Phosphate-binding tag, a new tool to visualize phosphorylated proteins

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).     

Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions

We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) in Zn(2+)-Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10-200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris-HCl and Tris-acetic acid (Tris-AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10-200 kDa cellular proteins, the Bis-Tris-HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn(2+)-Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris-AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.     

Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin

We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.     

Detection of heavy metal ions using protein-functionalized microcantilever sensors

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg(2+) and Zn(2+). AgNt84-6, a protein that has the ability to bind multiple atoms of Ni(2+), Zn(2+), Co(2+), Cu(2+), Cd(2+) and Hg(2+) was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl(2) and 0.1 mM ZnCl(2) solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl(2) solution did not result in a similar bending indicating a weak or no interaction of Mn(2+) ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn(2+) ions but not with Mn(2+) ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.     

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.     

Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents

Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.     

Manganese enhances phosphorylation of a 47 kD protein in retinoic acid-induced HL-60 cells

We previously observed that HL-60 cells treated with manganese (Mn) during differentiation displayed an enhanced oxidative burst. Since a Mn-dependent kinase has been identified and phosphorylation is involved in burst activation, the objective of this research was to identify proteins in retinoic acid-induced HL-60 cells whose phosphorylation after phorbol myristate acetate (PMA) stimulation was affected by Mn treatment. Cells received Mn during differentiation and were then harvested, labeled with [32]P-orthophosphate, and stimulated with PMA. Cytosolic proteins were separated by isoelectric focusing, SDS-PAGE, and two-dimensional (2-D) gel electrophoresis. Time studies showed that Mn treatment did not alter the rate of PMA activated phosphorylation. Isoelectric focusing revealed that PMA stimulation resulted in the appearance of three phosphoproteins at pI's of 6.8, 7.3, and 7.8. Size separation gels showed a 200% increase in phosphorylation of a 47 kD protein in Mn-treated cells after stimulation. The 2-D gels showed that the pI of this protein was 6.8. Therefore, Mn treatment resulted in greater phosphorylation of a 47 kD protein, pI 6.8, in phorbol ester-stimulated cells. J. Cell. Physiol. 176:188–195, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803

The dxr gene encoding the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The K(m)'s for the metal ions Mn(2+), Mg(2+), and Co(2+) were determined for native DXR for the first time, with the K(m) for Mg(2+) being approximately 200-fold higher than the K(m)'s for Mn(2+) and Co(2+).     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates

The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading dye solution were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated Shc and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

     

Identification of lamin B and histones as 1,25-dihydroxyvitamin D3-regulated nuclear phosphoproteins in HL-60 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)-induced differentiation of HL-60 leukemia cells is accompanied by a number of cellular changes including regulation of oncogene expression and induction of terminal differentiation. We investigated the mechanism by which 1,25-(OH)2D3 induces these changes. We detected 10 nuclear phosphoproteins, designated p66, p45, p36, p33, p32, p27, p22, p19, p18 and p17, that show alterations in phosphorylation within 6-40 h of 1,25-(OH)2D3 treatment. When phosphorylation reactions were performed with isolated nuclei (in vitro), three of these proteins were phosphorylated in a calcium and phospholipid dependent manner: p66, p36, and p19 P66 was phosphorylated in response to 1,25-(OH)2D3 and purified in a manner similar to that used for nuclear lamins. Western blot analysis of 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels confirmed its identity as lamin B. Phosphorylation of p17 and p18 decreased following 1,25-(OH)2D3 treatment. We separated p17 and p18 by SDS-PAGE and obtained N-terminal amino acid sequence to identify these phosphorproteins as histones H2b and H3, respectively. P19 and p22 were both DNA-cellulose binding proteins whose phosphorylation was altered by 1,25-(OH)2D3 treatment. Increased phosphorylation of p27 was detected using 2-dimensional SDS-PAGE. Phosphorylation of nuclear proteins in the intact cell (in vivo), revealed increases in p66, p45, p36, and p33 phosphorylation and a decrease in p17 phosphorylation following 1,25-(OH)2D3 treatment. We detected an increase in phosphorylation of p32, which was extracted with salt from nuclei and migrated on SDS-PAGE similar to histone H1. Thus, we have identified 1,25-(OH)2D3-sensitive nuclear phosphoproteins, including lamin B and several histones. We have also detected and characterized several less abundant nuclear DNA binding phosphoproteins whose phosphorylation was affected by 1,25-(OH)2D3.     

Apoptotic DNA degradation: evidence for novel enzymes

Apoptosis is characterized by multiple morphological and biochemical changes. One biochemical change that has been primarily associated with apoptosis is the cleavage of chromatin in the internucleosomal regions. We have taken two independent approaches to investigating the enzyme(s) responsible for such cleavage. First, using SDS-PAGE gels with (32)P-labelled DNA incorporated into the matrix, we identified a nuclease activity (termed NUC18) from apoptotic thymocytes. This enzyme has been purified to homogeneity and the activity of the pure protein is dependent on Ca(2+) and Mg(2+) while inhibited by Zn(2+) and aurintricarboxylic acid. This protein is found in the nucleus of apoptotic and nonapoptotic cells but is maintained in nondying cells in a large-molecular-weight inactive complex. NUC18 has a denatured molecular weight of 18 Kd but elutes from gel filtration columns with a native molecular weight of approximately 25 Kd. Although an exhaustive search has not been performed, NUC18 has been identified in several cell lines and tissues. Our second approach is designed specifically to detect internucleosomal cleavage of DNA, an obvious requirement for an apoptotic nuclease. By examining the degradation of HeLa chromatin, we have identified a low-molecular-weight of approximately 23 Kd native molecular weight) internucleosomal cleavage enzyme active in nuclear extracts from glucocorticoid-treated thymocytes. This activity is also dependent upon Ca(2+)and Mg(2+) and is inhibited by Zn(2+) as well as aurintricarboxylic acid. It is present in a variety of cell lines and tissues and is maintained in control cells in a latent state prior to apoptosis. In addition to similarities in physical properties, the two enzymes appear to be immunologically related to one another by virtue of their ability to interact with the same antibody. Overall, using independent approaches, we have identified two nucleases with similar biochemical properties whose activity correlates with apoptosis. The current work suggests that these are novel and perhaps closely related enzymes.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Site-specific effects of zinc on the activity of family II pyrophosphatase

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.     

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.     

新鲜大蒜中蒜氨酸酶的分离纯化及性质

用葡聚糖凝胶G-200层析柱分离纯化了新鲜大蒜(Allium sativum)中的蒜氨酸酶, SDS-PAGE结果为单一条带, 分子量为53 kD在35 ℃下以蒜氨酸为底物, Km为0.693 mmol.L-1, Vmax 为0.353 mmol.min-1, 最适反应温度为30 ℃, 热稳定的温度在50 ℃以下。Zn2+对酶有抑制作用, Mn2+使酶活力增加。