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1.
The effects of electrical stimulation, γ-aminobutyric acid (GABA), acetylcholine (ACh), norepinephrine (NE), 5-hydroxytryptamine (5-HT), GABA agonists and bicuculline were studied on spontaneous movements of isolated rat oviduct. The tissue did not respond to electrical stimulation or to GABA, NE and 5-HT when added to the incubation medium. ACh produced contractions related to its concentration which were maximal at the diestrous-1 phase when GABA caused a 20% rise in the ACh contraction. This effect was mimicked by GABA agonists whereas it was suppressed by bicuculline. β-Estradiol benzoate (EB) increased ACh contractions in diestrous-1 and in the late proestrous phases. GABA did not modify the EB effect. Progesterone did not modify ACh contractions in any of the studied phases. These findings suggest a possible modulatory role for GABA on ACh responses in the isolated rat oviduct. 相似文献
2.
The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 106. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T1-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins. 相似文献
3.
A prototype analytical device incorporating an immobilized oxidase enzyme reactor is described. The monitor assembly involves the continuous measurement of dissolved oxygen in the sample stream and uses a commercially available Clark electrode mounted into a miniature vortex mixer. Utilizing glucose oxidase covalently coupled to porous glass particles a reagentless analytical procedure is presented for glucose in both simple and complex biological fluids. Operational limitations are described and characterized for both kinetic and endopoint methods of analysis. 相似文献
4.
Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation. 相似文献
5.
A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966). 相似文献
6.
The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones. 相似文献
7.
Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney 总被引:1,自引:0,他引:1
M D Maines 《Archives of biochemistry and biophysics》1982,216(1):17-26
The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane. 相似文献
8.
[3H]Muscimol binding at 23°C and muscimol stimulated [3H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [3H]muscimol binding sites can be observed. At 23°C [3H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [3H]muscimol binding and they increase the affinity of muscimol as a stimulator of [3H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [3H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [3H]flunitrazepam binding. Binding of [3H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag+, Hg+ and Cu2+ in μM concentrations, Ag+ being most potent. The effects of 100 μM AgNO3 persist after repeated washing of the membranes. When membranes are pretreated with AgNO3 only one apparent population of [3H]muscimol binding sites with high affinity (Kd: 6–8 nM) is found. In AgNO3 pretreated membranes, the affinity of muscimol as stimulator of [3H]flunitrazepam binding is increased 18 times (EC50 14 nM) when compared to control membranes, (EC50 253 nM). In AgNO3 pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [3H]muscimol binding or baseline and muscimol stimulated [3H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [3H]dihydropicrotoxinin binding. 相似文献
9.
A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced. 相似文献
10.
11.
Arnold Boersma Michel Lhermitte Geneviève Lamblin Pierre Degand 《Carbohydrate research》1981,95(2):227-236
The carbohydrate chains of the human-parotid, proline-rich glycoprotein are linked through a single type of carbohydrate-peptide linkage (asparaginyl-N-acetyl-glucosamine). The structure of the internal part of the carbohydrate chains, determined by chemical, enzymic, and g.l.c.-m.s. methods, includes the trimannosyl-di-N-acetylchitobiose core involved in the carbohydrate-peptide linkage. Furthermore, an L-fucose residue is linked to the 2-acetamido-2-deoxy-d-glucosyl residue linked to the L-asparaginyl residue. The sequence of the peripheral part of the chains has also been determined as α-L-Fucp→β-d-Galp→β-d-GlcpNAc→α-d-Manp, suggesting a double-branched, basic carbohydrate structure. 相似文献
12.
Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system 总被引:4,自引:0,他引:4
The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented. 相似文献
13.
The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O2 mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxy-dopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75–100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus Superoxide dismutase). After the H2O2, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O2 and H2O2, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction. 相似文献
14.
Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase 总被引:3,自引:0,他引:3
Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [14C]Asp C-Asp exchange is much faster than [32P]carbamyl phosphate Pi exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound Pi or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: Ea (>15 °C) = 6.3 kcal/ mole and Ea (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with Ea ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme. 相似文献
15.
Hirofumi Ogawa Yoshihisa Ishiguro Motoji Fujioka 《Archives of biochemistry and biophysics》1983,226(1):265-275
Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The Km values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C. 相似文献
16.
One-step molybdate method for rapid determination of inorganic phosphate in the presence of protein 总被引:1,自引:0,他引:1
A colorimetric method for the determination of γ-carboxyglutamic acid (Gla) is presented. The procedure is based on the following results. (a) Gla is quantitatively converted into a proline derivative by reaction with acetaldehyde. (b) This derivative is spectrophotometrically detected by the secondary amine reagent: nitroprusside and acetaldehyde in alkaline medium. Under the reported conditions, Beer's law is obeyed for concentrations of Gla varying from 0.5 to 5 × 10?4m. The method has been used to determine the Gla content in urine samples. 相似文献
17.
Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation 总被引:5,自引:0,他引:5
Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a. 相似文献
18.
Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline 总被引:2,自引:0,他引:2
The threonine analog beta-hydroxynorvaline is an inhibitor of asparagine-linked glycosylation. In the presence of the analog human fibroblasts synthesized cathepsin D molecules containing two, one, or no oligosaccharides. The nonglycosylated cathepsin D precursor was but a minor species and was degraded within 45 min of its synthesis, presumably in the lumen of the endoplasmic reticulum. The polypeptides with one or two oligosaccharides were normally segregated into lysosomes and their proteolytic maturation was not affected. The stability of mature glycosylated and nonglycosylated cathepsin D polypeptides within the lysosomes, however, was markedly decreased. The recovery of cathepsin D polypeptides was increased in the presence of inhibitors of cysteine and aspartyl-proteinases. These data suggest that the absence of carbohydrate side chains in cathepsin D results in an enhancement of the degradation rate of the precursor in the endoplasmic reticulum, and the replacement of threonine by beta-hydroxynorvaline in an enhanced degradation of the mature cathepsin D in lysosomes. 相似文献
19.
Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat 总被引:5,自引:0,他引:5
The myo-inositol level of plasma was determined during pre- and postnatal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly 10-fold. Immediately after birth, the myo-inositol level decreased but was maintained at values 3–4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 mM.The biosynthesis of myo-inositol from glucose 6-phosphate is catalyzed by glucose 6-phosphate:l-myo-inositol-1-phosphate cyclase and l-myo-inositol-1-phosphate phosphatase. The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that the fetal liver accounted for over 48% of the total carcass cyclase and 26% of the total carcass phosphatase activity. Developmental changes correlated well with the pattern of myo-inositol in fetal rat plasma. Similarly, the enzymes of the myo-inositol biosynthetic pathway increased in rat mammary gland in close agreement with the myo-inositol content of milk and diminished to prelactation activities within 24 hr after the onset of involution.The myo-inositol level of colostrum and milk of five human subjects was highest (2.8 mM) before birth and decreased to 40% of that level 5 days postpartum, where it remained for at least 3 weeks. Even after 7 months of lactation, the milk of one subject contained 3–4-fold more myo-inositol than all commercial infant formulas analyzed. 相似文献
20.
Y Fujiwara T Okayasu T Ishibashi Y Imai 《Biochemical and biophysical research communications》1983,110(1):36-41
The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5. 相似文献