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51.
Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase. 相似文献
52.
We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility. 相似文献
53.
54.
Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution 总被引:2,自引:0,他引:2
Seifert MH Georgescu J Ksiazek D Smialowski P Rehm T Steipe B Holak TA 《Biochemistry》2003,42(9):2500-2512
Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant. 相似文献
55.
Morales-Ruiz T Birincioglu M Jaruga P Rodriguez H Roldan-Arjona T Dizdaroglu M 《Biochemistry》2003,42(10):3089-3095
A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics. 相似文献
56.
The diploid number 2n = 46 and the chromosome arm number NF = 74 are described in Lithobius forficatus from Olsztyn (Poland). Analyses of silver and CMA3-stained mitotic chromosomes suggest that a single chromosome pair has active NORs which correspond to G-C-rich (CMA3-positive) chromatin. Heteromorphism of the largest metacentric chromosome pair was observed. The sex chromosomes were not identified. Size polymorphism of the first chromosome pair was found. 相似文献
57.
Arima K Hines ER Kiela PR Drees JB Collins JF Ghishan FK 《American journal of physiology. Gastrointestinal and liver physiology》2002,283(2):G426-G434
We sought to characterize expression of an apically expressed intestinal Na-P(i) cotransporter (Na-P(i)-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P(i) uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-P(i)-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P(i) uptake and Na-P(i)-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P(i) uptake and Na-P(i)-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P(i) uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P(i)-IIb mRNA and protein expression. We also demonstrate that Na-P(i)-IIb is an N-linked glycoprotein and that glycosylation is age dependent. 相似文献
58.
Barylko B Wlodarski P Binns DD Gerber SH Earnest S Sudhof TC Grichine N Albanesi JP 《The Journal of biological chemistry》2002,277(46):44366-44375
Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme. 相似文献
59.
Wei YJ Sun HQ Yamamoto M Wlodarski P Kunii K Martinez M Barylko B Albanesi JP Yin HL 《The Journal of biological chemistry》2002,277(48):46586-46593
Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells. 相似文献
60.