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31.
The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl2 (5 mm) and a Ca2+ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca2+ (K0.5 ~0.4 μm); but not by similar concentrations of Mn2+, Zn2+, Co2+, or La2+. This stimulation was specific for ATP; there was little or no effect of Ca2+ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca2+ (10 to 100 μm) in the presence or absence of Mg2+ stimulated transfer of 32P from [γ-32P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K+. The specific activity of Ca2+-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca2+-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization. 相似文献
32.
Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules 总被引:5,自引:0,他引:5
Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of Mr 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the Km ATP is 1.1 mm but the Km for l-glutamate is not altered. The effect of pH on the Km for ammonium indicates that NH3 rather than NH4+ may be the true substrate. At 10 mm Mg2+, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg2+ concentrations produce progressively more acidic optima while lower Mg2+ concentrations raise the pH optimum. The rate-response curve for Mg2+ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (Ki, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting Ki values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested. 相似文献
33.
Barbara Mc. Chrisley 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,563(2)
The vitamin B6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B6 vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate, PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12–15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B6 and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B6 vitamer and 4-PA concentrations (nmol/1) were: PLP, 40.9–122.2; PNP, non-detectable (ND)—16.1; PMP, ND—8.1; PL, ND—15; PN, ND—21.9; PM, ND—17.8; and 4-PA, ND—55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 μmol/mmol of creatinine. B6 vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B6 status parameters. 相似文献
34.
Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzye immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30 degrees C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases. (c) 1992 John Wiley & Sons, Inc. 相似文献
35.
Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA)
in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell
P content (q
p). Both the photosynthetic capacity (Pmax) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was
apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the
culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51%
of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level,
ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA
depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the
dark showed a strong correlation with14C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their14C-fixation, with the same initial saturation intensity,I
k. The ratios of Pmax to the maximum ARA (ARAmax), and α to the slope of ARA (αara) were identical. A comparison of gross to net photosynthesis and N2 fixation suggested that there was little leakage or excretion of fixed C or N. 相似文献
36.
Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD+. The chloroplast enzyme shows low activity with NADP+ relative to NAD+ but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP+. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed. 相似文献
37.
A comparative study of nitrate-limited growth and nitrate uptake was carried out in chemostat cultures of Ankistrodesmus falcatus (Corda) Ralfs., Asterionella formosa Hass., and Fragilaria crotonensis Kit. In each species growth rate (μ) was related to total cell nitrogen or cell quota (q) by the empirical Droop growth function. Nitrate uptake was a function of both external N concentration and q. The apparent maximum uptake rate (Vm') at a given μ was inversely related to q – q0, where q0 is the minimum quota. The apparent half-saturation constant for uptake, (Km') appears to show a slight inverse trend with μ, although statistical analysis shows that this trend is inconclusive. When q approaches q0, Vm' is several orders of magnitude greater than μq, the calculated steady-state uptake rate. As q increases, however, the difference between these two variables decreases sharply until q approaches qm, the cell quota for nitrogen-rich cells. At this point the difference between μq and Vm' disappears. This behavior is explained by the feedback regulation of N uptake. The inverse relationship between Vm' and q – q0 can be described by an empirical three-parameter equation. 相似文献
38.
The optimum atomic ratio of N to P, the ratio at which one nutrient limitation changes over to the other, was determined in seven species of freshwater planktonic algae. The ratio varied over a wide range among species; the average for these species was 17. If the cellular nutrient ratios in marine species are comparable with those in freshwater organisms, Redfield's ratio of 15 is remarkably close to the average. Cellular N:P ratios varied over a 24-h period under a light:dark cycle. The variation of the optimum ratio between species and diel change in cellular N:P ratios within a species could play an important role in population dynamics by enhancing the probability of coexistence of species. 相似文献
39.
Antibodies against purified ( from the rectal gland of Squalus acanthias, as well as against its catalytic subunit, inhibited ouabain binding by as much as 50%. However, antibodies against the glycoprotein subunit did not inhibit ouabain binding. These data suggest that binding of antibody against the catalytic subunit to the enzyme either covers the ouabain binding site or destroys its conformation, while binding of antibody against the glycoprotein has no such effect. 相似文献
40.
A stopped-flow spectrometer is used for ATP assay by firefly luciferase-luciferin method. It allows one to record initial rise of the light intensity and to differentiate the light produced due to the conversion of ADP to ATP by nucleoside diphosphokinase in the firefly lantern when other nucleoside triphosphates are present. Addition of luciferin (0.27 mm) to luciferase extract increases the light intensity by a factor of 50–100. This method can be used to measure ATP in the picomole range. 相似文献