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11.
William R. Kem Chiang-Kuang Tu Robert W. Williams Arazdordi Toumadje W. Curtis Johnson Jr. 《The protein journal》1990,9(4):433-443
The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation. 相似文献
12.
13.
Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM)
2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester)
- BTB
bis-trispropane
- CDCF (CDCF-DA)
5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative)
- DAPI
4,6-diamidino-2 phenylindole dihydrochloride
- DMSO
dimethylsulfoxide
- HEPES
N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid]
- MES
2-[N-morpholino]ethane-sulfonic acid
- SNAFL-1 (SNAFL-1-DA)
carboxyl SNAFL-1 (its diacetate)
- SNARF-1 (SNARF-1-AM)
carboxyl SNARF-1 (its acetoxymethyl acetate) 相似文献
14.
Ke Zeng John P. Rose Hong-Chi Chen Corey L. Strickland Chen-Pei D. Tu Bi-Cheng Wang 《Proteins》1994,20(3):259-263
A chimeric enzyme (GST121) of the human α-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P212121, with cell dimensions a = 49.5, b = 92.9, c = 115.9 Å, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution resulting in a change of its solution properties. © 1994 Wiley-Liss, Inc. 相似文献
15.
Corn root plasma membrane catalyzed NADH reduction of ferricyanideand cytochrome c over a wide pH range. At pH 7.5, apparent Kmsof NADH-cytochrome c pair were significantly lower than thoseof NADH-ferricyanide pair. FMN and polylysine respectively enhancedthe reduction of ferricyanide and cytochrome c. Yet, polyaspartatedecreased the ferricyanide reduction. NADH oxidation observedin the presence of both ferricyanide and cytochrome c was significantlyslower than the sum of rates obtained with individual acceptors.The results suggest that the membrane may contain differentbut not totally independent reduction sites for cytochrome cand ferricyanide. (Received April 13, 1993; Accepted August 23, 1993) 相似文献
16.
Divalent cation and lipid-protein interactions of biomembranes 总被引:1,自引:0,他引:1
Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg2+-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H+-ATPase, (2) a proper transmembrane Ca2+ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca2+ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca2+-ATPase activity. 相似文献
17.
The type 1 insulin-like growth factor receptor (IGF-IR) and its docking protein, insulin receptor substrate-1 (IRS-1), play important roles in cell transformation, cell differentiation and aging. IRS-1 and other IRS proteins can, under certain conditions, localize to the nuclei of cells, where they undergo interactions with nuclear and nucleolar proteins. In this study, we confirm and extend these observations, demonstrating that IRS-1 is preferentially nuclear in growing cells. Differentiation and inhibition of ribosomal RNA synthesis cause subcellular redistribution of IRS-1 and other nuclear proteins to the cytoplasm. 相似文献
18.
The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na++ K+) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H+-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na++ K+) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na++ K+) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe. 相似文献
19.
REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae. 总被引:12,自引:2,他引:10 下载免费PDF全文
Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway. 相似文献
20.
Shui-Tein Chen Shiah-Yun Chen Chen-Chen Tu Shyh-Horng Chiou Kung-Tsung Wang 《Journal of Protein Chemistry》1995,14(4):205-215
The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents. 相似文献