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21.
Protein stability and electrostatic interactions between solvent exposed charged side chains 总被引:6,自引:0,他引:6
To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability. 相似文献
22.
The mutation of Scenedesmus obliquus strain C-6D is expressed in the dark only. Under these conditions, this strain synthesizes acyclic poly-cis carotenes. Cyclic carotenoids like -carotene or xanthophylls are absent. In the light the normal cyclic carotenes and xanthophylls are synthesized in the all-trans configuration. The poly-cis carotencs present in dark cultures have been analysed and quantitated. It could be shown that these poly-cis carotenes are identical with the poly-cis carotenes synthesized by the tomato mutant Lycopersicon esculentum var. Tangella. The poly-cis pathways, however, are different regulated in the two organisms. The tomato mutant accumulates prolycopene as the major carotene, whereas the mutant C-6D accumulates mainly pro--carotene. Furthermore, the mutation in Tangella is constitutive in light in contrast to Scenedesmus C-6D. Besides that, Scenedesmus C-6D synthesizes a further cis-carotene isomer of phytofluene as well as of -carotene. The configuration of these carotenes still have to be elucidated. The occurrence of this poly-cis carotene biosynthetic pathway by a mutation of only one enzyme, the phytoene desaturase which, however, is only expressed in darkness under heterotrophic conditions, is discussed. 相似文献
23.
用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。 相似文献
24.
Lalit K. Srivastava Samina B. Bajwa Rodney L. Johnson Ram K. Mishra 《Journal of neurochemistry》1988,50(3):960-968
The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
25.
Vincent A. Murphy Quentin R. Smith Stanley I. Rapoport 《Journal of neurochemistry》1988,51(6):1777-1782
Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3. 相似文献
26.
Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines. 相似文献
27.
28.
Benign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myocyte ultrastructure and consequent effects these might have in modifying the response of the detrusor to mechanical stimulus, we studied differences in dynamic nuclear-cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which specifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2-3 week) and old (8-12 mon) guinea pigs. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracellular stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti-alpha-smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor myocytes showed dose dependent disruption of the actin component of the cytoskeleton. These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the detrusor in response to outflow obstruction in infancy and adulthood. Furthermore, they suggest that 1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; 2) there appears to be a decrease in myocyte nuclear elasticity with ageing; 3) release of nuclear template restrictions increases activity of DNA polymerase alpha in young, but not old, detrusor myocytes; and 4) mechanico-chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that 1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and 2) integrity of nuclear actin may function to maintain the spatial template restriction on DNA polymerase alpha activity. 相似文献
29.
Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein. 相似文献
30.
P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines
MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings. 相似文献