Effect of Nerve Growth Factor on Glucose Utilization and Nucleotide Content of Pheochromocytoma Cells (Clone PC12)

The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7-fold increase in glucose utilization and a 4.7-fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP-glucose, UDP-galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90-120% for AMP, GMP, UDP-glucose, and UDP-galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neuronal-like cell population.     

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.

Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].     

Permeability of Rickettsia prowazekii to NAD.

Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

The development of gluconeogenesis in rat liver. Controlling factors in the newborn

1. Measurements in livers of rats delivered by Caesarian section show a rapid change in the relative proportion of adenine nucleotides. By 20min the ATP/ADP ratio had increased from 1.76 to 8.7 and the value of the relationship [ATP][AMP]/[ADP](2) increased from 1.0 to 4.4. These changes are dependent on the availability of oxygen to the animal. 2. The free [NAD(+)]/[NADH] ratio in the liver cytosol increases from 180 after delivery to reach a maximum of 1010 at 2h, before falling to 540 in the 24h-old animal. 3. The mitochondrial NAD redox potential also shows a sharp increase towards a more oxidized state in livers of delivered rats. 4. These results probably indicate that the foetal liver is hypoxic, with oxygenation occurring in the first hour after delivery. 5. Measurements in livers of naturally born rats 2min after birth also suggest that this tissue is hypoxic with an ATP/ADP ratio of 1.83 and a free [NAD(+)]/[NADH] ratio of 117. 6. Concentrations of intermediates in the gluconeogenic pathway have been determined in livers of foetal, 1h-old and 1-day-old rats. These experiments imply a facilitation of lactate dehydrogenase and glucose 6-phosphatase activities by 1h after birth, and a stimulation of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase steps by 1 day after birth. 7. The appearance of gluconeogenesis in livers of newborn rats seems therefore to involve an oxygenation stage followed by an increase in phosphoenolpyruvate carboxykinase activity.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Effect of urea on proteolysis of histone H2A in calf thymus nuclei

The 1 hour incubation of calf thymus nuclei at 37 degrees C leads to a proteolysis of the histones H1, H3 and H2B. Urea does not influence the histone degradation while 1.5 and 2.0 M NaCl lead to the proteolysis of the H2A histone. On this background, 2 M urea restrains the degradation of the H2A histone. It is assumed that hydrogen bonds are very important for the activity of the proteinases and its interaction with the H2A histone.     

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-microns Lichrosorb RP-18 column with uv detection at 254 nm.     

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin.     

Independence of the (NAD+):(NADH) ratio from the adenylic system in the liver cytoplasm of the developing chick embryo]

No dependence was found between the index of the adenylic system phosphorylated state (ATP) : (ADP) (HPO2-4), the ratios (ATP) : (ADP) and (ATP : (ADP + AMP), on one hand, and the ratio (NAD+) : (NADH) in the cytoplasm, on the other one. The maximum value of the ratio (ATP) : (ADP) (HPO2-4) was observed on the 17th day of development and correlated with the maximum rate of gluconeogenesis. The ratio (NAD+) : (NADH) in the cytoplasm suffered no changes until hatching and decreased twice thereafter.     

Accessibility and structural role of histone domains in chromatin. biophysical and immunochemical studies of progressive digestion with immobilized proteases

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.     

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.     

Changes in Acid-soluble Nucleotides in Cut-injured Sweet Potato Root Tissue

ATP and ADP increased in cut-injured sweet potato root tissue during the 3 to 6-hr incubation period, and showed the maximum for the 9 to 18-hr, and 6 to 9-hr incubation periods, respectively, then decreased. ATP was present in the highest amount among ATP, ADP and AMP throughout the 72-hr incubation period, while AMP was in the lowest. Total acid-soluble nucleotides increased gradually, and showed the peak content at the 12-hr incubation period, and decreased thereafter. Adenine mononucleotides such as ATP, ADP and AMP occupied about 40 to 65% of total acid-soluble nucleotides.     

Inhibitory effects of adenine nucleotides and related substances on UDP-glucuronosyltransferase: structure-effect relationships and evidence for an allosteric mechanism

The inhibitory effects of nucleotides and related substances on rat hepatic UDP-glucuronosyltransferase (UGT) were studied. ATP and NADP+ markedly reduced 4-methylumbelliferone (4-MU) UGT activity only when detergent-treated rat liver microsomes were used as the enzyme source. The IC50 values of adenine, ATP, NAD+ and NADP+ were estimated to be below 20 microM, whereas AMP had no inhibitory effect. From the kinetic behavior observed, these adenine-related compounds were assumed to inhibit UGT activity non-competitively without competing with either 4-MU or UDP-glucuronic acid. Among guanine, cytosine and their related nucleotides, only triphosphate nucleotides (CTP and GTP) exhibited potent UGT inhibition, although the effect of GTP was weak. Estradiol 3- and 17-glucuronidation were also inhibited by the inhibitors of 4-MU UGT. The only exception was that estradiol 17-glucuronidation activity was inhibited by AMP (IC50=31 microM). In addition, AMP antagonized the inhibitory effects of adenine, ATP, and NADP+ on 4-MU and estradiol 3- glucuronidation activities. These results suggest that (1) a number of cellular nucleotides present within the endoplasmic reticulum regulate UGT function; and (2) these substances bind to a common allosteric site on UGT to reduce catalytic function.     

Regulation of citrate synthase activity of Saccharomyces cerevisiae.

Citrate synthase activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product, 14C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADH were most inhibitory to the citrate synthase invitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and alpha-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.