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61.
W B Knight B G Green R M Chabin P Gale A L Maycock H Weston D W Kuo W M Westler C P Dorn P E Finke 《Biochemistry》1992,31(35):8160-8170
Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket. 相似文献
62.
Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water. 相似文献
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The interactive effects of increased carbon dioxide (CO2) concentration and ultraviolet-B (UV-B, 280–320 nm) radiation on Acacia karroo Hayne, a C3 tree, and Themeda triandra Forsk., a C4 grass, were investigated. We tested the hypothesis that A. karroo would show greater CO2-induced growth stimulation than T. triandra, which would partially explain current encroachment of A. karroo into C4 grasslands, but that increased UV-B could mitigate this advantage. Seedlings were grown in open-top chambers in a greenhouse in ambient (360 μmol mol-1) and elevated (650 μmol mol-1) CO2, combined with ambient (1.56 to 8.66 kJ m-2 day-1) or increased (2.22 to 11.93 kJ m-2 day-1) biologically effective (weighted) UV-B irradiances. After 30 weeks, elevated CO2 had no effect on biomass of A. karroo, despite increased net CO2 assimilation rates. Interaction between UV-B and CO2 on stomatal conductance was found, with conductances decreasing only where elevated CO2 and UV-B were supplied separately. Increases in water use efficiencies, foliar starch concentrations, root nodule numbers and total nodule mass were measured in elevated CO2. Elevated UV-B caused only an increase in foliar carbon concentrations. In T. triandra, net CO2 assimilation rates were unaffected in elevated CO2, but stomatal conductances and foliar nitrogen concentrations decreased, and water use efficiencies increased. Biomass of all vegetative fractions, particularly leaf sheaths, was increased in elevated CO2. and was accompanied by increased leaf blade lengths and individual leaf and leaf sheath masses. However, tiller numbers were reduced in elevated CO2. Significantly moderating effects of elevated UV-B were apparent only in individual masses of leaf blades and sheaths, and in total sheath and shoot biomass. The direct CO2-induced growth responses of the species therefore do not support the hypothesis of CO2-driven woody encroachment of C4 grasslands. Rather, differential changes in resource use efficiency between grass and woody species, or morphological responses of grass species, could alter the competitive balance. Increased UV-B radiation is unlikely to substantially alter the CO2 response of these species. 相似文献
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Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques 总被引:16,自引:0,他引:16
Jhy-Jhu Lin Jonathan Kuo Jin Ma James A. Saunders Hunter S. Beard Margaret H. MacDonald William Kenworthy George N. Ude Benjamin F. Matthews 《Plant Molecular Biology Reporter》1996,14(2):156-169
Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability
to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars
were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic
bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease
restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished
on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained
with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in
those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands
using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of
the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish
polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP
is the most useful. 相似文献
69.
Stephanie Wengert Watts Susan L. Gackenheimer Donald R. Gehlert Marlene L. Cohen 《Neurochemistry international》1994,24(6):565-574
Although the density and distribution of 5-HT2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized 125I-lysergic acid diethylamide ([125I]LSD) to quantify and compare 5-HT2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [125I]LSD binding from 5-HT2A and D2 binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist. 相似文献
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