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1.
Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with -[32P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.Abbreviations IP
inhibitor protein
- NR
NADH-nitrate reductase
- NRA
nitrate reductase activity
- NRPK
nitrate reductase protein kinase
- NRPP
nitrate reductase protein phosphatase
- PK
protein kinase
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251). 相似文献
2.
Peter J Kennelly 《FEMS microbiology letters》2002,206(1):1-8
For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism. 相似文献
3.
Rhythmic changes in the expression of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) were investigated during hexamethylene bisacetamide (HMBA) induced differentiation of murine erythroleukaemic (MEL) cells. Cell extracts were analysed by SDS-PAGE and western immunoblotting using specific antibodies. An immunospecific band of molecular mass 36 kDa (catalytic subunit) was detected for PP1. For PP2A, two immunospecific bands of 32 kDa (proteolytically cleaved catalytic subunit) and 36 kDa (catalytic subunit) were observed. Comparisons of proliferating and differentiating cells using only one time point showed no significant differences between mean values for the expression of the PP1 or PP2A enzyme proteins. This kind of analysis, implying that HMBA had little effect, proved misleading, as comparisons using multiple time points showed rhythmic patterns of protein expression which were modulated by the differentiating agent. The effects were complex affecting both the frequency and phasing of rhythms. The results add further support for the view that live cells are multi-oscillators and for the concept that differentiation depends on changes in temporal organization of complex autodynamic feedback loops and multiple interactions between control circuits performing in parallel. In particular, modulation of the dynamics of key proteins, such as PP1 and PP2A, may be a possible mechanism for controlling cellular function and reversing transformation in accordance with long standing theoretical and other experimental data. 相似文献
4.
Moncho-Amor V Galardi-Castilla M Perona R Sastre L 《Differentiation; research in biological diversity》2011,81(3):199-207
Dual-specificity protein phosphatases participate in signal transduction pathways inactivating mitogen-activated protein kinases (MAP kinases). These signaling pathways are of critical importance in the regulation of numerous biological processes, including cell proliferation, differentiation and development. The social ameba Dictyostelium discoideum harbors 14 genes coding for proteins containing regions very similar to the dual-specificity protein phosphatase domain. One of these genes, mkpB, additionally codes for a region similar to the Rhodanase domain, characteristic of animal MAP kinase-phosphatases, in its N-terminal region. Cells that over-express this gene show increased protein phosphatase activity. mkpB is expressed in D. discoideum ameba at growth but it is greatly induced at 12h of multicellular development. Although it is expressed in all the cells of developmental structures, mkpB mRNA is enriched in cells with a distribution typical of anterior-like cells. Cells that express a catalytically inactive mutant of MkpB grow and aggregate like wild-type cells but show a greatly impaired post-aggregative development. In addition, the expression of cell-type specific genes is very delayed, indicating that this protein plays an important role in cell differentiation and development. Cells expressing the MkpB catalytically inactive mutant show increased sensitivity to cisplatin, while cells over-expressing wild type MkpB, or MkpA, proteins or mutated in the MAP kinase erkB gene are more resistant to this chemotherapeutic drug, as also shown in human tumor cells. 相似文献
5.
A series of bromo-retrochalcones was designed, synthesized and evaluated as PTP1B inhibitors based on licochalcone A and E. Compounds 6, 12, 13, 14, 25, 36, 37, 39, and 41 showed potent inhibitory effects against PTP1B, and compound 37, the most potent among the series, had an IC50 value of 1.9 μM, about two-fold better than that of the positive control, ursolic acid. 相似文献
6.
A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity 总被引:1,自引:0,他引:1
Boriyana Tuleva Evgenia Vasileva-Tonkova Danka Galabova 《FEMS microbiology letters》1998,161(1):139-144
In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg2+ ions and an apparent Km of 3.6×10−5 M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation. 相似文献
7.
Erdodi F Kiss E Walsh MP Stefansson B Deng JT Eto M Brautigan DL Hartshorne DJ 《Biochemical and biophysical research communications》2003,306(2):382-387
Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways. 相似文献
8.
Hélène-Marie Thérien Walter E. Mushynski 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(2):188-200
Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high Km and a low Km protein kinase activity while only the high Km activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides. 相似文献
9.
Reaction of radicals in the presence of O2, or singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. They can be detected in cells and are poorly removed by enzymatic defenses. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, with this resulting in inactivation of some thiol-dependent enzymes. In light of these data, we hypothesized that inactivation of protein tyrosine phosphatases (PTPs), by hydroperoxides present on oxidized proteins, may contribute to cellular and tissue dysfunction by modulation of phosphorylation-dependent cell signaling. We show here that PTPs in cell lysates, and purified PTP-1B, are inactivated by amino acid, peptide, and protein hydroperoxides in a concentration- and structure-dependent manner. Protein hydroperoxides are particularly effective, with inhibition occurring with greater efficacy than with H2O2. Inactivation involves reaction of the hydroperoxide with the conserved active-site Cys residue of the PTPs, as evidenced by hydroperoxide consumption measurements and a diminution of this effect on blocking the Cys residue. This inhibition of PTPs, by oxidized proteins containing hydroperoxide groups, may contribute to cellular dysfunction and altered redox signaling in systems subject to oxidative stress. 相似文献
10.
Caixia Yuan Yanbo Wu Maolin Guo Shu Xing Miaoli Zhu 《Journal of inorganic biochemistry》2010,104(9):978-12934
Seven new mixed-ligand vanadyl complexes, [VIVO(5-Br-SAA)(NN)] and [VIVO(2-OH-NAA)(NN)] (1-7) (5-Br-SAA for 5-bromosalicylidene anthranilic acid, 2-OH-NAA for 2-hydroxy-1-naphthaldehyde anthranilic acid and NN for N,N′-donor heterocyclic base, namely, 2,2′-bipyridine (bpy, 1 and 5), 1,10-phenanthroline (phen, 2 and 6), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 3 and 7), dipyrido[3,2-a:2′,3′-c]phenazine (dppz, 4)), were synthesized and characterized. X-ray crystal structure of [VIVO(5-Br-SAA)(phen)] revealed a distorted octahedral geometry with the Schiff base ligand coordinated in a tridentate ONO-fashion and the phenanthroline ligand in a bidentate fashion. Density-functional theory (DFT) calculations suggest a similar structure and the same coordination mode for all the other oxovanadium complexes synthesized. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of protein tyrosine phosphatase 1B (PTP1B), with IC50 values approximately 41-75 nM. Kinetics assays suggest that the complexes inhibit PTP1B in a competitive manner. Notably, they had moderate selectivity of PTP1B over T-cell protein tyrosine phosphatase (TCPTP) (about 2-fold) and good selectivity over Src homology phosphatase 1 (SHP-1) (about 4∼7-fold). Thus, these mixed-ligand complexes represent a promising class of PTP1B inhibitors for future development as anti-diabetic agents. 相似文献
11.
Organo-vanadium compounds (OVC) have been shown to be more effective than inorganic vanadium compounds in ameliorating glucose homeostasis and insulin resistance in rodent models of diabetes mellitus. However, the precise molecular mechanism of OVC efficiency remains poorly defined. Since inorganic vanadium compounds have been found to activate several key components of the insulin signaling cascade, such as protein kinase B (PKB), the objective of the present study was to investigate if stimulation of PKB and its downstream target glycogen synthase kinase-3 (GSK-3), are responsible for the more potent insulinomimetic effects of OVC. Among several vanadium compounds tested, vanadium (IV) oxo bis (acetylacetonate) and vanadium (IV) oxo bis(maltolato) markedly induced the phosphorylation of PKB as well as GSK-3beta compared to vanadyl sulfate (VS), an inorganic vanadium salts in Chinese hamster ovary cells overexpressing the insulin receptor (IR). Furthermore, the OVC were stronger inhibitors of protein tyrosine phosphatase (PTPase) activity than VS. The higher PTPase inhibitory potential of the OVC was associated with more robust tyrosine phosphorylation of several cellular proteins, including the IRbeta subunit and insulin receptor substrate-1 (IRS-1). In addition, greater IRS-1/p85alpha interaction was elicited by the OVC than by VS. These data indicate that the higher PTPase inhibitory potential of OVC translates into greater phosphorylation of PKB and GSK-3beta, which, in turn, may contribute to a more potent effect of OVC on glucose homeostasis. 相似文献
12.
Hong SB Lubben TH Dolliver CM Petrolonis AJ Roy RA Li Z Parsons TF Li P Xu H Reilly RM Trevillyan JM Nichols AJ Tummino PJ Gant TG 《Bioorganic chemistry》2005,33(1):34-44
Mitogen-activated protein kinase phosphatase-4 (MKP-4) is a dual specificity phosphatase, which acts as a negative regulator of insulin-stimulated pathways. Here, we describe expression, purification, and biochemical characterization of MKP-4. We used the Baculovirus expression system and purification with a combination of affinity and gel filtration chromatography to generate pure MKP-4 and MKP-4/p38 complex. Both MKP-4 and the MKP-4/p38 complex exhibited moderate activity toward the surrogate substrates p-nitrophenyl phosphate, 6, 8-difluoro-4-methylumbelliferyl phosphate, and 3-O-methylfluorescein phosphate. The phosphatase activity could be inhibited by peroxovanate, a potent inhibitor of protein tyrosine phosphatases. We further determined kinetic parameters for the MKP-4 and the MKP-4/p38 by using spectrophotometric and fluorescence intensity methods. The MKP-4/p38 complex was found to provide substantially higher phosphatase activity than MKP-4 alone, similar to what has been shown for MKP-3. Our data allow the configuration of screens for modulators of MKP-4 activity. 相似文献
13.
Yuzhong Duan Fanglin Chen Anmei Zhang Bo Zhu Jianguo Sun Qichao Xie Zhengtang Chen 《BMB reports》2014,47(1):45-50
Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE2 in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE2 upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE2 levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE2 levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE2 upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE2. Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI. [BMB Reports 2014; 47(1): 45-50] 相似文献
14.
The regulation of tyrosine phosphorylation is recognized as an important developmental mechanism. Both addition and removal of phosphate moieties on tyrosine residues are tightly regulated during development. Originally, most attention focused on the role of tyrosine kinases during development, but more recently, the developmental importance of tyrosine phosphatases has been gaining interest. Receptor protein tyrosine phosphatases (RPTPs) are of particular interest to developmental biologists because the extracellular domains of RPTPs are similar to those of cell adhesion molecules (CAMs). This suggests that RPTPs may have functions in development similar to CAMs. This review focuses on the role of RPTPs in development of the nervous system in processes such as axon guidance, synapse formation, and neural tissue morphogenesis. 相似文献
15.
Serine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation-dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia-reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress. We studied PP1 complexes in response to IR stress and IT in brain using proteomic characterization of PP1 complexes in animal models of IR and IT. PP1alpha and PP1gamma were immunoprecipitated and resolved by 2-D. DIGE analysis detected 14 different PP1-interacting proteins that exhibited significant changes in their association with PP1alpha or PP1gamma. These proteins were identified by MALDI-TOF MS. Seven had the PP1-binding RVxF motif. IR altered the interaction of heat shock cognate 71 kDa-protein, creatine kinase B, and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) with both PP1alpha and PP1gamma, and the interaction of phosphodiesterase-6B, transitional ER ATPase, lamin-A, glucose-regulated 78 kDa-protein, dihydropyrimidinase-related protein-2, gamma-enolase, neurofilament-L, and ubiquitin ligase SIAH2 with PP1gamma. IT prevented most of the IR-induced effects. This study identifies novel PP1alpha- and PP1gamma-interacting proteins and reveals an in vivo modularity of PP1 holoenzymes in response to physiological ischemic stress. It supports a potential role of PP1 in IR stress and as a target of the endogenous protective mechanisms induced by IT. 相似文献
16.
17.
The production of recombinant proteins in Escherichia coli involves substantial optimization in the size of the protein and over-expression strategies to avoid inclusion-body formation. Here we report our observations on this so-called construct dependence using the catalytic domains of five Drosophila melanogaster receptor protein tyrosine phosphatases as a model system. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N- and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study we observe that inclusion of a 45 residue stretch at the N-terminus was crucial for over-expression of the enzymes, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order promoting residues or aliphatic index did not correlate with the improved biochemical characteristics. These findings thus suggest the inclusion of additional parameters apart from rigid domain predictions to obtain domain constructs that are most likely to yield soluble protein upon expression in E. coli. 相似文献
18.
19.
Yasunori Sugiyama Sho Yamashita Yuuki Uezato Yukako Senga Syouichi Katayama Naoki Goshima Yasushi Shigeri Noriyuki Sueyoshi Isamu Kameshita 《Analytical biochemistry》2016
To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca2+/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities. 相似文献
20.
Caixia Yuan Liping Lu Xiaoli Gao Yanbo Wu Maolin Guo Ying Li Xueqi Fu Miaoli Zhu 《Journal of biological inorganic chemistry》2009,14(6):841-851
Abstract A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic
acid (SAA)], and a bidentate NN ligand [viz., 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[3,2-a:2′,3′-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2′,3′-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry,
UV–vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of
both complexes, [VIVO(SAA)(bpy)]·0.25bpy and [VIVO(SAA)(phen)]·0.33H2O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen
atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO3N3). The oxidation state of V(IV) with d
1 configuration was confirmed by EPR spectroscopy. The speciation of VO–SAA–bpy in aqueous solution was investigated by potentiomtreic
pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0–7.4, and one
is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine
phosphatase 1B (PTP1B) (IC50 approximately 30–61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly,
the [VIVO(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against
SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive
and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents.
Graphical Abstract
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献