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81.
In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany  相似文献   
82.
Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.  相似文献   
83.
Georg Kaiser  Ulrich Heber 《Planta》1984,161(6):562-568
Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent Km of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg2+ present, nor by protonophores and valinomycin (with K+ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday  相似文献   
84.
(L -Cys)n, (L -Lys)n, and (L -Glu)n were studied by ir spectroscopy in terms of their degree of deprotonation or protonation. It is shown that structurally symmetrical, easily polarizable SH ?S? ? ?S ?HS, N+H ?N ? N ?H+N, and OH ?O? ? ?O ?HO hydrogen bonds are formed between the side chains. The different wave number distributions of the ir continua caused by these hydrogen bonds show that the barrier in the double-minimum proton potential decreases in the series of these hydrogen bonds. The stability of these hydrogen bonds against hydration increases in this series. The OH ?O? ? ?O ?HO bonds are not broken by small amounts of water. With (L -Cys)n the formation of easily polarizable hydrogen bonds and a β-structure–coil transition are strongly interdependent. As a result of this coupling effect, the β-structure–coil transition becomes cooperative. With (L -Glu)n, the formation of the polarizable hydrogen bonds and the observed conformational change are independent processes. The (L -Glu)n conformation changes from α-helix to coil only if more than 80% of the residues are deprotonated. Finally, on the basis of the various types of easily polarizable hydrogen bonds, charge shifts in active centers of enzymes and the proton-conducting mechanism through hydrophobic regions of biological membranes are discussed.  相似文献   
85.
Summary All of ourEscherichia coli C mutants blocked in the first step of D-arabitol catabolism (D-arabitol dehydrogenase) became unable to grow in the presense of D-arabitol. We have shown that this sensitivity is eliminated by a defect in the second enzyme of the pathway (D-xylulokinase), leading to a pattern of toxicity and its relief which has not been previously reported. We have found a similar pattern of toxicity and its relief in the closely related ribitol pathway. The evolutionary significance of these findings is discussed.  相似文献   
86.
Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.  相似文献   
87.
Thylakoids of Oscillatoria chalybea are able to split water. The Hill reaction of these thylakoids is sensitive to DCMU. Diphenylcarbazide can substitute for water as the electron donor to photosystem II with these fully functioning thylakoids. However, the diphenylcarbazide photooxidation is completely insensitive to 3-(3,4-dichlorophenyl)-N-N-dimethyl urea (DCMU) at high diphenylcarbazide concentrations. In with Tris-treated Oscillatoria thylakoids the water splitting capacity is lost and diphenylcarbazide restores electron transport through photosystem II as occurs with higher plant chloroplasts. However, also these photoreactions are insensitive to DCMU. If diphenylcarbazide acts in Oscillatoria as an electron donor to photosystem II the result suggests that diphenylcarbazide feeds in its electrons behind the DCMU inhibition site. This in turn indicates that in Oscillatoria the site of inhibition of DCMU is on the donor side of photosystem II.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-N-N-dimethyl urea - DPC diphenylcarbazide - DCPiP 2,6-dichlorophenol indophenol - TMB tetramethyl benzidine - A-2-sulf anthraquinone-2-sulfonate  相似文献   
88.
-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl2 nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.  相似文献   
89.
Various human Burkitt lymphoma and LCL lines established in vitro and their derived somatic cell hybrids were tested for their comparative EBV receptor levels in a virus binding assay. Their graded C3b and C3d complement receptor expression was estimated simultaneously by means of isotope labeled rosette marker cells. The receptor concentration of each cell line was related to Raji as the standard of comparison, K 562, P3HR-1, and YACUT were used as negative controls. In general, the charging curves for EBV and C3d receptors parallelled each other (r = 0.97) while C3b receptor charging showed no correlation (r < 0.60). In the Raji hybrids between the C3b receptor positive Raji parent and various patents that were negative for this receptor, C3b receptor expression was low or negative. In contrast, the C3d negative P3HR-1 line gave rise to hybrids, after fusion with receptor-positive cells, that were intermediate with regard to their C3d receptor expression. The host range restriction of the Epstein-Barr virus is determined at the receptor level. The close relationship between the EBV receptor and the C3d receptor, a B-lymphocyte-specific moiety, suggests that the moderate interaction with EBV with the B lymphocytes may have had a selective advantage, favoring the presence of EBV. Since EBV causes lytic infections after artificial introduction into nonnatural host cells, it may represent a B-lymphocyte-specific host range mutant, derived from an originally lytic herpesvirus with a much broader target cell range.  相似文献   
90.
Electron transfer in milk xanthine oxidase as studied by pulse radiolysis   总被引:1,自引:0,他引:1  
Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.  相似文献   
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