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991.
Previously, tau protein kinase I/glycogen synthase kinase-3/kinase FA(TPKI/GSK-3/FA) was identified as a brain microtubule-associated tau kinase possibly involved in the Alzheimer disease-like phosphorylation of tau. In this report, we find that the TPKI/GSK-3/FA can be stimulated to phosphorylate brain tau up to 8.5 mol of phosphates per mol of protein by heparin, a polyanion compound. Tryptic digestion of32P-labeled tau followed by high-performance liquid chromatography and high-voltage electrophoresis/thin-layer chromatography reveals 12 phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and peptide sequence analysis further reveals that TPKI/GSK-3//FA after heparin potentiation phosphorylates tau on sites of Ser199, Thr231, Ser235, Ser262, Ser396, and Ser400, which are potential sites abnormally phosphorylated in Alzheimer tau and potent sites responsible for reducing microtubule binding possibly involved in neuronal degeneration. The results provide initial evidence that TPKI/GSK-3/FA after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau and neuronal degeneration in Alzheimer disease brains.Abbreviations FA
the activating factor of type 1 protein phosphatase
- GSK-3
glycogen synthase kinase-3
- TPKI
tau protein kinase I
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis
- PHF
paired helical filaments
- HPLC
high-performance liquid chromatography 相似文献
992.
The occurrence of -d-mannosidase II activity in insect cells was studied using pyridylaminated oligosaccharides as substrates and two-dimensional HPLC and glycosidase digestion for the analysis of products. GlcNAcMan5GlcNAc2 was converted to GlcNAcMan3GlcNAc2 by each of the three cell lines investigated (Bm-N, Sf-21, and Mb-0503). The respective activity was highest in Bm-N cells which were used for further experiments. Man5GlcNAc2 was not degraded by the Bm-N cell homogenate. Thus, this -mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi -mannosidase II. The -mannosidase II-like activity from Bm-N cells exhibits a pH optimum of 6.0–6.5, has no requirement for divalent metal ions, and is highly sensitive to swainsonine. The 1,6-linked mannosyl residue is removed first as deduced from the elution time on reversed phase HPLC of the intermediate product. The same branch preference was found with -mannosidase II from mung bean seedlings andXenopus liver. Upon ultracentrifugation of Bm-N cell homogenate, 72% of the mannosidase acting on the GlcNAcMan5GlcNAc2 substrate was found in the microsomal pellet indicating the enzyme to be membrane-boundAbbreviations Endo H (Endo D)
endo--N-acetylglucosaminidase H (D)
- GlcNAc
N-acetylglucosamine
- M3, M5, M5Gn etc.
oligosaccharides — for an explanation see Table 1
- -PA
-pyridylamine. 相似文献
993.
Tadashi II Masayuki Kubota Takashi Hirano Mamoru Ohashi Keiichi Yoshida Sakaru Suzuki 《Glycoconjugate journal》1995,12(3):282-289
The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS
fast atom bombardment mass spectrometry
- CID
collision induced dissociation
- MS/MS
mass spectrometry/mass spectrometry
- UA2S-GalNAc6S
2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose
- UA-GalNAc4S
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose
- UA-GalNAcDiS
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose 相似文献
994.
A number of N- and C-terminal deletion and point mutants of bovine -1,4 galactosyltransferase (-1,4GT) were expressed inE. coli to determine the binding regions of the enzyme that interact withN-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1–129, do not show any significant difference in the apparentK
ms toward NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15mm NAG and 50mm EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130–402 of bovine -1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130–257 of the -1,4GT, binds to, and elutes with 15mm NAG and 50mm EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15mm NAG. The C-terminus fragment GT-257UDP, containing residues 258–402 of -1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5–10% of the bound protein, can be eluted from the UDP-agarose column with 50mm EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of -1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+. 相似文献
995.
1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed - a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans — a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer. 相似文献
996.
997.
Sujoy Ghosh John W. Kyle Sara Dastgheib Francois Daussin Zhixiong Li Subhash Basu 《Glycoconjugate journal》1995,12(6):838-847
A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK
mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl2, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS
central nervous system
- GM1
monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer
- GM2
monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer
- DSS
detergent solubilized supernatant
- ECB
embryonic chicken brain
- TBS
Tris-buffered saline 相似文献
998.
Molecular dynamics simulations and energy analysis have been carried out to study the structural mobility and stability of the four -helix bundle motifs. The simulation results as well as the X-ray data show that the atomic RMS fluctuation is larger at the loop region for four representative proteins investigated: methemerythrin, cytochrome b-562, cytochrome c, and bovine somatotropin. The loop-loop, helix-helix, and loop-helix interactions are computed for the unfolded and folded proteins. In the folded and solvated protein structures the loop-helix interaction is stronger than the helix-helix interaction, especially in the electrostatic component. But the stabilization energies of both the loop-helix and the helix-helix interactions relative to the those of an unfolded structure are of the same order of magnitude. The stabilization due to protein-solvent interaction is greater in the helix region than in the loop region. The percentage of hydrophilic solvent accessible area for the four proteins studied was calculated with the method of Eisenberg and McLachlan. The percentage of the hydrophilic area is greater in the loops than in the helices. A Poisson-Boltzmann calculation shows that the potential from the loops acting on a helix is generally more negative than that from other helices. 相似文献
999.
Thomas J. Thekkumkara Jing Du John C. Zwaagstra Kathleen M. Conrad John Krupinski Kenneth M. Baker 《Molecular and cellular biochemistry》1995,152(1):77-86
G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent (EC50=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII. 相似文献
1000.
Anne L. Holleran Donald A. Briscoe Gary Fiskum Joanne K. Kelleher 《Molecular and cellular biochemistry》1995,152(2):95-101
A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of14C-glutamine to14CO2 and of14C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-14C]glucose, [1-14C]acetate and [5-14C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [14C]glutamine to14CO2, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-14C]glutamine into14C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells. 相似文献