共查询到20条相似文献,搜索用时 15 毫秒
1.
M Nemer 《Developmental biology》1979,68(2):643-647
A procedure is presented for the simultaneous measurement of the tissue pool size and specific activity of methyl-labeled S-adenosylmethionine (SAM). The method of Kopin and Baldessarini (1971) is used with the introduction of a reference amount of SAM, methyl-labeled with a second isotope to provide an isotope dilution by the tissue sample. The SAM pool sizes in two species of sea urchin embryo were approximately constant during development from blastula to gastrula, being 6.8 and 6.3 × 10?14 moles/embryo at these respective stages for Strongylocentrotus purpuratus and 15.7 and 14.2 × 10?14 moles/embryo for Lytechinus pictus. The rates of turnover of SAM in the gastrulae of these two species are at least 2.7 × 10?15 and 5.2 × 10?15 moles/min/embryo, respectively. 相似文献
2.
Intracellular specific radioactivity of an amino acid (leucine) was measured in preimplantation mouse ova. The measured specific activity along with leucine incorporation rates allow calculation of the protein synthetic rate in mouse ova. Unfertilized ova, fertilized ova, two-cell ova, and blastocysts convert amino acid to protein at the rate of 8.60, 7.36, 6.92, and 56.68 × 10?13 moles of amino acid per hour per ovum, respectively. The specific activity measurements also allowed the calculation of intracellular leucine pool size. For the unfertilized, fertilized, two-cell, and blastocyst stage, the endogenous leucine pool was 11.9, 15.9, 6.4, and 62.4 × 10?14 moles of leucine, respectively. Most of the increase in pool size in the blastocyst probably results from the increase in total volume of cells. Protein degradation measurements indicated a marked difference between protein turnover in the two-cell and blastocyst stage. Approximately 10% of the protein at the two-cell stage turns over with a half-life of 18.3 hr and 35% of the protein at the blastocyst stage turns over with a half-life of 11.2 hr. The large remaining fraction of protein turns over much more slowly. 相似文献
3.
Results from kinetic studies on the incorporation of 3H-5-uridine and 3H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10?5 M for uridine and 6 × 10?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA. 相似文献
4.
Samuel A. Sholl 《Steroids》1981,38(2):221-228
C17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey () fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [14C]17-hydroxyprogesterone to [14C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, Km and Vmax. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10?3 μmoles NADPH oxidized/min. × mg?1 protein with an apparent Km of 3.6 × 10?7 M and a Vmax of 2.2 × 10?3 μmoles/min. × mg?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis. 相似文献
5.
Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10?9 to 3.5 × 10?7M. Populations of 106 cells could amplify an initial cAMP concentration of 2.5 × 10?9M with a low probability, while an initial cAMP concentration of 5 × 10?8M always induced a response. An initial cAMP concentration of 1.2 × 10?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 107 molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10?7M or more. The amplification of a 10?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 104 cells/cm2, both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10?13–10?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation. 相似文献
6.
The kinetic mechanism of α-acetohydroxy acid isomeroreductase from Salmonella typhimurium has been investigated by initial velocity kinetic and product inhibition studies. The results of the initial velocity studies are consistent with a sequential reaction. The product inhibition studies suggest an ordered reaction with NADPH and the acetohydroxy acid adding in that order, and dihydroxy acid release before NADP release.NADPH binding has been studied both by fluorimetric techniques and difference spectroscopy. From these investigations it has been calculated that 4 moles of NADPH bind per mole of enzyme; the first molecule of NADPH binds with a dissociation constant of 1.7 × 10?6m, the subsequent 3 moles of NADPH bind with a constant of 6 × 10?6m. Biphasic kinetics have been demonstrated at a wide range of NADPH concentrations. The occurrence of biphasic kinetics and two separate binding constants are discussed in terms of negative cooperativity. 相似文献
7.
Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of 14C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10?5 to 5 × 10?7 M, and to 110% of controls at 5 × 10?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10?5 M and to 115% at 5 × 10?6 M but had no effect at 5 × 10?7 or 5 × 10?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion. 相似文献
8.
Agneta L. Sunehag Chiara Dalla Man Gianna Toffolo Morey W. Haymond Dennis M. Bier Claudio Cobelli 《Obesity (Silver Spring, Md.)》2009,17(2):233-239
Given the increase in the incidence of insulin resistance, obesity, and type 2 diabetes in children and adolescents, it would be of paramount importance to assess quantitative indices of insulin secretion and action during a physiological perturbation, such as a meal or an oral glucose‐tolerance test (OGTT). A minimal model method is proposed to measure quantitative indices of insulin secretion and action in adolescents from an oral test. A 7 h, 21‐sample OGTT was performed in 11 adolescents. The C‐peptide minimal model was identified on C‐peptide and glucose data to quantify indices of β‐cell function: static φs and dynamic φd responsivity to glucose from which total responsivity φ was also measured. The glucose minimal model was identified on glucose and insulin data to estimate insulin sensitivity, SI, which was compared to a reference measure, SIref, provided by a tracer method. Disposition indices, which adjust insulin secretion for insulin action, were then calculated. Indices of β‐cell function were φs = 51.35 ± 8.89 × 10?9min?1, φd = 1,392 ± 258 × 10?9, and φ = 82.09 ± 17.70 × 10?9min?1. Insulin sensitivity was SI = 14.19 ± 2.73 × 10?4, not significantly different from SIref = 14.96 ± 3.04 × 10?4 dl/kg·min per µU/ml, and well correlated: r = 0.98, P < 0.0001, thus indicating that SI can be accurately measured from an oral test. Disposition indices were DIs = 1,040 ± 201 × 10?14 dl/kg/min2 per pmol/l, DId = 33,178 ± 10,720 × 10?14 dl/kg/min per pmol/l, DI = 1,844 ± 522 × 10?14 dl/kg/min2 per pmol/l. Virtually the same minimal model assessment was obtained with a reduced 3 h, 9‐sample protocol. OGTT interpreted with C‐peptide and glucose minimal model has the potential to provide novel insight regarding the regulation of glucose metabolism in adolescents, and to evaluate the effect of obesity and interventions such as diet and exercise. 相似文献
9.
Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos 总被引:1,自引:0,他引:1
Mouse embryos from the one-cell to the blastocyst stage were cultured for 2 hr in the presence of 5 μM [3H]uridine or 10 μM [3H]adenosine, and the size and specific activity of the UTP and ATP pools were determined by an Escherichia coli RNA polymerase assay using synthetic poly(dA-dT) as template. The total UTP pool increased in size and specific activity with development from 0.05 pmole (0.06% labeled) in the one-cell stage to 0.54 pmole (27% labeled) in the blastocyst stage. The total ATP pool remained relatively constant in size at about 1 pmole/embryo, but increased in specific activity from 2.6 to 52% from one-cell to blastocyst. The turnover of the [3H]UTP pool was also examined under pulse-chase conditions in eight-cell and morula-stage embryos. The UTP pool decayed with approximately first-order kinetics up to 20 hr of chase, but the rate of decay was slower in eight-cell embryos (t0.5 = 5.5 hr) than in morulae (t0.5 = 2.8 hr). The observed specific activities of the UTP pools were used to calculate the overall rates of uridine incorporation into acid-precipitable material during early development. The rate of uridine incorporation per embryo increased from 3.6 × 10?3 pmole/2 hr in the two-cell embryo to 1.8 × 10?1 pmole/2 hr in the blastocyst. The rate of RNA synthesis per cell over a 2-hr period was estimated at 2.5 pg in the two- to four-cell embryo, 5 pg in the eight-cell, and 10 pg in the morula-early blastocyst. 相似文献
10.
P Thomopoulos B Dastugue N Defer 《Biochemical and biophysical research communications》1974,58(2):499-506
incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 107 M?1 h?1 and the rate constant of dissociation was 0.015 h?1. The dissociation constant Kd was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10?9 M. The maximum binding capacity was 10?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone. 相似文献
11.
Bacterial dynamics in two high-arctic lakes 总被引:1,自引:0,他引:1
The heterotrophic planktonic bacteria in two high-arctic lakes were studied by direct microscope count and the enzymatic uptake of 14C labelled glucose which generally conformed to Michaelis-Menten kinetics. Bacterial numbers and activity in oligo-trophic Char Lake ranged from 0.1 to 2.0×10?3 bacteria/l and a maximum uptake velocity (Vmax) of 1.8 × 10?3μg glucose l? h?1. Nearby Meretta Lake received waste water from the Department of Transport Base at Resolute and this eutrophication was reflected in higher bacterial numbers of 2-80 × 108/1 and Kmax of 0.1 × 10?1-7.5 × 10?1 fig glucose l?1 h?1 The Kmax per cell in Char Lake was 3 × 10?11μg glucose l?1 h?1 and changed little between the period of solid ice cover in May and ice-free conditions in August. Bacterial cycles could not be related to phytoplankton cycles in either lake. Comparison of kinetic data from several lakes suggests a relationship between the bacterial uptake rate of glucose and phytoplankton production. Both bacterial numbers and activity in Char Lake may be very close to the minima to be expected in undisturbed freshwater environments. 相似文献
12.
In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [14C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [32P]PO4 from [γ-32P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed. 相似文献
13.
M D Hollenberg 《Archives of biochemistry and biophysics》1975,171(2):371-377
Epidermal growth factor (EG factor) and insulin stimulate the incorporation of thymidine into contact-inhibited rabbit lens epithelial cells in culture. The maximal stimulation observed with EG factor is greater than with insulin. Half-maximal stimulation by EG factor is observed at 6 × 10?10m and for insulin at 1 × 10?9m. [125I]-labeled EG factor (2000 Ci/mmol, about 1 g atom of iodine per mol) is equipotent with native EG factor in stimulating DNA synthesis. Both insulin and EG factor bind to distinct high-affinity sites in intact lens cell monolayers; half-maximal binding is observed at about 10?9m for both polypeptides. A maximum of approximately 8 × 104 insulin molecules and 4 × 104 EG factor molecules are bound per cell. These observations indicate that cultured rabbit lens cells possess receptors for insulin and EG factor by biological and physicochemical criteria and raise the possibility that both peptides may play a role in lens growth and development. 相似文献
14.
Using a specific protein binding assay we have shown that a spermatozoon of the ascidian Ciona intestinalis contains 1.58 ± 0.74 × 10?19 moles of inositol 1,4,5-tri-phosphate (InsP3), while a human spermatozoon contains 6.4 ± 0.14 × 10?19 moles. Induction of the acrosome reaction (AR) in both species, by exposure to the calcium ionophore A23187, does not significantly alter levels of InsP3, suggesting that phosphatidylinositol (PI) turnover is not necessary for the calcium ionophore induced AR. Furthermore, PI turnover in ascidian spermatozoa appears to be insensitive to lithium and phorbol ester. The high intracellular concentration of InsP3 in spermatozoa, corresponding to 50–200 μM, suggests it may play a role in egg activation. © 1993 Wiley-Liss, Inc. 相似文献
15.
Simple techniques are described for quantitating dansyl amino acids by direct fluoresence scanning and by photo-copying and densitometry. This technique is accurate between 1.0 to 10 × 10?12 moles with a lower limit of detection around 1 × 10?14 moles. It is possible to obtain amino acid compositions on very small quantities of peptides and it seems likely that this method will be useful for automatic peptide sequenators which use subtractive dansyl-Edman degradation as a detection procedure. 相似文献
16.
A general model of the kinetics of microbial growth has been developed involving the kinetics of incorporation of substrate into biomass and the maintenance energy requirements. Results obtained from batch cultures of the yeast Saccharomyces cerevisiae growing in synthetic media at pH 5.1 and 30°C permitted all biological parameters in the model to be calculated. Values obtained for these parameters were: maximum specific glucose uptake rate (μSm), 2.08 g/g biomass/hr; apparent Michaelis constant for glucose (KS), 0.1 g/liter (5.5 × 10?4M) apparent Michaelis constant for oxygen (KL), 1.4% O2 (3.2 × 10?6 M) quantitative index of the Pasteur effect (b), 4.9 × 10?4%?1 O2 (207 M ?1). Under conditions of strongly substrate-repressed respiration the values obtained for YATP and P/O were constant over the course of the exponential phase of growth (YATP = 10.4 g biomass/mole ATP; P/O = 3 moles ATP/atom 0). Mass balances for aerobic and anaerobic cultures confirmed the results obtained form the generalized model. Results presented suggested the operation of a mechanism for regulating energy-yielding metabolism which involved an equilibrium between the systems of oxidative phosphorylation and dephosphorylation and was dependent upon the level of catbolite repression. 相似文献
17.
3H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the fish. The Kd for binding to intact and solubilized rat brain membranes was 5.6 × 10?9 M and 1.1 × 10?8M respectively, and the binding capacity 2.0 × 10?14 and 3.0 × 10?13 moles /mg protein respectively. The Kd for Torpedo membranes was 3.1 × 10?7M and the binding capacity 6.8 × 10?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with 3H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain. 相似文献
18.
Noriko Ooya 《Journal of plant research》1974,87(3):253-259
InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10?6–5×10?8 g/ml kinetin. NAA, IAA, GA3, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10?7–5×10?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10?6 g/ml kinetin had a tendency to become diploid. 相似文献
19.
The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10−8 moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10−7 moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with 3H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells. 相似文献
20.
K. P. Krishnan Christabelle E. G. Fernandes Sheryl Oliveira Fernandes P. A. Loka Bharathi 《Geomicrobiology journal》2013,30(1):31-36
Bacteria isolated from cobalt–enriched ferromanganese crusts on the Afanasiy Nikitin Seamounts in the Equatorial Indian Ocean were examined for their ability to tolerate, and immobilize cobalt in unamended seawater and seawater amended with 0.01% glucose. Retrievable bacterial counts in the form of CFU (colony forming units) on media supplemented with 1 mmol Co l?1 (58 mg Co l?1) and 1 mmol Mn l?1 (54 mg Mn l?1) were in the range of 1.71 × 104 to 1.05 × 105 gm?1 (wet wt) of crust, respectively. Most of the isolates (14/24) were pigmented and showed taxonomic affinities to Flavobacterium sp. Two representative isolates were tested for their tolerance of cobalt. We observed that in amended medium, the isolates tolerated up to 1 mmol Co l?1, whereas in unamended medium they tolerated upto 10 mmol Co l?1. Microscopic observations of cultures incubated with 10 mmol Co l?1 showed the occurrence of an extracellular slime layer, which may be responsible for immobilizing the cobalt from the liquid phase. In the unamended medium, the tolerance and stimulation in total cell counts was similar to that in amended medium or sometimes greater. Total cell counts peaked at 100 μmol Co l?1 for incubations in unamended medium (1.1–2.5 × 1011 cells l?1) and at 0.1–1 μmol Co l?1 for incubations in amended medium (1.5–2.6 × 1011 cells l?1). Counts of formazan-stained respiring cells of both the isolates in the unamended medium reached up to a maximum of 2.9–7.8 × 1010 l?1 after incubation for 10 days at 23(±1)°. In the amended medium cell counts of respiring cells attained a maximum in the range of 4.6–15.8 × 1010 l?1 at 100 μmol Co l?1. The Co immobilization rate was on average 82 (± 87.9, n = 24) μmol of Co d?1. Since the isolates were naturally occurring bacteria from crusts, they could be more environmentally acceptable and safe if used for metal recovery and bio-leaching. 相似文献