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1.
Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a Cot12 = 0.03 while 65% of the particulate (microsomal) DNA reassociated with a Cot12 = 0.14 Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.  相似文献   

2.
The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, t12 = 12 days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, t12 = 3.3 days, 17α-hydroxylase, t12 = 2.3 days and the C17–C20 lyase, t12 = 3.4 days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C17–C20 lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, t12 = 4.5 days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at 0.1182 testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C17–C20 lyase and 5α-reductase, but not cytochrome b5, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals.  相似文献   

3.
Respiration (O), ammonium (NH4), phosphate (PO4), total nitrogen (NT) and phosphorus (PT) excretions were measured on mixed zooplankton during 3-, 6-, 9-, 12-, 21-, and 24-h incubation periods at 20–23 C. The excretion rates of PO4, NT. and PT decrease during a 21-h period, while rates of respiration and excretion of NH{IN4} are constant. The percentage of inorganic nitrogen excreted increases regularly from 3 h (30–40% of total nitrogen) to 21 h (70–80%) and it could be either due to a bacterial activity which was measured or to a decrease with time of organic nitrogen excreted because of starvation. ONT, OPO4, OPT, and NH4PO4 ratios increase during the first 9 h of incubation; the percentage of inorganic phosphorus excreted is higher at the very beginning and then remains constant from 6 to 24 h. ONH4 and NTPT ratios are constant during a 24-h term, which makes them useful metabolic indexes.  相似文献   

4.
Catalytic activity of thymidylate synthase, as measured in, vivo, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured in, vitro, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from G0G1 to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from G0G1 phase to S phase, coincided strongly with the level of in, vivo activity of the enzyme.  相似文献   

5.
Stable isotope labeled methadone (pentadeuteromethadone) has been used in conjunction with gas chromatography-chemical ionization mass spectrometry to study plasma disappearance rates and urinary excretion of pharmacologically active R-(?)-methadone (l-methadone) and inactive S-(+)-methadone (d-methadone) in three adult methadone maintenance patients. In all three cases, the analgesically active enantiomeric form of the drug had a significantly longer elimination half-life (t12β) when studied in a steady state than did the inactive form (t12β for active R-(?)-methadone, 51.7 to 61.8 hours; t12β for inactive S-(+)-methadone, 31.8 to 37.0 hours). The ratio of drug elimination half-lives } R-(?)-/S-(+)- ranged between 1.40 and 1.94. In the two cases so studied, slower plasma disappearance of active R-(?)-enatiomer than the inactive S-(+)-enantiomer was also observed (t1 R-(?)-, 42.8 and 52.5 hours; t1 S-(+)-, 38.3 and 41.3 hours).  相似文献   

6.
The cost of assays using one or two coupling enzymes is optimized by using equations to calculate the minimum amount(s) of enzyme(s) which should be used to obtain a given time (t99) in which 99% of the rate V0 of the first reaction is obtained. Using two coupling enzymes and given a value of t99, the induction period L = L1 + L2 fulfills the requirement t99 2124.6 ≥ L ≥ t994.6, allowing one to choose a cost lower than that derived from the until-now generally applied assumption of t99 = 4.6L. Being α = L1L2, in optimized assays the values α, t99, and L are related by T99=4.6(1+α)121+αL, thus allowing (graphical) calculation of the amounts of coupling enzymes which will minimize the cost for every chosen t99 or L. Maximum practical rates, allowed in some supposed interesting cases, have been calculated.  相似文献   

7.
We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation (t12≈10–15 min for monoacetylated histone H4) compared to the slower rate (t12≈140–200 min for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.  相似文献   

8.
《Insect Biochemistry》1987,17(3):433-437
We have studied the metabolism of the hyperglycemic/cardioacceleratory peptide periplanetin CC-2 (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2), in Periplaneta americana using both in vitro and in vivo methods. We focused on metabolism in hemolymph, and homogenates of fat body (a presumed target organ) and Malpighian tubules (previously suggested to degrade the nonpurified cardioacceleratory factors). Despite using low concentrations (6 nM) of [4-3H-Phe]CC-2, enzymatic degradation in homogenates of fat body and Malpighian tubules proceeded with t12 of ∼ 1 hr, while degradation in hemolymph was much slower. We injected low doses (1–2 pmol) of [3H]CC-2 into adult female cockroaches and analyzed hemolymph samples at various times; we determined the t12in vivo to be about 1 hr. We conclude that rate of secretion may be more important in controlling physiological levels of this peptide than rate of degradation.  相似文献   

9.
In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes ? 4 · 10?5. A minimal concentration of about 6 · 1011 dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method Ntopt is compared with the concentrations Ntel obtained from simultaneous electrical relaxation measurements. Ntopt and Ntel agreed at low dipicrylamine concentrations (10?8–10?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10?6 M). In the saturation range Ntopt was maximally four times higher than Ntel. The significance of this difference is discussed together with general aspects of the saturation phenomenon.  相似文献   

10.
(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by 31P-NMR. (2) 14:014:0 PE remains in the lamellar phase up to 90°C. 18:1t18:1t PE exhibits a lamellar to hexagonal (HII) transition between 60°C and 63°C. For 18:1c18:1c PE, the lamellar to hexagonal (HII) transition occurs between 7 and 12°C, whereas for 18:2c18:2c PE, the hexagonal (HII) phase is the preferred structure above ?15°C. (3) Mixtures of 18:1c18:1c PE and 18:1t18:1t PE exhibit near-ideal miscibility behaviour. For mixtures of 18:1c18:1c PE and 14:014:0 PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the 14:014:0 PE component. Mixtures of 18:2c18:2c PE and 18:1t18:1t PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) 31P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.  相似文献   

11.
12.
Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. E. coli DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a Rot12 of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a Tm of 73°C. The probe reacts with total rat nuclear RNA with a Rot12 of 1.0.  相似文献   

13.
The distribution of HCO3?-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3?-ATPase activity. Approximately 85 % of the total HCO3?-ATPase activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial HCO3?-ATPase were not distinguishable from those of the various microsomal HCO3?-ATPase previously described by other investigators.  相似文献   

14.
The turnover of acetylcholine receptors labeled with 125I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical (t12 = 30 hr). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower (t12 ≥ 5 days) and different than the rate of extrajunctional receptor degradation (t12 = 30 hr). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.  相似文献   

15.
Peter Nicholls 《BBA》1976,430(1):13-29
1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome aa3 (a3+a33+) and in the half-reduced species (a2+a33+). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a32+ minus a33+, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome a33+) and azide (which induces peak shifts of cytochrome a2+ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome a2+a33+-HCOOH is faster than its rate of dissociation from a3+a33+-HCOOH, especially in the presence of cytochrome c. The Ki for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2.5. Formate inhibition of ascorbate plus N,N,N′,N′-tetramethyl-p-phenyl-enediamine oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome c reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome aa3 level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.  相似文献   

16.
According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.  相似文献   

17.
Highly purified divalent and monovalent antibodies against cytochrome b5, anti-b5 immunoglobulin G (IG) and anti-b5 Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-b5 IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti-b5 Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome b5 in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.  相似文献   

18.
In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t12) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t12 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These t12 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t12 values for cholesterol efflux from these cell lines.  相似文献   

19.
An α1-mantitrypsin-like material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma α1-antrypsin. The liver material, in contrast to a previous report1 has the same molecular weight as the corresponding normal plasma α1-antrypsin. The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma α1-antrypsin. Amino acid and carbohydrate compositions of the liver material are similar to those of α1-antrypsin obtained from the plasma. The α1-antrypsin-like material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy.  相似文献   

20.
Synthetic somatostatin-28 (S-28) as well as a related endogenous rat hypothalamic somatostatin-like compound (3K SLI) were incubated with hypothalamic extracts from which endogenous somatostatin-like immunoreactivity (SLI) had been removed by immunoabsorption. The reaction products were analyzed by gel chromatography, HPLC as well as two different radioimmunoassays for tetradecapeptide somatostatin (S-14) in which S-28 crossreacted either 100% (RIA R149) or < 0.001% (RIA S39). The results indicate that incubation of S-28 with SLI free hypothalamic extracts results in a rapid decrease of total immunoreactivity measured with RIA R149 (t12 = 14 min). By contrast, with RIA S39 a rise from zero to a peak value at 8 min was measured suggesting the formation of S-14. This was confirmed by subsequent analysis by gel chromatography and HPLC. Using endogenous 3K SLI a decrease of total R149-immunoreactivity with a similar time course (t12 = 17 min) was observed simultaneously with the emergence of material that corresponded to S-14. This converting activity seems to be specific for SLI-containing tissues since similar rates of conversion were observed with extracts from cerebral cortex and cerebellum but not with lung and liver extracts.It is concluded that (1) S-28 is converted to S-14 by hypothalamic enzymes; (2) the processing of 3K SLI is similar, suggesting the two molecules are closely related, if not identical, and (3) the regulation of S-28 to S-14 conversion could represent an important mechanism for controlling the functional activity of somatostatinergic cells.  相似文献   

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