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51.
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53.
Abstract— The incorporation of NeuNAc from CMP-NeuNAc into endogenous glycolipids and glyco-proteins, and exogenously added GM1a (monosialoganglioside) and desialylated fetuin (DS-fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP-NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM1a, 0.20 mm ; DS-fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM1a and exogenous DS-fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM3, while exogenously added GM1a was converted to GD1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS-polyacrylamide gel electrophoresis. Neura-minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM3, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM1a. This was supported by the observation that exogenously added GM1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS-fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP-NeuNAc. 相似文献
54.
SIALYLTRANSFERASES IN RAT BRAIN: INTRACELLULAR LOCALIZATION AND SOME MEMBRANE PROPERTIES 总被引:4,自引:4,他引:0
Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM1a , 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants. 相似文献
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM
55.
Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum. 总被引:4,自引:0,他引:4
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Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex. 相似文献
56.
The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels. 相似文献
57.
A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h. 相似文献
58.
S C Koh P C Wong R Yuen S E Chua B L Ng S S Ratnam 《Journal of reproduction and fertility》1992,96(2):407-413
The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle. 相似文献
59.
M Brown M Stenzel-Poore S Stevens S K Kondoleon J Ng H P B?chinger M B Rittenberg 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(2):339-346
Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints. 相似文献
60.
Sulfate reduction and S-oxidation in a moorland pool sediment 总被引:3,自引:2,他引:1
In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO4
2– reduction rates in the sediment, (2) depletion of SO4
2– in the overlying water column and (3) release of35S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO4
2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core35SO4
2– injection. Rates of SO4
2– consumption in the overlying water were estimated by changes in SO4
2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m–2 d–1. Rates of SO4
2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m–2 d–1 (November) to 0.43–1.81 mmol m–2 d–1 (July, August and April). Maximum rates of oxidation to SO4
2– in July 1990 estimated by combination of SO4
2– reduction rates and rates of in situ SO4
2– uptake in the enclosed water column were 10.3 and 10.5 mmol m–2 d–1 at an organic rich and at a sandy site respectively.Experiments with35S2– and35SO4
2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO4
2– and (2) the presence of mainly non SO4
2–-S derived from reduced S transported from the sediment into the overlying water. A35S2– tracer experiment showed that about 7% of35S2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author 相似文献