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31.
Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts 总被引:8,自引:0,他引:8
We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm). Upon stimulation, the raft-preferring receptors are clustered, inducing larger, stabilized rafts, probably by coalescing small, unstable rafts or cholesterol-glycosphingolipid complexes in the receptor clusters. This receptor-cluster-induced conversion of raft types may be caused by suppression of alkyl chain isomerization and the lipid lateral diffusion in the cluster, with the aid of exclusion of cholesterol from the bulk domain and the boundary region of the majority of transmembrane proteins. We critically inspected the possible analogy to the boundary lipid concept. Finally, we propose a hypothesis for the coupling of GPI-anchored receptor signals with lipid-anchored signaling molecules in the inner-leaflet raft. 相似文献
32.
Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana 总被引:1,自引:0,他引:1
Shimada T Yamada K Kataoka M Nakaune S Koumoto Y Kuroyanagi M Tabata S Kato T Shinozaki K Seki M Kobayashi M Kondo M Nishimura M Hara-Nishimura I 《The Journal of biological chemistry》2003,278(34):32292-32299
The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing. 相似文献
33.
Yoshimura T Sagami I Sasakura Y Shimizu T 《The Journal of biological chemistry》2003,278(52):53105-53111
The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme. 相似文献
34.
Mizuguchi H Hayashi H Miyahara I Hirotsu K Kagamiyama H 《Biochimica et biophysica acta》2003,1647(1-2):321-324
Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases. 相似文献
35.
Hasegawa Y Ishida Y Kato K Ijiri R Miyake T Nishimata S Watanabe T Namba I Hayabuchi Y Kigasawa H Tanaka Y 《Acta cytologica》2003,47(4):679-684
BACKGROUND: Pancreatoblastoma (PBL) is a rare neoplasm that generally occurs in the pediatric age group and shows unique histopathology, including squamoid corpuscles that may contain tumor cells with optically clear nuclei (OCN) rich in biotin. In the English-language literature there have been two reports on the cytology of PBL, but neither of them refers to the cytologic features of squamoid corpuscles. CASE: A 3-year-old boy with nausea and general fatigue was referred to our center. Imaging studies showed an approximately 7.5-cm, left-sided abdominal mass and multiple metastases in the lung. The abdominal mass was biopsied, and its histology showed solid cellular nests with occasional acinar differentiation and squamoid corpuscles. Imprint cytology of the biopsied sample displayed cellular epithelial nests with focal acinar structures and foci composed of larger cells with a low nuclear/cytoplasmic ratio. These foci contained a few tumor cells with biotin-rich OCN and were determined to be squamoid corpuscles. CONCLUSION: Detection of occasional squamoid corpuscles with biotin-rich OCN can be useful in making a diagnosis of PBL on cytologic samples. 相似文献
36.
Shimizu A Asakawa S Sasaki T Yamazaki S Yamagata H Kudoh J Minoshima S Kondo I Shimizu N 《Biochemical and biophysical research communications》2003,309(1):143-154
We identified a novel giant gene encoding a transmembrane protein with CUB and sushi multiple domains on the human chromosome 8q23.3-q24.1 in which benign adult familial myoclonic epilepsy type 1 (BAFME1/FAME, OMIM:601068) has been mapped. This giant gene consists of 73 exons and spans over 1.2Mb on the genomic DNA region. It showed significant homology to two genes, CSMD1 gene on 8p23 and CSMD2 gene on 1p34, at reduced amino acid sequence level and hence we designated as CSMD3. The CSMD3 gene was expressed mainly in adult and fetal brains. We performed mutation analysis on the CSMD3 gene for seven patients with BAFME1/FAME, but no mutation was found in the coding sequence of the CSMD3 gene. Comparative genomic analysis revealed a conserved family of CSMD genes in the mouse and fugu genomes. Possible functions of the CSMD gene family are discussed. 相似文献
37.
Sakuraba H Tsuge H Shimoya I Kawakami R Goda S Kawarabayasi Y Katunuma N Ago H Miyano M Ohshima T 《The Journal of biological chemistry》2003,278(12):10799-10806
38.
39.
Sato A Sasakura Y Sugiyama S Sagami I Shimizu T Mizutani Y Kitagawa T 《The Journal of biological chemistry》2002,277(36):32650-32658
The heme environments of Met(95) and His(77) mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(CO) plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the nu(Fe-His) band but gave a nu(Fe-Im) band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a five-coordinate heme complex. Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2). Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS. 相似文献
40.
Takagaki K Iwafune M Kakizaki I Ishido K Kato Y Endo M 《The Journal of biological chemistry》2002,277(21):18397-18403
We previously found that endo-beta-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-beta-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-beta-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-beta-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides. 相似文献