Purification and characterization of an adenosine cyclic 3':5' monophosphate-dependent protein kinase from human erythrocyte membrane.

A cAMP dependent protein kinase was extracted from human erythrocyte membrane with hydrosoluble fraction and partially purified by ammonium sulfate-precipitation and DEAE-cellulose chromatography. The pH of optimal activity is 6.5; the enzyme has an absolute requirement of Mg²⁺ ions at the concentration of 10 mM and is strongly inhibited by Ca²⁺. It uses ATP as phosphate donor with a Km of 3.7 × 10^?6 M. Cyclic AMP stimulates the activity with an apparent Ka of 5 × 10^?8 M; cIMP and cGMP also acts as activators. Enzyme activity is thermolabile and not protected by Mg ATP complex. The enzyme purified from erythrocyte membrane is a type I protein-kinase as proven by DEAE cellulose chromatography and dissociation of the subunits in presence of NaCl 0.5 M and histone.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.     

Inactivation of E. coli RNA polymerase by pyridoxal 5'-phosphate: identificationof a low pKa lysine as the modified residue.

The inactivation of E. coli RNA polymerase (3.3 × 10^?7M) by pyridoxal 5′-phosphate (1 × 10^?4M to 5 × 10^?4M) is a first order process with respect to the remaining active enzyme. Studies of the variation of the first order rate constant with the concentration of pyridoxal 5′-phosphate show that the inactivation reaction follows saturation kinetics. The formation of a reversible enzyme-inhibitor intermediate is postulated. Kinetic studies at different pH values indicate that the inactivation rate constant depends on the mole fraction of one conjugate base with pK_a 7.9. The apparent equilibrium constant (association) for the inactivation reaction is independent of the pH and is 1.8 × 10⁴ M^?1. By electrophoretic and chromatographic analysis of enzyme hydrolyzates after pyridoxal 5′-phosphate and NaBH₄ treatment only N-ε-pyridoxyllysine was found. It is postulated that a lysine ε-amino group with a low pK_a is critical for the activity of the enzyme.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

The isolation of pig liver monoamine oxidase

Monoamine oxidase from pig liver has been isolated and purified approximately three hundred-fold. This enzyme has a molecular weight of 1,200,000, is highly polymeric, and contains subunits of molecular weight 146,000, as determined by Sephadex chromatography. The apparent K_m at 25°C is 1.28 × 10^?6 M at pH 9.0 (0.05 M glycine) and 1.74 × 10^?5 M at pH 7.2 (0.2 M phosphate) using benzylamine as a substrate. This enzyme contains approximately 8 copper(II) ions per 1,200,000 molecular weight.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Fatty acid esters of testosterone in rat brain: identification, distribution, and some properties of enzymes which synthesize and hydrolyze the esters

[4-¹⁴C]Testosterone was converted to an unknown compound with a much higher R_f on thin layer chromatogram than the substrate when it was incubated with a rat brain microsomal preparation. Evidence from its mass, infrared, and ultraviolet spectra indicated that the enzymic product is a mixture of fatty acid esters of testosterone. Saponification of the product yielded testosterone and a mixture of C_12:0, C_14:0, C_16:0, C_18:0, and C_18:1 fatty acids. The enzymic product was identical to testosterone laurate and testosterone stearate which were synthesized chemically. The enzyme system had a pH optimum at 4.9 with acetate buffer. The apparent K_m was 8.3 × 10^?5m for testosterone and 5.0 × 10^?5m for palmityl CoA. An enzyme which hydrolyzes testosterone[1-¹⁴C]oleate was also detected in rat brain. Most of this activity was in the nuclear and mitochondrial fractions. This enzyme had an optimum pH at 6.5 with phosphate buffer and its apparent K_m was 2.1 × 10^?4m. A low level of synthetic activity was found in fetal brain tissue which increased and reached a maximum at 3 weeks of age. The synthetic activity rapidly decreased with further increase in age. Hydrolytic activity was nearly undetectable in fetal rat brain, increased gradually until the animal reaches 3 weeks old, and remained at this level. Both synthetic and hydrolytic enzyme activities were higher in the brain than in other tissues examined.     

Corticotropin stimulates cyclic nucleotide independent protein kinase activity of intact adrenocortical cells

Intact adrenocortical cells possess cyclic nucleotide-independent protein kinase activity which is capable of phosphorylating endogenous proteins and casein when incubated in the presence of [γ-³²P]ATP. The cyclic nucleotide-independent enzyme was dependent on cell number and temperature and had an apparent K_m for ATP of 6.5 × 10^?5 M and a V_max of 12.5 pmol/3 min/2 × 10⁵ cells at 37°C. Phosphorylation of endogenous proteins by this kinase was increased by treatment of intact cells with corticotropin (2.2 nM) for 24 h. In control cells, two endogenous proteins of apparent molecular weights of 39,000 and 76,000 were phosphorylated. In corticotropin-treated cells, another protein of apparent molecular weight of 87,000 was also phosphorylated. Thus, this protein kinase activity, which appears to be located on the plasma membrane, may be involved in mediating longer term actions of corticotropin on the adrenal cortex.     

The assay of Met-enkephalin aminopeptidase with [125I]Met-enkephalin

Presented here are procedural modifications whic permit the utilization of ¹²⁵I-labeled Met-enkephalin as substrate in the assay of rat brain enkephalin amipeptidase. The hydrolysis of enkephalin is monitored by the release of [¹²⁵I]tyrosine separated on Porapak Q. The release of tyrosine is proportionate with both increasing time and tissue concentration. The estimated K_m is near 10^?4 M and the enzyme activity can be inhibited more than 95% with puromycin. The majority of the enzyme activity remains in the 100 000 × g supernatant following differential centrifugation.     

Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Effect of Nucleotides and Related Compounds on Glutamine Synthetase Activity in Chick Embryo Retina: A Biochemical and Immunohistochemical Study

Abstract: The effect of a number of nucleotides and related compounds on glutamine synthetase (GS) induction in retina from 12-day chick embryo was studied with both biochemical and immunohistochemical techniques. A number of these compounds gave rise to GS activity comparable to that induced by treatment with cortisol, which is known to give rise to precocious induction of the enzyme in this system. Of the cyclic nucleotides examined, cAMP (0.5-1.2 × 10^?3 M) gave essentially no increase in GS activity. In contrast, dibutyryl cAMP (0.8 × 10^?3 M) had a more significant effect on GS activity, as did 8-bromo-cAMP and cGMP at the same concentration. The activity elicited by these nucleotides was generally half that obtained by treatment with 2.8 × 10^?7 M-cortisol for the same length of time, 8-Bromo-cGMP (0.8 × 10^?3 M) had an effect comparable to the aforementioned concentration of cortisol. Since phosphodiesterase activity is minimal in the chick retina at 12 days of development, addition of isobutylmethylxanthine (1 × 10^?5 M) to this system had, as would be expected, little effect on GS activity. Of the noncyclic compounds, 8-bromoguanosine often gave rise to GS activity comparable to that obtained with cortisol. The other compounds (8-bromo-5′-GMP, guanosine, adenosine, and 5′-AMP) generally had less of an effect on GS. In general, the degree of staining in the immunohistochemical localization of GS corresponded well with the biochemical results and showed the enzyme to be present in regions consistent with the distribution of Muller cells and their processes. Thin-layer chromatography and radioimmunoassay of the nucleotides did not show any steroid impurity in any of the compounds used in the study, even when determinations were carried out at five times the concentration of nucleotide used in the experiments.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin.

We have studied the affinity of methylmalonyl CoA mutase for its required cofactor, adenosylcobalamin, in extracts of control and mutant human cultured fibroblasts. Control enzyme has an apparent K_m for adenosylcobalamin of 6–7 × 10^?8 M. Five mutant cell lines from patients with methylmalonicacidemia due to a mutase apoenzyme defect were studied. Three have undetectable mutase activity (<0.15% of control) at all cofactor concentrations. Two others, however, have markedly altered K_m's for adenosylcobalamin of 2.8 × 10^?4 M and 1.7 × 10^?5 M. These mutant lines synthesize adenosylcobalamin normally and, by complementation analysis, are genetically identical to all other mutase apoenzyme mutants tested. We conclude that the mutase deficiency in these two cell lines results from structurally altered mutase apoenzymes with markedly reduced affinities for adenosylcobalamin.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

Purification and Properties of Hypoxanthine Phosphoribosyltransferase from Streptomyces cyanogenus

Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) of a strain of Streptomyces cyanogenus was purified 1,900-fold to an apparent homogenity from cell-free extracts. The enzyme had a molecular weight of 150,000 and consisted of eight identical subunits with a molecular weight of 18,000. The isoelectric point was at pH 4.4. The enzyme required Mg²⁺ or Ma²⁺ for activity and had a pH optimum at 8.5. Hypoxanthine and guanine were good substrates for the enzyme. Xanthine was a very poor substrate and adenine was not a substrate. Apparent Km values of the enzyme for hypoxanthine, guanine and 5-phosphoribose-1-pyro-phosphate were 1.6 × 10^?8, 2.7 × 10^?6 and 6.3 × 10^?5 m, respectively. All purine nucleotides tested inhibited the activity significantly, apparently by competing with 5-phosphoribose-1-pyrophosphate.