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151.
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153.
Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.  相似文献   
154.
Human neutrophils stimulated by ionophore A23187 incorporate [3H]acetate into platelet-activating factor and an additional product which is chromatographically similar to phosphatidylethanolamine and accounts for approximately 25% of the [3H]acetate-containing lipids. Three general approaches indicated the sn-1 moiety of the unknown phospholipid is primarily alk-1'-enyl-linked: 1) approximately 80% of the intact phospholipid as well as its derivatives was highly sensitive to hydrolysis by HCl, 2) 80% of the product which resulted from treating the unknown with phospholipase C and acetylating the free hydroxyl group at the sn-3 position, chromatographed with authentic 1-O-alk-1'-enyl-2,3-diacetylglycerol, and 3) catalytic hydrogenation of the diacetylglycerol product described in 2) resulted in a product which chromatographed with alkyldiacetylglycerol and was not sensitive to strong acid. Treatment of the intact phospholipid with phospholipase A2 resulted in the release of 88% of the radiolabel into the acidified aqueous phase of the extraction mixture, indicating the moiety in the sn-2 position remained as acetate and had not been elongated to fatty acid. The head group was determined to be phosphoethanolamine based upon its complete conversion to the dinitro- and trinitrophenyl derivatives by the amine-derivatizing reagents fluorodinitrobenzene and trinitrobenzenesulfonic acid, respectively. From these data is was concluded that the unknown product is 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (80%), and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (10%). Sonicates prepared from neutrophils stimulated with ionophore A23187 contained an acetyltransferase activity capable of utilizing 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine and [14C]acetyl-CoA to produce the product identified as 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine.  相似文献   
155.
Angiotensin II receptor agonist (125I-angiotensin II) and antagonist (125I-[Sar1,Ile8]angiotensin II) bind in a specific and saturable manner to rat ovarian membranes. Agonist and antagonist binding affinity (KD approximately 0.5 nM) and the number of sites estimated (Bmax approximately 60 fmol/mg of protein) were similar. Dissociation of receptor-bound agonist was more rapid than the dissociation of receptor-bound antagonist, and agonist, but not antagonist, dissociation from the receptor was accelerated by GTP gamma S. A 0-150 mM increase in Na+ produced a 27% increase in the KD of agonist binding. Antagonist binding was not modified by Na+. These studies suggest that both agonist and antagonist identify putative angiotensin II receptors in the ovary but that the properties of agonist and antagonist binding are distinct. Angiotensin II antagonist binding sites are present on the granulosa cell layer of rat ovarian follicles (Speth, R. C., Bumpus, F. M., and Husain, A. (1986) Eur. J. Pharmacol. 130, 351-352). To determine the role of angiotensin II in ovarian function, we examined angiotensin II receptors and function during the onset of puberty. High affinity and low capacity angiotensin II receptors were present in ovaries from immature rats. After pregnant mare's serum gonadotropin induced ovulation in immature rats, antagonist binding to total ovarian membranes increased over 3-fold. In vitro incubation of peripubertal ovaries with 1 microM angiotensin II produced a stimulation of estrogen, but not progesterone, secretion. This steroidogenic effect of angiotensin II was most pronounced in the luteal phase of the estrus cycle. These studies point toward the involvement of angiotensin II in the regulation of ovarian function, possibly through modulation of follicular estrogen levels.  相似文献   
156.
The three-dimensional structure of acarbose bound to glycogen phosphorylase   总被引:2,自引:0,他引:2  
Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.  相似文献   
157.
DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.  相似文献   
158.
The entire proton NMR spectrum of the aminoglycoside antibiotic neomycin B has been assigned at physiological pH by a combination of two-dimensional J-resolved and J-correlated and nuclear Overhauser enhancement difference spectroscopy. Unambiguous assignment of all four ring systems is possible without recourse to model or derivative compounds by observing nuclear Overhauser enhancements between as well as within rings. The subsequent assignment of the carbon 13 spectrum is simply achieved using two-dimensional heteronuclear J-correlated techniques. The proton NMR spectrum of a sonicated aqueous dispersion of the intracellular second messenger precursor phosphatidylinositol 4,5-bisphosphate is reported for the first time. The spectrum is consistent with a high degree of side chain unsaturation and a conformation for the myo-inositol head group, which appears highly mobile, in which all bulky substituents are equatorial (except the 2-hydroxyl). Addition of aliquots of phosphatidylinositol 4,5-bisphosphate to an aqueous buffered solution of neomycin B induces complex changes in the whole spectrum of the latter, including downfield shifts of differential magnitude for several well-resolved signals, viz. the anomerics, and the pair of methylene protons of the substituted cyclohexane. The complexation kinetics are fast on the NMR time scale at 25 degrees C. The binding results are discussed in terms of a tentative complexation geometry.  相似文献   
159.
The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.  相似文献   
160.
Exposure of macrophages to phorbol esters or the calcium ionophore A23187 increases the number of several surface receptors due to recruitment of receptors from internal pools (Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38, 569-576). We have examined the mechanism by which these agents increase surface receptor number. Cells which were preloaded with either fluid phase or receptor-mediated ligands did not lose ligand following exposure to ionophore or phorbol ester. The rate of movement of ligands to the lysosome was also unaffected. These results suggest that A23187 does not induce the fusion of ligand-containing compartments with the cell surface. Ionophore treatment did, however, produce a severalfold increase in the rate at which unoccupied receptors reappear on the cell surface. These results suggest that the compartment of receptors affected by the ionophore formed subsequent to the dissociation of ligand from receptor. The altered rate of receptor reappearance was transitory (90 s), and the increase in receptor number was subsequently maintained by a decrease in the rate of internalization. Changes in the rate of receptor internalization did not correlate with changes in the rate of fluid phase pinocytosis, suggesting that the effect on receptor internalization was selective.  相似文献   
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