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101.
Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction. 总被引:1,自引:1,他引:0 下载免费PDF全文
J. J. Lennon K. A. Walsh 《Protein science : a publication of the Protein Society》1997,6(11):2446-2453
Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data. 相似文献
102.
Monitoring calcium-induced conformational changes in recoverin by electrospray mass spectrometry. 总被引:1,自引:1,他引:0 下载免费PDF全文
T. A. Neubert K. A. Walsh J. B. Hurley R. S. Johnson 《Protein science : a publication of the Protein Society》1997,6(4):843-850
Recoverin is a calcium-binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N-acylated by myristoyl and related fatty acyl residues that are thought to act as "calcium-myristoyl switches," whereby, in the presence of Ca2+, the N-terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium-binding sites. In addition to supporting the calcium-myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N-acyl group (N-lauroyl or N-myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X-ray crystallography. 相似文献
103.
The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK. 相似文献
104.
A slide staining device is described that performs Gram and Wright stains during space flight. Reagents and liquid wastes are contained within a closed system. 相似文献
105.
Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine 总被引:16,自引:0,他引:16
F H Chilton J T O'Flaherty C E Walsh M J Thomas R L Wykle L R DeChatelet B M Waite 《The Journal of biological chemistry》1982,257(10):5402-5407
1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids. 相似文献
106.
Dihydrocytochalasin B disorganizes actin cytoarchitecture and inhibits initiation of DNA synthesis in 3T3 cells 总被引:9,自引:0,他引:9
Dihydrocytochalasin B (H2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H2CB (2-10 X 10(-7) M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H2CB is effective only when added to cells within 8-10 hr after stimulation. Low doses of H2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off. 相似文献
107.
Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle. 总被引:2,自引:1,他引:1 下载免费PDF全文
The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 x 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 x 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant. 相似文献
108.
109.
110.
Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD+-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD+-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K+, Rb+, Cs+. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na+ or Li+. Na+ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K+. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD+. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria. 相似文献