共查询到20条相似文献,搜索用时 9 毫秒
1.
Alisha Stephens-Jarnagin William K. Sietsema Duncan A. Miller Hector F. DeLuca 《Archives of biochemistry and biophysics》1983,220(2):502-508
Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-3H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [3H]retinoic acid. The biological activity of all discernible metabolities was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-β-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action. 相似文献
2.
B D Kabakoff N C Kendrick D Faber H F Deluca S Yamada H Takayama 《Archives of biochemistry and biophysics》1982,215(2):582-588
Oxygen-dependent calcium uptake by chick duodenal discs has been characterized and used to assay the relative activities of 1,25-dihydroxyvitamin D3 and its 24,24-difluoro analog. The calcium uptake was found to be stimulated by low sodium (30 mm) and phosphate (0.01–0.3 mm). The rate of oxygen-dependent calcium uptake was found half-maximal at a calcium concentration of 5 mm. At a concentration of 5 mm calcium, the uptake was linear for at least 15 min with approximately a threefold stimulation by prior administration of 1,25-dihydroxyvitamin D3 (125 ng). This determination, as well as increase in serum calcium and percentage bone ash, was used to assess the biological activities of 1,25-dihydroxyvitamin D3 and its 24,24-difluoro analog. The difluoro analog is about four to five times more active than 1,25-dihydroxyvitamin D3 as measured in each of these systems. 相似文献
3.
Sodium dodecyl sulfate was shown to promote both the inactivation and proteolytic degradation of the yeast glycoprotein, carboxypeptidase Y, with the former effect occurring six times faster than the latter. Although the proteolysis, as judged by polyacrylamide gel electrophoresis, was inhibited by pepstatin, which implicates the presence of proteinase A, the possibility of autodigestion could not be ruled out. A contributing role of the enzyme's carbohydrate moiety to these two processes was revealed by treating carboxypeptidase Y with endo-β-N-acetylglucosaminidase H. This treatment removes all four of the enzyme's Oligosaccharide chains in sodium dodecyl sulfate and as a consequence increases the rate of inactivation of the resulting carboxypeptidase Y by twofold and its proteolytic degradation by threefold relative to that of untreated enzyme. It thus appears that carboxypeptidase Y is a glycoprotein whose structural integrity and functional activity are influenced by its associated carbohydrate component. 相似文献
4.
Y. Tanaka J.K. Wichmann H.F. De Luca Y. Kobayashi N. Ikekawa 《Archives of biochemistry and biophysics》1983,225(2):649-655
To evaluate possible functional roles for 24,25-dihydroxyvitamin D3, 24,24-difluoro-25-hydroxyvitamin D3 has been synthesized and shown to be equally as active as 25-hydroxyvitamin D3 in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D3 was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D3, while large amounts were found when 25-hydroxyvitamin D3 was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D3, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D3 from 24,24-difluoro-25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D3 is less active than 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D3 is more active than 25-hydroxyvitamin D3 which is itself more active than 24R,25-dihydroxyvitamin D3. The 24,24-difluoro-1,25-dihydroxyvitamin D3 is equal to 1,25-dihydroxyvitamin D3, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D3 in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D3 cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds. 相似文献
5.
Y Tanaka H F DeLuca Y Kobayashi N Ikekawa 《Archives of biochemistry and biophysics》1984,229(1):348-354
A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism. 相似文献
6.
To study general stimulatory effects of 1,25-dihydroxyvitamin D3 on intestinal protein synthesis, slices of duodenal villi from 1,25-dihydroxyvitamin D3-treated and vitamin D-deficient rats were incubated in vitro for 90 min at the surface of medium containing [3H]leucine. Incorporation of the [3H]leucine into TCA-precipitated protein, which was shown to be linear for 12 h and 90% inhibited by cycloheximide, was increased by 50-60% at 26 h after a single injection of 125 ng of 1,25-dihydroxyvitamin D3 (three experiments, P less than 0.001). The increase, which was not due to circadian rhythm fluctuations of the intestine, was in synchrony with the second Ca2+ transport response observed by Halloran and DeLuca (Arch. Biochem. Biophys. 208, 477-486, 1981). However, no significant difference in [3H]leucine incorporation was observed before or during the initial Ca2+ transport response observed by Halloran and DeLuca, i.e., at 1.0, 3.0, and 6.5 h following an injection of 1,25-dihydroxyvitamin D3. The late onset of the 1,25-dihydroxyvitamin D3-induced increase in total protein synthesis implies that it is an indirect rather than a direct effect of the hormone. 相似文献
7.
Subcellular location of rat liver calciferol-25-hydroxylase 总被引:9,自引:0,他引:9
8.
Hideo Kurebayashi Akira Tanaka Tsutomu Yamaha 《Archives of biochemistry and biophysics》1982,215(2):433-443
The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of 18O2 or in a medium containing H218O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate () and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia. 相似文献
9.
S J Pilkis M R El-Maghrabi J Pilkis T H Claus 《Archives of biochemistry and biophysics》1982,215(2):379-389
Rat hepatic 6-phosphofructo-1-kinase (ATP:d-fructose-6-phosphate 1-phosphotransferase) was purified to homogeneity and its phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase examined. Up to 4 mol of phosphate could be incorporated per mole of tetrameric enzyme, and the phosphate was incorporated into seryl residues. Phosphorylation did not alter the affinity of the enzyme for fructose 6-phosphate or fructose 2,6-bisphosphate. The rate of phosphorylation was enhanced by allosteric activators of 6-phosphofructo-1-kinase such as AMP and fructose 2,6-bisphosphate, and it was decreased by the allosteric inhibitors ATP and H+. The phosphopeptide region of the enzyme subunit was susceptible to limited proteolysis by trypsin. Removal of the phosphopeptide did not affect the subunit molecular weight nor the maximum activity of the enzyme, but it enhanced the apparent affinity of the enzyme for both fructose 6-phosphate and fructose 2,6-bisphosphate. It is concluded that the phosphopeptide region of the enzyme subunit is an important determinant of the affinity of the enzyme for its substrate as well as for the allosteric activator fructose 2,6-bisphosphate. 相似文献
10.
Solubilized components of the vitamin D3-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D3in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b5 or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: Km(NADPH) = 3.14 μM, Km(NADH) = 31.25 μM, and Km(cyt c) = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, 3H-labeled vitamin D3, and soluble cytosolic supernatant, full vitamin D3-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D3-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D3 to 25-hydroxyvitamin D3. 相似文献
11.
The osmotic shrinking rate of unsonicated egg phosphatidylcholine (PC) liposomes in hypertonic NaCl was studied by determining the initial time rate of change of the reciprocal of the optical density, , in a stopped-flow kinetics apparatus, was found to be a linear function of reciprocal OD and reciprocal PC concentration, where the linear parameters were quite different depending on the size distribution of liposomes in the dispersion. An approximate theoretical calculation of relative shrinking rates suggests that the larger liposomes mask the osmotic activity of smaller liposomes in the same dispersion. It is concluded that this method should only be used for comparing osmotic permeabilities of liposomes dispersions when both the OD and liposome size distribution of the dispersions are the same. 相似文献
12.
Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 109m?1 s?1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations. 相似文献
13.
Heidrun Böck Susanne Abler Xian-Yang Zhang Hans Fritton Tibor Igo-Kemenes 《Journal of molecular biology》1984,176(1):131-154
The location of nucleosomes on rat satellite I DNA has been investigated using a new approach. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1. From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined. The location of nucleosomes along the satellite sequence was found to be non-random. Our results show that nucleosomes occupy a number of positions on satellite I DNA. About 35 to 50% of all nucleosomes are positioned in two corresponding major sites, the remainder in about 16 less preferred sites. The major nucleosome positions are apparently strictly defined with the precision of a single base-pair. These results were confirmed by other approaches, including restriction nuclease digestion experiments. There are good indications of a defined long-range organization of the satellite chromatin fiber in two or more oligonucleosomal arrays with distinct nucleosome configurations. 相似文献
14.
Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3 总被引:3,自引:0,他引:3
Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3. 相似文献
15.
16.
The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [14C]CCNU (1-(2-[14C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [14C]CCU (1-(2-[14C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [14C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [14C]CCNU was shown to undergo denitrosation to [14C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450. 相似文献
17.
Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes 总被引:2,自引:0,他引:2
E F Hildebrandt P C Preusch J L Patterson J W Suttie 《Archives of biochemistry and biophysics》1984,228(2):480-492
Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM. 相似文献
18.
19.
A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates 总被引:2,自引:0,他引:2
N Horiuchi T Shinki S Suda N Takahashi S Yamada H Takayama T Suda 《Biochemical and biophysical research communications》1984,121(1):174-180
A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals. 相似文献