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131.
Peters RL Bossen M van Kooten O Vredenberg WJ 《Journal of bioenergetics and biomembranes》1983,15(6):335-346
The P515 absorbance change upon single-turnover light flashes has been studied in intact leaves and isolated chloroplasts from spinach. A comparative study of the effects of preillumination on the kinetics of the P515 response and on the activity of the chloroplast ATPase has been made. The slow component (reaction 2) in the flash-induced P515 response normally present in dark-adapted chloroplasts is reduced or even absent under conditions in which the ATPase is activated by preillumination. This suppression of reaction 2 appeared to be temporary in leaves and chloroplasts; its duration in chloroplasts is shown to be dependent on the amount of ATP present. Tentoxin inhibits the preillumination-dependent suppression of reaction 2. 相似文献
132.
Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit 总被引:3,自引:0,他引:3
W H Peters A M Fleuren-Jakobs J J Schrijen J J De Pont S L Bonting 《Biochimica et biophysica acta》1982,690(2):251-260
(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits. 相似文献
133.
The localization in rat liver of alkaline phosphodiesterase to a discrete organelle implicated in ligand internalization 总被引:4,自引:0,他引:4
The subcellular distribution of alkaline phosphodiesterase and NADH pyrophosphatase, two activities thought to be expressed by the same enzyme, was investigated. Although the two activities share a localization to a low-density vesicular membrane (equilibrium density = 1.12 g.cm-3), little NADH pyrophosphatase activity, in contrast to alkaline phosphodiesterase, was found in plasma membrane (equilibrium density = 1.18 g.cm-3), as reflected by the distribution of 5'nucleotidase. The binding and uptake of 125I-labelled insulin in perfused rat liver was also investigated. This ligand was found to bind to sinusoidal plasma membrane at 4 degrees C, but was rapidly internalized at 37 degrees C to the low-density membrane, which is rich in alkaline phosphodiesterase and NADH pyrophosphatase. These vesicular membranes were shown to belong to none of the enzymatically characterized subcellular bodies, and it is proposed that they represent discrete organelles participating in the subcellular processing of receptor-ligand complexes. 相似文献
134.
G Wicker V Prill D Brooks G Gibson J Hopwood K von Figura C Peters 《The Journal of biological chemistry》1991,266(32):21386-21391
The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease. 相似文献
135.
p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV- 40 T antigen proteins 总被引:12,自引:0,他引:12
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D A Jans M J Ackermann J R Bischoff D H Beach R Peters 《The Journal of cell biology》1991,115(5):1203-1212
136.
Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila. 总被引:6,自引:5,他引:1
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S A Raybuck S E Ramer D R Abbanat J W Peters W H Orme-Johnson J G Ferry C T Walsh 《Journal of bacteriology》1991,173(2):929-932
The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria. 相似文献
137.
Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis. 总被引:4,自引:2,他引:2
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sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E. 相似文献
138.
Andrey A. Rosenkranz Sergey V. Yachmenev David A. Jans Natalya V. Serebryakova Vitaly I. Murav''ev Reiner Peters Alexander S. Sobolev 《Experimental cell research》1992,199(2):323-329
A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor. 相似文献
139.
J M Peters J R Harris A Lustig S Müller A Engel S Volker W W Franke 《Journal of molecular biology》1992,223(2):557-571
We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48. 相似文献
140.
Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA
Indole-3-acetic acid
- MES
2-[N morpholino-ethane-sulfonic acid
- TRIS
2-Amino-2-hydroxymethyl-1,3-propanediol
We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft. 相似文献