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Query: UNIPROT:P06889 (Mol)
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Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.
Mol Cell Biochem 1980 Dec 10
PMID:Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate. 720 67

The hydrogen photoevolution was studied to compare the efficiency of chloroplasts or solubilized chlorophyll in the presence of hydrogenase from Clostridium butyricum and methylviologen which links the electron transfer from photosystems to the exogenous enzyme. The hydrogen evolution by chloroplasts in the absence of exogeneous electron donors (or in the presence of irreversibly oxidized dithiotreitol or cysteine) is probably limited by cyclic electron flow shot-circuiting the photosystem 1. Efficiency of hydrogen photoproduction when ascorbate or NADP.H are used as electron donors is probably limited by reverse reaction of photoreduced methylviologen with the oxidized electron donor. The combination of both dithiotreitol and ascorbate prevents the shot-circuiting of photosystem 1 by methylviologen; in this case the maximum efficiency of hydrogen photoevolution was achieved up to 400 mumol H2 per 1 mg chlorophyll per hour.
Mol Biol (Mosk)
PMID:[Conditions for effective hydrogen photoevolution by chloroplasts in the presence of bacterial hydrogenase]. 738 27

Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.
Biochem Mol Biol Int 1995 Apr
PMID:Mitochondria-bound hexokinase from rabbit reticulocytes is resistant to the inactivation induced by Fe(II)/ascorbate. 754 32

We investigated the oxidative degradation pathway of 5CH3-H4PteGlu, the main extracellular folate and the predominant form of the vitamin found in food and blood. 5CH3-H4PteGlu is oxidized to 5CH3-5,6-H2PteGlu which subsequently undergoes C9-N10 bond cleavage yielding a pteridine residue and P-ABG, the latter step resulting in irreversible loss of vitamin activity. Under moderately acid conditions typical of the postprandial gut (pH 3.5) 5CH3-H4PteGlu is fairly stable (t1/2 = 273.6 min), while 5CH3-5,6-H2PteGlu is rapidly degraded (t1/2 = 16.9 min). In a neutral environment (pH 6.4) stability is reversed; 5CH3-H4PteGlu t1/2 = 12.0 mins, 5CH3-5,6-H2PteGlu t1/2 = 1504.6 min. Ascorbic acid was efficacious in the facile salvage of 5CH3-H4PteGlu from 5CH3-5,6-H2PteGlu which occurred rapidly and with significant efficiency (100% conversion) under acid (pH 3.5) conditions, t1/2 = 1.3 min (1 mmol/liter ascorbate), but was less efficient under neutral (pH 6.4) conditions t1/2 = 273.6 min (36% conversion). The presence of zinc and iron broadly maintains the pattern of effect, but increases all reaction rates. PteGlu was stable under all conditions studied. These results obtained in an artificial environment were supported by findings in human gastric juice: at a gastric pH of 1.47 with low endogenous ascorbate (7.0 mumol/liter), 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu both degrade instantly via C9-N10 bond cleavage to yield an equimolar amount of P-ABG. If the same gastric juice is spiked at 58.0 mumol/liter ascorbate (moderate endogenous concentration), 5CH3-H4PteGlu is stable (t1/2 = 334.7 min), while 5CH3-5,6-H2PteGlu is instantly salvaged to 5CH3-H4PteGlu with 43.3% efficiency, and the remaining 5CH3-5,6-H2PteGlu is degraded to P-ABG. In gastric juice with an elevated pH of 7.0 and no endogenous ascorbate, 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu are both stable, with no C9-N10 bond cleavage. This, for 5CH3-H4PteGlu, is in apparent contrast to findings at pH 6.4 in an artificial environment. The same gastric juice spiked to 50 mumol/liter ascorbate did not result in 5CH3-H4PteGlu salvage from 5CH3-5,6-H2PteGlu.(ABSTRACT TRUNCATED AT 400 WORDS)
Biochem Mol Med 1995 Jun
PMID:Nonenzymatic degradation and salvage of dietary folate: physicochemical factors likely to influence bioavailability. 755 25

The present study was conducted to investigate the effects of aging and medium supplements on steroid-induced ovalbumin mRNA in primary cultures of tubular gland cells from the chicken oviduct. In experiment 1, the effect of aging was examined by comparing the responsiveness to administration of estrogen and corticosterone in cells derived from laying hens and estrogen-primed chicks. In experiment 2, the effect of supplementing a culture medium with various compounds on the responsiveness to the steroid treatment was examined. In experiment 3, the effect of supplementing with ascorbic acid was tested in the presence or absence of the steroid hormones. The results indicated that the oviduct cells from immature chicks had clearer induction of ovalbumin mRNA by the steroid treatment than did those from laying hens. Among medium supplements, ascorbate increased the steroid responsiveness to a great extent, and fetal calf serum had modest, but long lasting, induction of ovalbumin mRNA. The drastic induction of ovalbumin mRNA by ascorbic acid supplementation was exerted only when the steroid hormones were present in the medium, implying that the effect of ascorbic acid may be auxiliary in steroid-induced transcription of the ovalbumin gene.
Comp Biochem Physiol B Biochem Mol Biol 1995 Oct
PMID:Induction of ovalbumin mRNA by ascorbic acid in primary cultures of tubular gland cells of the chicken oviduct. 758 52

The relationship between the metabolism of alpha-tocopherol (alpha-T) (vitamin E) and that of ascorbic acid (vitamin C) was examined in cultured hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Unlike vitamin E, the cellular content of vitamin C did not decline after overnight culturing of freshly prepared hepatocytes. In addition, this basal vitamin C content was not affected by the presence of alpha-T phosphate in the overnight culture medium. Supplementation of the overnight culture medium with vitamin C (10 microM to 10 mM) increased the cellular content of vitamin C by > 1 order of magnitude. Increasing the cellular content of ascorbate increased the protection against TBHP toxicity, with or without the presence of a physiological content of vitamin E. In vitamin E-supplemented cells, a loss of alpha-T occurred within 15 min of exposure to TBHP and before the decrease in cellular ascorbate content. The vitamin C content declined in parallel with the loss of cell viability. Supplementation of the overnight culture medium with increasing concentrations of ascorbate progressively spared the depletion of alpha-T while preventing the cell killing. Pretreatment with the ferric iron chelator deferoxamine or the antioxidant N,N'-diphenyl-1,4-phenylenediamine prevented the loss of ascorbate and the cell killing by TBHP in hepatocytes either sufficient or deficient in alpha-T. However, neither alpha-T nor ascorbate prevented the accumulation of DNA single-strand breaks caused by TBHP, indicating that these vitamins do not effectively scavenge the TBHP-derived radicals responsible for DNA damage. The data in the present study indicate that vitamins E and C act as independent antioxidants and that ascorbate does not regenerate alpha-T in cultured rat hepatocytes.
Mol Pharmacol 1995 Jul
PMID:Relationship of the metabolism of vitamins C and E in cultured hepatocytes treated with tert-butyl hydroperoxide. 762 78

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1-36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1-7-month-old infants (group 1) and 12-36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 mumol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean +/- SE: 6.76 +/- 1.86, 7.37 +/- 1.82, 61.9 +/- 1.86, 5.75 +/- 1.74 and 8.51 +/- 3.23 pmol/10(6) cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 +/- 1.15, 1.50 +/- 2.75, 1.44 +/- 2.75, 0.78 +/- 1.74 and 3.23 +/- 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 +/- 0.89, 13.5 +/- 1.17 and 51.7 +/- 3.23), while progesterone secretion remained unchanged (2.82 +/- 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins.(ABSTRACT TRUNCATED AT 400 WORDS)
J Steroid Biochem Mol Biol 1995 Jun
PMID:Human prepubertal testicular cells in culture: steroidogenic capacity, paracrine and hormone control. 762 44

It is established that acetaminophen exhibits oxidative behaviour. The effects of acetaminophen (0.3-14.5 microM) on methemoglobin levels, superoxide dismutase and Na(+)-K+ ATPase activities of normal and vitamin E or vitamin C pretreated erythrocytes were investigated. In acetaminophen incubated erythrocytes, methemoglobin concentration and superoxide dismutase activity were increased in a dose and incubation-time dependent manner, the activity of Na(+)-K+ ATPase was decreased by acetaminophen treatment. Vitamin E (1mg/dl of erythrocyte suspension) or vitamin C (1mg/dl of erythrocyte suspension) provided partial protection of hemoglobin, superoxide dismutase and Na(+)-K+ ATPase against acetaminophen action. Vitamin E was more effective than vitamin C.
Biochem Mol Biol Int 1995 Apr
PMID:Effects of acetaminophen on methemoglobin, superoxide dismutase and Na(+)-K+ ATPase activities of human erythrocytes. 762 22

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.
Plant Mol Biol 1995 Jul
PMID:Nature and regulation of pistil-expressed genes in tomato. 764 1

Substantial evidence supports the theory that free radicals, especially oxygen radicals, are involved in the process of aging. The human organisms have two ways to fight them: an enzymatic way with enzymatic intervention like superoxide dismutase, catalase... and a chemical way with the intervention of scavengers such as vitamins, cysteine, methionine, gluthatione... The aim of this work was to determine that an intakes of vitamins association: vitamin E, vitamin C and beta carotene induce an increase of singlet oxygen protection of erythrocytes' subjects. The method was based on the haemolytic effect of singlet oxygen which is generated by irradiation of hematoporphyrine at 365 nm, in 22 p. cent suspension of erythrocytes' subjects. Results show that a supply of beta carotene (15 or 30 mg/day), vitamin E (15 mg/day) and vitamin C (30 mg/day) involves an increase of singlet oxygen protection of erythrocytes of subjects. This protection appears very quickly after 15 days of treatment.
Biochem Mol Biol Int 1995 Feb
PMID:Increase of singlet oxygen protection of erythrocytes by vitamin E, vitamin C, and beta carotene intakes. 766 92


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