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151.
Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide" 总被引:20,自引:0,他引:20
C Harter P James T B?chi G Semenza J Brunner 《The Journal of biological chemistry》1989,264(11):6459-6464
Toward elucidating molecular details of virus-induced membrane fusion, we have studied the low pH-triggered interaction of the bromelain-solubilized ectodomain of influenza hemagglutinin with liposomes. Polypeptide segments which insert into the apolar phase of the lipid bilayer were first labeled specifically using either of the two membrane-restricted carbene-generating reagents, 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] undecanoyl]-sn-glycero-3-phosphorylcholine, and were then identified on the basis of cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine-skatole fragment analysis and Edman degradations. Here, we demonstrate that the hydrophobic interaction is mediated solely by the so-called "fusion peptide" which corresponds to the NH2-terminal segment of the BHA2 subunit of nature influenza hemagglutinin. Predominant sites of labeling within that segment were Phe-3, Ile-6, Phe-9, Trp-14, Met-17, and Trp-21. The average 3-4 residue spacing between consecutive labeled amino acid side chains suggests a helical structure of that segment with an amphiphilic character. 相似文献
152.
153.
1-5-D-Gluconolactone is a competitive inhibitor of both sucrase and isomaltase. Substitution of the 1H and 2H at C1 of the glucosyl moiety in p-CL-phenyl-alpha-D-glucopyranoside leads to a decrease in kcat of both sucrase and isomaltase, the k1H/k2H ranging between 1.14 and 1.20. Treatment of the association constants and of the kcat values for a number of p-substituted phenyl-alpha-D-glucopyranosides on the basis of the Hammet-Hansch equation has allowed the estimation of the importance of hydrophilicity-hydrophobicity as well as of the magnitude of the p values for both substrate-enzyme interaction and catalysis in both sucrase and isomaltase. The magnitude of the secondary deuterium effect as well as the low values of p in both sucrase and isomlatase are strongly indicative of the rate-limiting step going through the formation of an oxocarbonium ion. In conjunction with other observations reported previously, the data presented here led to the suggestion of the main lines of a reaction mechanism for the two glucosidases: prptonation of the glycosidic oxygen is followed by the liberation of the "aglycone" with formation of an oxocarbonium ion, which is temporarily stabilized by a carboxylate group. 相似文献
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157.
Summary At
0,d-glucose influx into, and efflux out of, membrane vesicles from small-intestinal brush borders are affected by trans Na+ and transd-glucose to different extents.d-glucose influx and efflux respond to
(negative at the trans side) to different extents. The small-intestinal Na+,d-glucose cotransporter, is thus functionally asymmetric. This is not unexpected, in view of the structural asymmetry previously found. The characteristics of the
of transinhibition byd-glucose are compatible with the mobile part of the cotransporter bearing a negative charge of at least 1 (in the substrate-free form). They are not compatible with its mobile part being electrically neutral. Pertinent equations are given in the Appendix. Partial Cleland's kinetic analysis and other criteria rule out (Iso) Ping Pong mechanisms, and makes likely a Preferred Ordered mechanism, with Na
out
+
binding to the cotransporter prior to the sugarout. A likely model is proposed aimed at providing a mechanism of flux coupling and active accumulation. 相似文献
158.
In brush border vesicles from guinea pig small intestine l-ascorbate transport is Na+-dependent and electroneutral (in the presence of Na+, as shown by its lack of response to either positive or negative Δψ across the membrane).l-Ascorbate transporter has the kinetic characteristics of a mobile carrier (Km for l-ascorbate, 0.3 mM). d-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter.l-Ascorbate transport is subjected to heterologous inhibition by d-glucose. 相似文献
159.
A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles. 相似文献
160.
Carl Tannenbaum Gerhard Toggenburger Markus Kessler Aser Rothstein Giorgio Semenza 《Journal of cellular biochemistry》1977,6(4):519-533
In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd ? 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein. 相似文献