Aspirin as a quantitative acetylating reagent for the fatty acid oxygenase that forms prostaglandins.

A selective acetylation of the prostaglandin-forming fatty acid oxygenase (part of the prostaglandin "synthetase" system) occurs with 100 muM concentrations of aspirin (acetylsalicylic acid). The amount of acetylation, measured by counting the [3H]acetyl-protein formed, was proportional to the amount of active, functional oxygenase in a sample. When samples were aged to allow spontaneous inactivation of the oxygenase, the amount of acetylation was proportional to the remaining measurable activity rather than the initial amount of oxygenase protein in the sample. Diethyl dithiocarbamate inhibited the oxygenase activity, but did not interfere with the subsequent acetylation by aspirin. Indomethacin, on the other hand, appeared to inactivate the oxygenase in a manner that interfered only partially with the action of aspirin as an acetylating reagent. The amount of acetylation appeared to be dependent upon the amount of native, undenatured enzyme. The results suggest that the acetylation may be dependent upon an essential functional group or conformation of groups in the catalytic peptide chain(s) that can be destroyed during spontaneous inactivation of the oxygenase, and altered by indomethacin.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

The effects of some salicylate analogues on human blood platelets. 2. The role of platelet acetylation in the inhibition of platelet aggregation

Aspirin, 2,3-diacetoxybenzoic acid, 2,6-diacetoxybenzoic acid and 2-propoxybenzoic acid were incubated in human platelet-rich plasma at 37°C for 5 and 10 min and the effects upon collagen induced platelet aggregation and the uptake by platelets of radioactive acetate and propionate groups from 14C-labelled analogues were studied to determine if a correlation existed between acylation of the platelet and inhibition of aggregation. Inhibition of aggregation and the uptake of radioactive label were both concentration-dependent and both increased with the time of incubation. Potency re inhibitors of aggregation was, in decreasing order, aspirin, 2,propoxybenzoic acid, 2,3-diacetoxybenzoic acid and 2,6-diacetoxybenzoic acid. Uptake of radioactive label however, was greatest with aspirin, intermediate with 2,3- and 2,6-diacetoxybenzoic acid, and lowest with 2-propoxybenzoic acid. Platelets exposed to a metabolic inhibitor (oligomycin, 10^?5M for 15 min) showed reduced uptake of labelled acetate and propionate and the degree of uptake did not correlate with the degree of inhibitory activity of the analogues on platelet aggregation. Platelet fragments produced by sonification did not take up radioactive label and chloroform: methanol extraction removed about 50% of the label from intact platelets. The results do not support the hypothesis that acetylation of platelets by aspirin is solely responsible for its inhibitory effects on aggregation but do not conflict with the suggestion that acetylation of platelets may be responsible for the persistent $\text{in}$$\text{vivo}$ effects of aspirin.     

Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin.

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating an active site portion of the enzyme, prostaglandin synthetase. In the current study, the site of acetylation has been demonstrated to be a seryl residue at the NH2 terminus of the enzyme. Purified [3H]acetyl enzyme was prepared from seminal vesicle homogenates treated with [acetyl-3H]aspirin. The [3H]acetate to protein bond was stable to hydroxylamine, indicating an N-acetyl linkage. The [3H]acetyl enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. The 3H material isolated from the pronase digest was identified as N-acetylserine. This finding indicates that the oxygenase portion of prostaglandin synthetase has an NH2-terminal serine which is involved in enzymatic activity and is susceptible to acetylation by aspirin.     

Stimulation of GABA-Induced Ca2+ Influx Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Neurons

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca²⁺ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca²⁺ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca²⁺ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca²⁺ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca²⁺ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca²⁺ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.     

Preventive aspirin treatment of streptozotocin induced diabetes: blockage of oxidative status and revertion of heme enzymes inhibition

Some late complications of diabetes are associated with alterations in the structure and function of proteins due to glycation and free radicals generation. Aspirin inhibits protein glycation by acetylation of free amino groups. In the diabetic status, it was demonstrated that several enzymes of heme pathway were diminished. The aim of this work has been to investigate the in vivo effect of short and long term treatment with acetylsalicylic acid in streptozotocin induced diabetic mice. In both treatments, the acetylsalicylic acid prevented delta-aminolevulinic dehydratase and porphobilinogen deaminase inactivation in diabetic mice and blocked the accumulation of lipoperoxidative aldehydes. Catalase activity was significantly augmented in diabetic mice and the long term treatment with aspirin partially reverted it. We propose that oxidative stress might play an important role in streptozotocin induced diabetes. Our results suggest that aspirin can prevent some of the late complications of diabetes, lowering glucose concentration and probably inhibiting glycation by acetylation of protein amino groups.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin.

Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100- or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (+/-)-2-fluoro-alpha-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acetyl]NAI indicated incorporation of 1.7 +/- 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo- and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. The different biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Lack of covalent modification of prostaglandin synthetase (cyclo-oxygenase) by indomethacin

We have previously shown that aspirin irreversibly inhibits prostaglandin synthetase (cyclo-oxygenase) by acetylating the active site of the enzyme. By utilizing ¹⁴C-labeled indomethacin and a close analogue, we now show that indomethacin, unlike aspirin, does not covalently modify cyclo-oxygenase. Furthermore, indomethacin binding to the enzyme may be reversible since even though indomethacin can inhibit acetylation by aspirin, when enzyme inhibited by indomethacin (1 μM) is treated with 200 μM aspirin 3 times for 1 hour each, complete acetylation of cyclo-oxygenase is achieved.     

Activation of sucrose-phosphate synthase from Prosopis juliflora in light: Effects of protein kinase and protein phosphatase inhibitors

Sucrose‐phosphate synthase (SPS) activity measured under limiting substrate and in the presence of inorganic phosphate as an allosteric inhibitor (V_lim activity) from the leaves of Prosopis juliflora was earlier observed to respond rapidly and reversibly to light/dark transitions ( Sinha et al. 1997b,c ). The experiments therefore, were conducted to study the potential regulation of the enzyme by a mechanism of phosphorylation/dephosphorylation. The desalted extract of the enzyme prepared from irradiated leaves showed a time‐dependent spontaneous inactivation of the V_lim activity when the extract was preincubated and an additional inactivation when incubated with ATP. The spontaneous inactivation is not inhibited by phosphatase inhibitors but the ATP‐dependent inactivation was abolished when either 5′‐p‐fluorosulphonylbenzoadenosine (FSBA) or glucose‐6‐phosphate (G6P), (both reported as inhibitors for the SPS‐protein kinase from spinach) was included during preincubation. FSBA also prevented the dark inactivation of SPS in the leaves of P. juliflora when fed through the transpiration stream. The activity of SPS measured under the V_max condition remained relatively unaffected by ATP or FSBA. The desalted extract prepared from darkened leaves on the other hand, when preincubated at 25°C showed a time‐dependent increase in the V_lim activity and the activation state of the enzyme. The spontaneous activation observed during preincubation appears to be due to the dephosphorylation of the enzyme and is strongly inhibited by okadaic acid, a potent protein phosphatase inhibitor. Alternately, feeding okadaic acid to excised leaves in the dark also blocked the subsequent light activation of V_lim activity. These results are consistent with the assumption that the light/dark regulation of V_lim activity observed in the leaves of P. juliflora was mediated through a dephosphorylation/phosphorylation mechanism.     

A stoichiometric study of heme degradation catalyzed by the reconstituted heme oxygenase system with special consideration of the production of hydrogen peroxide during the reaction

In heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of hemin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a "peroxo" species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase reaction.     

In Streptomyces lividans,acetyl‐CoA synthetase activity is controlled by O‐serine and Nɛ‐lysine acetylation

Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK⁶¹⁰). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD⁺‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N^?‐Lys acetylation.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

SIRT3 deficiency decreases oxidative metabolism capacity but increases lifespan in male mice under caloric restriction

Mitochondrial NAD⁺‐dependent protein deacetylase Sirtuin3 (SIRT3) has been proposed to mediate calorie restriction (CR)‐dependent metabolic regulation and lifespan extension. Here, we investigated the role of SIRT3 in CR‐mediated longevity, mitochondrial function, and aerobic fitness. We report that SIRT3 is required for whole‐body aerobic capacity but is dispensable for CR‐dependent lifespan extension. Under CR, loss of SIRT3 (Sirt3 ^−/− ) yielded a longer overall and maximum lifespan as compared to Sirt3 ^+/+ mice. This unexpected lifespan extension was associated with altered mitochondrial protein acetylation in oxidative metabolic pathways, reduced mitochondrial respiration, and reduced aerobic exercise capacity. Also, Sirt3 ^−/− CR mice exhibit lower spontaneous activity and a trend favoring fatty acid oxidation during the postprandial period. This study shows the uncoupling of lifespan and healthspan parameters (aerobic fitness and spontaneous activity) and provides new insights into SIRT3 function in CR adaptation, fuel utilization, and aging.     

Photomodification of a serine at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase by vanadate

Irradiation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach in the presence of vanadate at 4 degrees C resulted in rapid loss of carboxylase activity. The inactivation was light and vanadate dependent. When the enzyme was irradiated in the presence of the substrate ribulose 1,5-bisphosphate or an analogue such as fructose 1,6-bisphosphate, the inactivation was greatly reduced. Sodium bicarbonate and phosphate also protected against inactivation. No additional protection was observed in the presence of Mg2+ nor did Mg2+ alone protect. Carboxylase activity could be partially restored by treatment with NaBH4, and the photomodified protein could be tritiated with NaB3H4. Amino acid analysis showed that the tritium had been incorporated into serine. The data suggest that an active-site serine is photooxidized by vanadate to an aldehyde which results in activity loss. Irradiation in the presence of vanadate also resulted in cleavage in the large subunit of the enzyme which was subsequent to inactivation.     

The ultraviolet light and photosensitized inactivation of tobacco mosaic virus

The quantum yield for the inactivation of tobacco mosaic virus has been determined at 253.7 mµ and found to be 4.3 x 10^–6. The possible significance of the observed one-hit process of inactivation has been discussed in terms of the kinetics and the rupture of model substances including nucleic acid. The ultraviolet light inactivation, which proceeds independent of oxygen, occurs without change in physicochemical properties, with the possible exception of an enhanced sensitivity to thermal denaturation. The photosensitized inactivation of virus by acriflavine has been found to proceed parallel with the destruction of the dye. The action was found to be dependent upon adsorbed dye, and the inactivation is enhanced by the presence of oxygen.     

Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full-length form that binds to bacterial membranes

A plasmid, pKK-RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233-2. Escherichia coli strain XL1-blue transformed with pKK-RHO produced a catalytically active, full-length heme oxygenase. The 32-kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane-binding properties of a hydrophobic segment in its C-terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane-associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C-terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30-kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH--cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin. The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.