Na+ + K+-ATPase activity and transport processes in toad corneal epithelium

1. Ascorbic acid, diamide and N-ethylmaleimide inhibit Na+ + K+-ATPase activity in toad corneal epithelium. 2. Ascorbic acid, diamide and N-ethylmaleimide increase alpha-aminoisobutyric acid accumulation in this tissue. 3. The effects of these compounds on corneal amino acid and ion transport are not mediated through alterations in Na+ + K+-ATPase activity.     

Mechanism of Inactivation of Bacteriophage J1 by Ascorbic Acid

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacillus casei, by ascorbic acid was investigated.

Bubbling air, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by ascorbic acid. In contrast, bubbling nitrogen gas, other reducing agents and radical scavengers prevented the inactivation. The results indicated that the inactivating effect of ascorbic acid was oxygen dependent and caused by free radicals formed during the autoxidation of ascorbic acid.

The target of ascorbic acid in the phage particle was not the tail protein but DNA. Ascorbic acid caused single-strand scissions in phage DNA, as exhibited by alkaline sucrose density gradient centrifugation analysis, and caused a slight decrease in the viscosity of DNA.     

Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint

Ascorbic acid, at physiological concentrations, can scavenge the myeloperoxidase-derived oxidant hypochlorous acid at rates sufficient to protect alpha 1-antiprotease against inactivation by this molecule. The rapid depletion of ascorbic acid at sites of inflammation, as in the inflamed rheumatoid joint, may therefore facilitate proteolytic damage.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.     

Biosynthesis of folic acid

Summary Biosynthesis of folic acid activity by Bacillus subtilis cell suspensions was studied with respect to substrates utilized as precursors. Among purine bases, adenine was utilized the best and not guanine although guanosine or guanylic acid were utilized efficiently. Among 3-C precursors, glyceraldehyde gave maximum synthesis of folate activity. The cells appeared to utilize p-aminobenzoate preferentially to p-aminobenzoyl-glutamate. Pteroic acid appears to be an intermediate in the synthesis of folate derivatives in this system. Ascorbic acid stimulates the synthesis to a great extent.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.     

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.     

Ascorbic acid and copper in linoleate oxidation. II. Ascorbic acid and copper as oxidation catalysts

Both ascorbic acid and copper were strong prooxidants in the oxidation of linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C. Minimum concentrations at which catalytic activity was detected were 1.3 x 10(-7) m for copper and 1.8 x 10(-6) m for ascorbic acid. For concentrations up to 10(-3) m, the increase in rate of oxidation with increase in concentration of catalyst was greater for ascorbic acid than for copper. Ascorbic acid had maximum catalytic activity at 2.0 x 10(-3) m, but was still prooxidant at the highest concentration tested (5.0 x 10(-2) m). Dehydroascorbic acid was a weaker prooxidant than ascorbic acid. Further degradation products of ascorbic acid were not prooxidant. In early stages of the oxidation autocatalytic behavior was observed with copper, but not with ascorbic acid. Ascorbic acid functioned as a true catalyst, i.e., it accelerated the reaction but it was not oxidized simultaneously with the linoleate. It is proposed that the dehydroascorbic acid radical initiates the linoleate oxidation reaction.     

Investigation of Helicobacter pylori ascorbic acid oxidating activity

Abstract Helicobacter pylori sonicate was shown to oxidize ascorbic acid. Ascorbic acid oxidation was determined by chromatography combined with electrochemical detection. Water soluble ascorbic acid oxidase activity was rather independent of pH with a pH optimum around 2. By gel filtration the oxidizing activity co-eluted with an absorbency peak at 408 nm. The relative molecular mass ( M _r) was approximately 14000. It is suggested that this oxidating activity was caused by a cytochrome c -like molecule. Ascorbic acid oxidating activity could also be extracted from bacterial membranes by detergents. Gel filtration showed several forms, the major one with a M _r= 19000. pH optimum was 6–7. Other oxidase-positive bacterial strains like Campylobacter coli, Enterobacter cloacae and Pseudomonas aeruginosa could degrade ascorbic acid. Since ascorbic acid oxidation by Helicobacter pylori whole bacterial lysates has a pH optimum in the acidic range corresponding to pH in gastric fluid, the activity of the cytochrome c -like water soluble oxidant of Helicobacter pylori seems to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients.     

Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia

Ascorbic acid (vitamin C) has been suggested to protect cerebral tissue in a variety of pathophysiological situations such as head trauma, ischemia or Alzheimer's disease. Most of these protective actions have been attributed to the antioxidative capacity of ascorbic acid. Besides the presence of elevated levels of oxygen radicals, prostaglandins produced by neurones and microglial cells seem to play an important role in prolonged tissue damage. We investigated whether ascorbic acid alone inhibits prostaglandin E2 (PGE2) synthesis and may augment the inhibitory effect of acetylsalicylic acid on prostaglandin synthesis. Ascorbic acid dose-dependently inhibited PGE2 synthesis in lipopolysaccharide-treated primary rat microglial cells (IC50 = 3.70 micro m). In combination with acetylsalicylic acid (IC50 = 1.85 micro m), ascorbic acid augmented the inhibitory effect of acetylsalicylic acid on PGE2 synthesis (IC50 = 0.25 micro m in combination with 100 micro m ascorbic acid). Ascorbic acid alone or in combination with acetylsalicylic acid did not inhibit cyclooxygenase-2 (COX-2) protein synthesis but inhibited COX-2 enzyme activity. Our results show that ascorbic acid and acetylsalicylic acid act synergistically in inhibiting PGE2 synthesis, which may help to explain a possible protective effect of ascorbic acid in various brain diseases.     

Two types of metabolic regulation of phenol oxidase activity in ontogenesis of house fly

Changes of the tyrosinase activity in ontogenesis of the house fly Musca domestica were shown to be phase-specific and ontogenetic changes of tyrosinase and dihydroxyphenylalanine oxidase activities proved to be coordinated. Ascorbic acid stimulated some ontogenetic stages of the house fly and physiological indices, such as fertility, survival at different stages, and weight of puparia. Also, ascorbic acid modulated the tyrosinase activity.     

Selective effects of ascorbic acid on acetylcholine receptor number and distribution

Ascorbic acid in soluble extracts of neural tissue can account for the increase in surface acetylcholine receptors (AChR's) seen on L5 myogenic cells treated with crude brain extract (Knaack, D., and T. R. Podleski, 1985, Proc. Natl. Acad. Sci. USA., 82:575-579). The present study further elucidates the nature of the response of L5 cells to ascorbic acid. Light autoradiography showed that ascorbic acid treatment affects both the number and distribution of surface AChR's. Ascorbic acid, like crude brain extracts, caused a three- to fourfold increase in average AChR site density. However, the number of AChR clusters induced by ascorbic acid was only one-fifth that observed with crude brain extract. The rate constant for degradation of AChR in ascorbic acid-treated cells of 0.037 +/- 0.006 h-1 (t1/2 = 19 h) was not significantly different from that in untreated controls of 0.050 +/- 0.001 h-1 (t1/2 = 14 h). The increase in AChR site density is primarily due to a 2.8-fold increase in the average rate of AChR incorporation. Ascorbic acid also stimulates thymidine incorporation and increases the total number of nuclei per culture. However, cellular proliferation is not responsible for the increase in AChR's since 10 microM cytosine arabinofuranoside blocks the mitogenic effect without affecting the AChR increase. The specificity of ascorbic acid on AChR expression was established by showing that (a) ascorbic acid produced only a slight increase in total protein, which can be accounted for by the mitogenic effect, and (b) the normal increase seen in creatine kinase activity during muscle differentiation was not altered by the addition of ascorbic acid. We conclude that the action of ascorbic acid on AChR number cannot be explained by changes in cell growth, survival, differentiation, or protein synthesis. Therefore, in addition to a minor stimulation of AChR clustering, ascorbic acid specifically affects some aspect of the AChR biosynthetic pathway.     

Metabolic regulation of two types of phenol oxidase activity in ontogenesis of house fly

Changes of the tyrosinase activity in ontogenesis of the house fly Musca domestica were shown to be phase-specific and ontogenetic changes of tyrosinase and dihydroxyphenylalanine oxidase activities proved to be coordinated. Ascorbic acid stimulated some ontogenetic stages of the house fly and physiological indices, such as fertility, survival at different stages, and weight of puparia. Also, ascorbic acid modulated the tyrosinase activity.     

Ascorbic acid modulation of calcium channels in pancreatic beta cells

We have studied the effect of ascorbic acid on voltage-dependent calcium channels in pancreatic beta cells. Using the whole-cell and perforated-patch variants of the patch clamp technique to record calcium tail currents, we have shown that the slowly deactivating (SD) calcium channel, which is similar to the T-type channel in other cells, is inhibited in a voltage-dependent manner by ascorbic acid (AA). The other channels that carry inward current in beta cells, FD calcium channels and sodium channels, are unaffected by AA. Ascorbic acid causes a voltage-dependent decrease in the magnitude of the SD channel conductance which can be explained by the hypothesis that approximately 50-60% of the channels have their voltage dependence shifted by approximately 62 mV in the depolarizing direction. Thus, ascorbate appears to modify only a fraction of the SD channels. The activation kinetics of the ascorbate-modified channels are slower than control channels in a manner that is consistent with this hypothesis. Deactivation and inactivation kinetics are unaffected by ascorbate. These effects of ascorbate require metal ions, and it appears that some of the activity of ascorbate is due to a product of its metal catalyzed oxidation, perhaps dehydroascorbate.     

L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases.

Ascorbic acid 2-sulphate has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 mumol/mg per min (150 mumol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes.?     

Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli

Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.     

Role of Ascorbic Acid in Bud Development

Ascorbic acid was found to increase bud development in Pisum sativum L. The interactions of ascorbic acid with indole-3-acetic acid, kinetin and gibberellic acid were studied. It was found that ascorbic acid promoted bud growth and overcame the inhibitory effect of auxin. When applied with gibberellin, bud growth was greatly enhanced. Ascorbic acid promoted bud development in red light only; it did not in far-red or dark.     

Influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media

Ascorbic acid rapidly decays in plant tissue culture media. Within 50 min to 3 h after preparing 100 mM solutions, ascorbic acid was destroyed. Autoclaving, shaking flasks, high light intensity and increasing pH over a range from 4.5–7 accelerated decay. Ascorbic acid was oxidized to dehydroascorbic acid which also underwent decay. Within 11 h and 15 min after adding ascorbic acid both ascorbic acid and its oxidation product, dehydroascorbic acid, disappeared from medium. Since ascorbic acid is rapidly destroyed in plant tissue culture media it may not exert its effect as an intact molecule. Instead its antioxidant/antibrowning role in plant cell, tissue and organ cultures may be mediated by some product of further oxidation.     

Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin

Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO(2)(-) + NO(3)(-)) production and l-arginine to l-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by approximately 50% without altering the K(m) for l-arginine. The effect of ascorbate was tetrahydrobiopterin (BH(4))-dependent, because ascorbate was ineffective with BH(4) concentrations >10 microm or in PAECs treated with sepiapterin to increase intracellular BH(4). The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH(4). These data suggest that ascorbic acid enhances NO bioactivity in a BH(4)-dependent manner by increasing intracellular BH(4) content.     

The influence of glucose, other monosaccharides, and ascorbic acid on tyrosine hydroxylase activity of rat striatal synaptosomes.

We investigated the action of glucose, other monosaccharides, and ascorbic acid on the activity of tyrosine hydroxylase in rat striatal synaptosomes. We found that glucose at 0.2 mM maximally activated enzyme activity by as much as 100 percent and caused half-maximal activation at 0.036 mM. Mannose, fructose and galactose also stimulated tyrosine hydroxylase activity, half-maximal activation occurring at 0.036, 8, and 50 mM, respectively; arabinose was inactive up to 100 mM. Ascorbic acid did not stimulate enzyme activity at 0.1 and 1 mM, and at 10 mM was inhibitory.The activating effect of glucose on tyrosine hydroxylase activity was blocked by 2-deoxyglucose and by glucosamine. We interpret the action of glucose to be dependent upon its metabolism and to be indirect, probably due to the maintenance of the cofactor in the reduced form in the synaptosomes.