As to LDH, none of the LDH subunit-deficient patients examined showed profoundly lowered urinary oxalate excretion. Based on the results obtained, the presumed efficacies in vivo of individual enzymes, as catalysts of glyoxylate oxidation, and the in vivo conditions assumed to allow their catalysis of oxalate production are discussed.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Reaction mechanism for automodification of poly(ADP-ribose) synthetase

The reaction mechanism of automodification of poly (ADP-ribose) synthetase was studied. The synthetase, bound to nicked DNA-cellulose in a small column, was pulse-labelled with [3H]NAD in the presence of Mg2+, and then chased with [14C]NAD under the same conditions after complete washing of [3H]NAD. The poly(ADP-ribose), synthesized on the synthetase molecule, was digested with snake venom phosphodiesterase and analyzed. The [3H]-labeled product (35% of the total product) was identified as isoADP-ribose but [3H]-labelled AMP was not detected. The average chain length was 16.0 and the terminal AMP was detected as [14C]-labelled AMP. These results indicate that the initially attached ADP-ribose unit at an automodification site was successively elongated by the addition of a new ADP-ribose unit to the terminal AMP moiety.