Ascorbic Derivative Research Articles

Enhancement of Plasmacytoma Cell Growth by Ascorbic Acid is Mediated Via Glucose 6-phosphate Dehydrogenase

We investigated the mechanism by which some types of cancer cells grow faster in the presence of ascorbic acid supplementation. Adj.PC-5, a mouse plasmacytoma cell, is known to show ascorbic acid-dependent growth and was chosen as a test system. The growth of cancer cells was measured by the colony number on soft agar or the cellular proliferation in suspension culture. The ascorbate level was measured by a high performance liquid chromatography system with an electrochemical detector. Glucose 6-phosphate dehydrogenase was analyzed both on the specific enzyme activity level and on the transcription level by performing Northern blot analysis. Ascorbyl 2-phosphate among the ascorbate derivatives was the most efficient in stimulating cell growth. The intracellular and extracellular ascorbate concentrations following treatment with either ascorbate or ascorbyl 2-phosphate suggest that the superiority of ascorbyl 2-phosphate for stimulating cell growth may be due to its slow conversion to ascorbate in the culture medium. The steady transformation to ascorbate ensures sustained levels of ascorbate in the culture medium and thereby maximizes the growth stimulatory effect of ascorbate. Ascorbyl 2-phosphate markedly enhanced, in a concentration-and time-dependent manner, mRNA synthesis as well as the enzymatic activity of glucose 6-phosphate dehydrogenase, which is known to be a rate-limiting enzyme in cell growth. On the other hand, simultaneous addition of dehydroisoandrosterone, a well- known inhibitor of glucose 6-phosphate dehydrogenase, to the culture medium abrogated the growth stimulation by ascorbyl 2-phosphate, and it also reduced the glucose 6-phosphate dehydrogenase activity proportionately. The results from this study suggest that enhanced glucose 6-phosphate dehydrogenase activity may at least in part explain the stimulation of cell growth by ascorbate or ascorbyl 2-phosphate.

Synthesis and Characterization of New Ascorbic Derivative: Sodium Isostearyl 2-O-L-Ascorbyl Phosphate

A novel alkyl ester of Vitamin C derivative, originated in a stable ascorbate derivative, sodium L-ascorbic acid 2-phosphate (VCP-Na), was chemically synthesized. The thermal stability, surface tension, distribution between organic and water phase, and in vitro skin permeability were evaluated. This monoalkyl ester derivative was identified as sodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-Na) by UV spectra, infrared spectra, mass spectra, and nuclear magnetic resonance spectroscopy. The reaction afforded VCP-IS-Na in a high yield (60%). VCP-IS-Na exhibited satisfactory stability in neutral solution comparable to that of a typical stable derivative, VCP-Na. Increased skin permeability was superior to those of VCP-Na and ascorbic acid (VC). VCP-IS-Na that is susceptible to the enzymatic hydrolysis by tissue esterase and/or phosphatase released VC in the skin tissues. Thus, these characteristics indicate that the novel Vitamin C derivative presented here, VCP-IS-Na, may be effective pro-Vitamin C for skin care application.

Ascorbyl palmitate augments hypoxic respiratory response in the cat.

The redox signaling is germane for the hypoxia-sensing mechanisms at the carotid body. This raises the strong possibility that agents possess reducing and antioxidant attributes, such as ascorbate, could influence the hypoxic respiratory response. However, water solubility of ascorbate makes its effectiveness at membrane-associated target sites dubious. In this study, we sought to determine the effect of ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbate which penetrates biomembranes, on hypoxic respiration in the anesthetized, paralyzed and ventilated cat. AP was given by gavage: 600 mg/kg daily for 6 days before the beginning of the acute experiment. Respiration was then assessed from the phrenic electroneurogram, from which peak phrenic amplitude, a surrogate of tidal component, respiratory frequency, and their product, the minute phrenic output, were quantified. The response to normocapnic hypoxia, 7% O(2) in N(2), in the AP-treated cats was compared with that in controls. We found that AP augmented hypoxic respiration, delayed the appearance of hypoxic depression and decreased it, although the stimulatory/depressant character was preserved. The results suggest that the ascorbate moiety of AP interacts with the hypoxia-sensing mechanisms. Ascorbate may affect hypoxic respiration at multiple stages of chemotransduction pathways, which are subject to continuing uncertainties. The study highlights the augmentative effect of AP, a redox modulator, on hypoxic respiration, which may have a therapeutic potential.

Ascorbyl Palmitate Augments Hypoxic Respiratory Response in the Cat

The redox signaling is germane for the hypoxia-sensing mechanisms at the carotid body. This raises the strong possibility that agents possess reducing and antioxidant attributes, such as ascorbate, could influence the hypoxic respiratory response. However, water solubility of ascorbate makes its effectiveness at membrane-associated target sites dubious. In this study, we sought to determine the effect of ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbate which penetrates biomembranes, on hypoxic respiration in the anesthetized, paralyzed and ventilated cat. AP was given by gavage: 600 mg/kg daily for 6 days before the beginning of the acute experiment. Respiration was then assessed from the phrenic electroneurogram, from which peak phrenic amplitude, a surrogate of tidal component, respiratory frequency, and their product, the minute phrenic output, were quantified. The response to normocapnic hypoxia, 7% O₂ in N₂, in the AP-treated cats was compared with that in controls. We found that AP augmented hypoxic respiration, delayed the appearance of hypoxic depression and decreased it, although the stimulatory/depressant character was preserved. The results suggest that the ascorbate moiety of AP interacts with the hypoxia-sensing mechanisms. Ascorbate may affect hypoxic respiration at multiple stages of chemotransduction pathways, which are subject to continuing uncertainties. The study highlights the augmentative effect of AP, a redox modulator, on hypoxic respiration, which may have a therapeutic potential.

Suppressive effects of ascorbate derivatives on ultraviolet-B-induced injury in HaCaT human keratinocytes.

The aging of skin, including sunburning, is caused by ultraviolet (UV) irradiation. Here, we examined the inhibitory effect of ascorbic acid (AsA) and its derivatives AsA 2-phosphate (AA-2P) and AsA 2-glucoside (AA-2G) on UV-B- induced cytotoxicity in HaCaT keratinocytes. Results show that cell viability significantly decreased when exposed to UV-B at 0.1-0.4 J/cm2 in a dose-dependent manner. In this study, AsA could not inhibit cytotoxicity, but AA-2P and AA-2G was able to cancel the harmful effect of UV-B when treated at high levels of 0.5-5 mM. These results indicate that the masking of the C-2 OH group may be an effective modification for AsA to inhibit UV-B-induced cytotoxicity in human keratinocytes.

Vitamin C activity in guinea pigs of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain.

A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C(12), 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.

Availability of Ascorbyl Palmitate, a Lipophilic Derivative of Ascorbate, for Redox Signalling in the Carotid Body

The exact mechanism of hypoxia sensing by the carotid body remains unknown, but the redox signalling seems to be involved, due to the presence of vitamin C, or ascorbate, in the organ. In my study, I hypothesize that ascorbyl palmitate, a lipophilic derivative of ascorbate, ought to be able to penetrate the neural biomembranes, as opposed to water-soluble ascorbate, and, acting as a carrier of the ascorbate moiety, would facilitate the chemosensory respiratory response. The results support this contention.

Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids, as skin antioxidants.

A series of novel monoacylated vitamin C derivatives were chemically synthesized with a stable ascorbate derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and acid anhydrides in pyridine. Their solubility in organic phase, thermal stability, radical scavenging activity, and in vitro skin permeability was evaluated. These monoacylated derivatives were identified as 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) by UV spectra, elemental analyses, and nuclear magnetic resonance spectroscopy. The reactions afforded 6-Acyl-AA-2G in high yields (30-60%). 6-Acyl-AA-2G exhibited satisfactory stability in neutral solution comparable to that of a typical stable derivative, AA-2G, and also showed the radical scavenging activity. The lipid solubility of 6-Acyl-AA-2G was increased with increasing length of their acyl group. Increased skin permeability was superior to those of AA-2G and ascorbic acid (AsA). 6-Acyl-AA-2G that is susceptible to enzymatic hydrolysis by tissue esterase and/or alpha-glucosidase produces AA-2G and AsA, which is in the skin tissues. Thus, these findings indicate that the novel vitamin C derivatives presented here, 6-Acyl-AA-2G, may be effective antioxidants in skin care and medicinal use.

Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation.

The present study was conducted to examine the effect of ascorbic acid 2-O-alpha-glucoside (AA-2G), a stable ascorbate derivative, on the sustenance of cytoplasmic maturation responsible for subsequent developmental competence after in vitro fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured for 44 h in North Carolina State University 37 medium supplemented with cysteine, gonadotropins, 10% (v:v) porcine follicular fluid, and 0-750 microM AA-2G. When oocytes were matured in the presence of 250 microM AA-2G, their ability to promote transformation of the sperm nucleus into the male pronucleus (MPN) was strongly enhanced after in vitro fertilization. Similarly, the presence of 25 microM beta-mercaptoethanol (ME) enhanced the degree of progression to MPN of penetrated sperm by associating with the increase in intracellular glutathione (GSH) content. Although the AA-2G treatment during oocyte maturation showed no influence on the GSH concentration, significantly higher levels of ascorbic acid (AsA) were detected in these oocytes than in those oocytes cultured without AA-2G (P < 0.05). The length of DNA migration encompassed by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase system, was not increased in the oocytes treated with AA-2G, whereas ME treatment could not block the DNA damage by ROS. These findings indicate that AA-2G in maturation medium can potentiate the cellular protection of oocytes against oxidative stress by continuously supplying AsA. The proportion of development to the blastocyst stage after in vitro insemination was significantly increased in oocytes matured with AA-2G (P < 0.05), and this proportion showed no difference in comparison with that of oocytes treated with ME. These findings suggest that a critical concentration of intracellular AsA, supplied by AA-2G during in vitro maturation, plays an important role in supporting the cytoplasmic maturation responsible for developmental competence after fertilization by prevention of oxidative stress against porcine oocytes.

Male Pronuclear Formation and Blastocyst Formation Are Improved by Supplementation of Ascorbic Acid 2-O-.ALPHA.-Glucoside During In Vitro Maturation Culture of Denuded Porcine Oocytes.

The present study was conducted to examine the effect of ascorbic acid (AsA) on the cytoplasmic maturation of cumulus-denuded porcine oocytes during in vitro maturation (IVM) culture. After mechanical removal of cumulus cells, cumulus-denuded oocytes (DOs) were cultured for 44 h in BSA-free NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and 0 to 750 μM ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative, and then assessed for their developmental changes, including the progression of meiosis, in vitro fertilization (IVF) parameters and subsequent embryo development. Treatment with 250 μM AA-2G during IVM culture in DOs showed no influence on the rates of maturation (60-61%), sperm penetration (86%), and polyspermy (69-71%) following IVF, but enhanced the ability (43%) to form male pronucleus (MPN) compared to that of DOs treated without AA-2G (20%) (P<0.05). Although this rate of MPN formation of DOs was still lower than that of COCs (66%) (P<0.05), the addition of 250 μM AA-2G to maturation medium in DOs apparently enabled IVM-IVF-derived zygotes to develop to the blastocyst stage (3%). The levels of intracellular AsA and glutathione in DOs matured with AA-2G were significantly higher than those in DOs matured without AA-2G (P<0.05). Moreover, the length of DNA migration, analyzed using the comet assay, was increased by oxidative damage in DOs treated without AA-2G during IVM culture, but treatment with AA-2G abated DNA damage, indicating that intracellular AsA supplied by AA-2G can potentiate the cellular protection of oocytes against oxidative stress through its scavenging activity during IVM culture. Thus, the present findings suggest that the preventing oxidative stress in oocytes during meiotic maturation supports the cytoplasmic maturation responsible for the subsequent developmental competence post-fertilization.

Influence of dietary ascorbate derivatives on tissue copper, iron and zinc concentrations in grass shrimp, Penaeus monodon

Effects of three ascorbate derivatives namely, l-ascorbyl-2-sulfate (C2S), l-ascorbyl-2-monophosphate–Mg (C2MP–Mg) and l-ascorbyl-2-polyphosphate (C2PP) on tissue (hepatopancreas and muscle) copper, iron and zinc concentration in juvenile grass shrimp, Penaeus monodon, were studied. The control diet contained 12 mg/kg copper, 318.1 mg/kg iron, 125.8 mg/kg zinc and 0 mg/kg ascorbate supplemented with either adequate or high (5× adequate) dietary levels of C2S, C2MP–Mg and C2PP. Each diet was fed to triplicate groups of shrimp (mean initial weight: 0.67±0.05 g) for 8 weeks. Significantly ( P<0.05) higher tissue iron and lower copper concentrations were observed in shrimp fed diets containing ascorbate and high ascorbate, respectively, than shrimp fed the control diet regardless of the ascorbate source. Shrimp fed C2MP–Mg- and C2PP-containing diets had higher tissue iron concentrations than shrimp fed C2S-containing diets regardless of ascorbate supplementation level. Lower hepatopancreas copper concentrations were observed in shrimp fed C2MP–Mg- and C2PP-containing diets than in shrimp fed C2S-containing diets at the high ascorbate supplementation level. Tissue zinc concentrations in shrimp were not affected by either ascorbate source or ascorbate supplementation level.

Ascorbate 6-palmitate protects human erythrocytes from oxidative damage

Lipid-soluble antioxidants, such as α-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane α-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the α-tocopheroxyl radical to α-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.

Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy- vitamin D3-induced differentiation of HL-60 cells.

1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1,25-(OH)2D3-induced differentiation of HL-60 cells can be explained by redox regulation of the cAMP pathway.

The Dual Roles of Nutrients as Antioxidants and Prooxidants: Their Effects on Tumor Cell Growth

The development of a beneficial or a detrimental cellular response by a nutrient will depend on the nutrient’s antioxidant or prooxidant characteristics, which in turn are a product of the cellular oxygen environment. Nutrients such as carotenoids, tocopherols or ascorbate derivatives will demonstrate an antioxidant or prooxidant characteristic depending on the redox potential of the individual molecule and the inorganic chemistry of the cell. Nutrients acting as chemopreventives, inhibit the continual growth of transformed clones of cells through their prooxidant activity. In contrast, when an antioxidant activity occurs in transformed cells an enhanced growth may result. In addition, when an inappropriate prooxidant activity develops in normal cells, the reactive oxygen metabolites generated could damage the DNA and cellular membranes. The cellular response is usually a loss of normal regulatory function and activity, depressing cellular intergrity. Therefore, the labile redox character of each nutrient must be considered in terms of the extracellular and intracellular microoxygen environment. To predict if a specific nutrient will have a beneficial or detrimental effect on a particular tissue or cell, it is important to identify markers that will characterize the biologic activities of each nutrient and elucidate a possible mechanism of action for that nutrient. In various tissues chemopreventive agents derived from nutrients have been shown in laboratory animal studies and in some human intervention trials to inhibit the growth and development of premalignant or malignant lesions. Examples of these tissues include oral tissues, esophagus, gastric cardia and lung tissues. Recently, some clinical studies demonstrated no reduction in the incidence of premalignant change, but, to the contrary, statistical evidence indicated an increase in cancer development. In general, the results of clinical intervention trials remains equivocal. The use of chemopreventive agents without considering their pharmacologic, oxygen-responsive characteristics will produce unwanted iatrogenic side effects or further cloud evidence of these nutrients’ biologic activities.

Detection of metabolic conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid in tiger prawn (Penaeus monodon) using high-performance liquid chromatography and colorimetry

Biochemical conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid by acid phosphatase and ascorbate-2-sulfate sulfohydrolase, respectively, were found in extracts of a hepatopancreas of Penaeus monodon, bovine liver and tilapia liver. Both enzymes were assayed with high-performance liquid chromatography (HPLC) and colorimetry. Colorimetry was based on the reduction of a color of 2,6-dichlorophenolindophenol (DCIP) when ascorbic acid was released from enzymatic activity. Assay of acid phosphatase either with HPLC or with colorimetry was found to be equally reliable. However, sensitivity of the HPLC assay was slightly higher than that of colorimetry; HPLC was able to detect activity as little as 1 nmol ascorbic acid released per min, whereas colorimetry was limited at 6–7 nmol/min. Assay of ascorbate-2-sulfate sulfohydrolase in crude extracts with the HPLC technique was found to be more specific than that with the colorimetric assay. The excess reduction of DCIP color not related to the sulfohydrolase activity was observed in the colorimetric technique. An accumulation of ascorbic acid in a hepatopancreas of P. monodon fed with feeds supplemented with phosphorylated or sulfated ascorbic acid was higher than that of the prawn fed with feed without ascorbic acid. The accumulated ascorbic acid was possibly from the activity of acid phosphatase or the sulfohydrolase that hydrolyzed phosphorylated or sulfated derivatives in vivo, respectively. Metabolism of the ascorbate derivatives in the prawn is discussed.

Accessibility and reactivity of ascorbate 6-palmitate bound to erythrocyte membranes

Lipophilic derivatives of ascorbic acid may protect lipid bilayers and micelles against lipid peroxidation. In this work the binding, accessibility, and reducing capacity of ascorbate 6-palmitate (A6P) were studied in human erythrocyte membranes. In contrast to less lipophilic carbon-6-modified ascorbate derivatives, A6P bound to erythrocyte membranes in a concentration-dependent manner. This binding was preserved following centrifugation washes, but was largely reversed by extraction with bovine serum albumin. Most of the ascorbyl groups of membrane-bound A6P were readily accessible to oxidation by water-soluble oxidants. Ferricyanide quantitatively oxidized membrane-bound A6P, but the latter spared endogenous tocopherols from destruction. In EPR studies, A61? was much more effective than ascorbate in reducing nitroxide spin labels positioned at either carbon-5 or carbon-16 of membrane-bound stearic acid in both intact cells and in membranes. A6P, thus, appears to intercalate into the erythrocyte membrane with the ascorbyl group located superficially, but with access to the hydrophobic membrane interior, and with the ability to recycle endogenous a-tocopherol during oxidant stress.

Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes

Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [ 14C]ascorbate to [ 14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.

E.p.r. Studies of Reduced Rat Liver Mitochondria incubated with Nitric Oxide

Conference Article| May 01 1995 E.p.r. Studies of Reduced Rat Liver Mitochondria incubated with Nitric Oxide MARILIA PAIXÃO LINHARES; MARILIA PAIXÃO LINHARES * 1Centre for the Study of Metals in Biology and Medicine, Division of Life Sciences, King's College, Campden Hill Road, London W8 7AH, U.K. Search for other works by this author on: This Site PubMed Google Scholar RICHARD CAMMACK RICHARD CAMMACK 1Centre for the Study of Metals in Biology and Medicine, Division of Life Sciences, King's College, Campden Hill Road, London W8 7AH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (2): 232S. https://doi.org/10.1042/bst023232s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation MARILIA PAIXÃO LINHARES, RICHARD CAMMACK; E.p.r. Studies of Reduced Rat Liver Mitochondria incubated with Nitric Oxide. Biochem Soc Trans 1 May 1995; 23 (2): 232S. doi: https://doi.org/10.1042/bst023232s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Properties of Ascorbate Oxidase Produced byAcremoniumsp. HI-25

The presence of copper and carbohydrate was demonstrated in an acidic ascorbate oxidase purified from Acremonium sp. HI-25. The enzyme was inhibited by cyanide, azide, and sulfide. The reaction product from L-ascorbic acid was identified to be dehydro-L-ascorbic acid. This enzyme did not oxidize other electron donors than ascorbate derivatives. Stoichiometric investigations on the oxidation reaction found that this enzyme belongs to the EC 1.10.3.3 group, using molecular oxygen as an electron acceptor. The Km for L-ascorbic acid was estimated to be 0.29 mM. The activation energy for inactivation of Acremonium ascorbate oxidase, 129 kcal/mol, was four and three times higher than those of ascorbate oxidases from cucumber and pumpkin, respectively.

Substrate Specificity of Ascorbate Oxidase: Unexpected Similarity to the Reduction Site of Dopamine β-Monooxygenase

A series of ascorbate derivatives has been used to examine the specificity of the reduction site of ascorbate oxidase. Replacement of the 6-OH group of ascorbic acid with either hydrogen or bromine does not alter the substrate activity significantly. 6-Amino-6-deoxy-L-ascorbic acid is a weak substrate for the enzyme, suggesting that positively charged groups at the 6-position are not well tolerated by the enzyme. The modification of the 5-OH reduces the effective interaction with the enzyme and the replacement of 6-OH with 6-S-phenyl- or 6-O-phenyl groups significantly increases the affinity for the enzyme. Both 2-Amino-6-S-phenyl-L-ascorbic acid and imino-D-glucoascorbic acid are not substrates for the enzyme. The stereoelectronic properties and alternate binding modes of these molecules are being considered to explain these observations. The substrate specificity of the enzyme is compared to the specificity of the reduction site of dopamine β-monooxygenase.