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71.
Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases 总被引:2,自引:0,他引:2
Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
72.
Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies 总被引:3,自引:0,他引:3
R J Nachman G M Holman B J Cook 《Biochemical and biophysical research communications》1986,137(3):936-942
Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK. 相似文献
73.
Different populations of DNA polymerase alpha in HeLa cells 总被引:3,自引:0,他引:3
Three different populations of HeLa DNA polymerase alpha have been distinguished using a novel preparation of chromatin isolated using an isotonic salt concentration, which contains intact DNA. One synthesizes DNA in vitro at 85% of the rate in vivo, is found only in S-phase nuclei tightly associated with the nucleoskeleton and requires unbroken DNA in the form of chromatin as a template: we assume this is the authentic S-phase activity. On incubation at 37 degrees C, this activity dissociates from the nucleoskeleton to give a soluble activity that prefers broken templates. This soluble activity is in turn heterogeneous, containing active complexes of about 0 X 75 X 10(6) and 3 X 10(6) Mr. The third activity is also soluble and released by lysing cells at any stage of the cell cycle. It, too, prefers broken templates. The authentic activity is obscured by the soluble ones if broken templates are provided. 相似文献
74.
75.
The sequential transfer of internalized, cell surface sialoglycoconjugates through the lysosomes and Golgi complex in HeLa cells 总被引:1,自引:0,他引:1
Surface sialoglycoproteins of HeLa cells were labeled by NaB[3H]4 reduction after oxidation with NaIO4, yielding seven major radioactive bands as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When labeled cells are reincubated in growth medium, all of these major classes of glycoproteins are internalized and all but one (105 kDa) are recycled, i.e. subsequently reappear on the surface. The surface-labeling patterns over time remain qualitatively similar, but changes in relative specific activity of the bands suggest some preferential degradation of individual glycoproteins. Analytical fractionation at various time points after labeling suggests that the surface molecules pass through the lysosomal compartment and subsequently accumulate in the Golgi and Golgi-related compartments before returning to the surface. Inhibition of lysosomal function with chloroquine or NH4Cl prevents the accumulation and subsequent recycling. The pathway is confirmed with preparative fractionation into surface membrane, prelysosomal, lysosomal, Golgi, and Golgi-related compartments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrates a degree of preferential handling of the glycoproteins on this pathway, e.g. the 180-kDa band is relatively reduced at the endocytic/prelysosomal stage and the 105-kDa band appears to be degraded in its first passage through the lysosomes. The other bands recycle 10-20 times before being degraded. 相似文献
76.
Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding. 相似文献
77.
Inhibition of glycine N-methyltransferase activity by folate derivatives: implications for regulation of methyl group metabolism 总被引:3,自引:0,他引:3
C Wagner W T Briggs R J Cook 《Biochemical and biophysical research communications》1985,127(3):746-752
Glycine N-methyltransferase, an enzyme that uses S-adenosylmethionine to methylate glycine with the production of sarcosine, was recently shown to be identical with a major folate binding protein of rat liver (Cook, R.J. and Wagner, C. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). We now present evidence that 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5) is bound with high specificity, and is a powerful inhibitor of the enzyme. It is proposed that this information may be used to modify the "methyl trap" hypothesis which describes how the availability of one-carbon units is regulated by folate, vitamin B12 and methionine. 相似文献
78.
Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s). 相似文献
79.
gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold. 相似文献
80.
The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group. 相似文献