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1.
A calcium-regulated protein kinase (CRPK) associated with the plasma membrane of beet root cells was purified by deoxycholate extraction of plasma membrane proteins followed by chromatography in DEAE- and phenyl-Sepharose columns. This procedure rendered a purified CRPK preparation with a specific activity higher than 2.2 μmol mg-1 min-1 using histone III-S as substrate. The molecular mass of CRPK, estimated by SDS-PAGE, was 58 kDa. Gel filtration and glycerol gradient centrifugation indicated that the native enzyme is a monomer. Phosphorylation of histone by CRPK displayed a broad pH optimum between 7.0 and 8.2, Syntide-2, a CaM-kinase II substrate, was phosphorylated much more efficiently than kemptide, Ac-MBP (4–14), CKII substrate or poly-(Glu, Tyr) 4:1. The kinetics of this enzyme can be described by a steady-state ordered mechanism in which four bound Ca2+ ions are required for the phosphorylation of the peptide substrate. After Ca2+ binding, the affinity of the enzyme for ATP was not affected while that for syntide-2 or histone decreased. Curves of kinase activity vs ATP at different syntide-2 concentrations indicated that the Km values for ATP or syntide-2 increased with increasing cosubstrate concentrations. Similar curves of kinase activity vs ATP at different histone concentrations indicated that the apparent affinities of CRPK for ATP or histone were not affected by the cosubstrate concentration. The purified CRPK was not recognized by antibodies against soybean or Arabidopsis calcium-dependent protein kinase (CDPK). These results suggest that CRPK and CDPKs could be different enzymes, although they show some similar properties. 相似文献
2.
Kentaro Kato Atsushi Sudo Kyousuke Kobayashi Tatsuki Sugi Yukinobu Tohya Hiroomi Akashi 《Parasitology international》2009,58(4):394-400
In Plasmodium berghei, the orthologous gene of P. falciparum calcium-dependent protein kinase 4 (PfCDPK4) was reported to be essential for the exflagellation of male gametocytes. To elucidate the role of PfCDPK4 in P. falciparum gametogenesis, we characterized the biological function of PfCDPK4 in vitro. PfCDPK4 was purified as a fusion protein that was labeled with [γ-32P]ATP; this labeling was then eliminated by phosphatase. Phosphorylation activity of PfCDPK4 was eliminated when its putative catalytic lysine residue was replaced with alanine. In biochemical analyses, PfCDPK4 was found to have characteristics that were similar to those of homologous proteins from plants. PfCDPK4 phosphorylation was activated when experimental conditions were changed from those characteristic of human blood (37 °C, pH 7.4) to those of the mosquito bloodmeal (at least 5 °C below 37 °C, pH 7.6, with xanthurenic acid (XA)). PfCDPK4 was overexpressed in day 15 gametocytes exposed to XA or human serum. Thus, PfCDPK4 phosphorylation is activated by an increase in Ca2+ concentration or pH and by a decrease in temperature, and is associated with the Ca2+ signals that facilitate P. falciparum gametogenesis. 相似文献
3.
4.
5.
We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the mossFunaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation,
DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at
a concentration of 50 (AM free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated
with Ca2+ and CaM. Underin vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with
the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco
CCaMK. We have characterized it as a moss CCaMK. 相似文献
6.
Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca2+ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca2+ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-32 P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca2+ , while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca2+ -stimulated phosphoprotein band at 64 kDa. 相似文献
7.
8.
Summary An NADH-cytochromeb
5 reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb
5 reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb
5 reductases.Abbreviations CHAPS
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate
- BCA
bicinchonicic acid
- EDTA
ethylenediamine tetraacetate acid disodium salt
- FeCN
ferricyanide
- HPLC
high-performance liquid chromatography
- NADH
nicotinamide adenine dinucleotide reduced form
- PMSF
-phenylmethylsulfonyl fluoride 相似文献
9.
Membranes isolated from pea buds contain protein-kinase activity which is greatly activated by low concentrations of calcium ions. This paper describes a simple purification of this enzyme with a novel means of detecting enzyme activity by Western blotting. The purified enzyme appears to autophosphorylate primarily on serine residues, is activated by bovine calmodulin and additional evidence from phase partitioning indicate most of this enzyme to be located in the plasma membrane.Abbreviations PAGE
polyacrylamide-gel electrophoresis
- SDS
sodium dodecyl sulphate 相似文献
10.
Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC50=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants. 相似文献
11.
McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydration-stress responsive. The enzyme
also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system,
we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2
in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of
the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for
McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function
also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin superfamily
that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and
fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton
and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTI1a, an Arabidopsis
v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that
McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma
membrane along actin microfilaments of the cytoskeleton.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
12.
A 67-kDa plasma-membrane-bound Ca2+-stimulated protein kinase active in sink tissue of higher plants
A novel 67-kDa protein kinase (p67
cdpk
) was identified in the microsomal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissues. Microsomal proteins were blotted
onto Nylon or polyvinylidenedifluoride membranes, and p67
cdpk
assayed by in situ-labelling renatured proteins with [γ-32P]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the 32P-labelled protein band indicated that serine and threonine, but not tyrosine residues were phosphorylated. A detailed analysis
of the ion-dependency of p67
cdpk
revealed that it was a Ca2+-stimulated, Mg2+-dependent protein kinase. However, p67
cdpk
was ten times more active in the presence of 10 mM Mn2+, and these assay conditions were used routinely to increase the sensitivity of the assay. Activity of p67
cdpk
was found at high levels in the plasma membrane, and solubilisation experiments with a number of detergents suggested that
p67
cdpk
is an integral membrane protein. A homologous protein kinase with similar biochemical properties was also present in cell-suspension
cultures of pear and maize. In maize (Zea mays L.) plants, sink tissues, such as young expanding leaves of both light-grown and etiolated plants, mature etiolated tissue
and roots all had high levels of p67
cdpk
activity. However, mature light-grown (source) tissues had barely detectable levels. In etiolated maize leaves and coleoptiles
the kinase activity was highest in expanding tissue and decreased as the cells expanded. When etiolated maize plants were
exposed to light, the activity of p67
cdpk
was reduced to background levels after 8 h. Although p67
cdpk
has biochemical properties similar to those of other plant calcium-dependent protein kinases, this is the first identification
of a membrane-bound calcium-dependent protein kinase which is specifically active in sink tissues.
Received: 14 July 1997 / Accepted: 25 September 1997 相似文献
13.
McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is induced transiently by salinity and water deficit stress and also McCPK1 undergoes dynamic subcellular localization changes in response to these same stresses. Here we have confirmed that low humidity is capable of causing a drastic change in McCPK1’s subcellular localization. We attempted to elucidate this phenomenon by isolating components likely to be involved in this process. McCAP1 (M. crystallinum CDPK adapter protein 1) was cloned in a yeast two-hybrid screen with a constitutively active McCPK1 as bait. We show that McCPK1 and McCAP1 can interact in the yeast two-hybrid system, in vitro, and in vivo as demonstrated by coimmunoprecipitation experiments from plant extracts. However, McCAP1 does not appear to be a substrate for McCPK1. DsRed–McCAP1 and EGFP–McCPK1 fusions colocalize in epidermal cells of ice plants exposed to low humidity. McCAP1 is homologous to a family of proteins in Arabidopsis with no known function. Computational threading analysis suggests that McCAP1 is likely to be an intermediate filament protein of the cytoskeleton. 相似文献
14.
Giovanna Carratù Anna Maria Carafa Maria Carmen Rucabado Matteo Giannattasio 《Phytochemistry》1984,23(12):2733-2735
A kinase phosphorylating casein and phosvitin has been purified from pine pollen by a three-step procedure involving DEAE-cellulose chromatography, affinity chromatography on casein-Sepharose and Sephadex G-100. A purification of about 2000 fold was obtained by this procedure. The kinase is affected neither by cyclic nucleotides nor by Ca2+-calmodulin, whereas it is strongly inhibited by heparin. Using this purification procedure, we have isolated protein kinase exhibiting phosphorylating activity towards casein in the pollen of many other Pinaceae species. 相似文献
15.
Timothy M. Chapman Simon A. Osborne Nathalie Bouloc Jonathan M. Large Claire Wallace Kristian Birchall Keith H. Ansell Hayley M. Jones Debra Taylor Barbara Clough Judith L. Green Anthony A. Holder 《Bioorganic & medicinal chemistry letters》2013,23(10):3064-3069
A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria. 相似文献
16.
Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco 总被引:1,自引:0,他引:1
By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 (accession number AY971376), which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone (fraction Ⅲ, abbreviated to histone Ⅲs) as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein. 相似文献
17.
Crude Ca2+-activated protein kinase from membranes of apple (Malus domestica L. Borkh., Cox's Orange Pippin) fruit can be partially purified to yield a Ca2+-dependent protein kinase whose activity is apparently not regulated by calmodulin. The autophosphorylating catalytic subunit of this protein kinase shows a Ca2+-dependent mobility shift of approx. 10 kilodaltons (kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis; in the absence of added Ca2+ or ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) its apparent molecular mass is approx. 50 kDa. The Ca2+-dependent protein kinase is inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide and trifluoperazine with IC50 values of approx. 45 M and 15 M, respectively. These similarities between the protein kinase and calmodulin indicate that the kinase may be a calmodulin-like protein.Abbreviations DEAE
diethylaminoethyl
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Hepes
4-(-2-hydroxyethyl)-1-piperazineethanesulphonic acid
- kDa
kilodalton
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulphate
- W7
N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide
- W5
N-(6-aminohexyl)-naphthalenesulphonamide 相似文献
18.
19.
The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56° C and pH 2.0. Its plasma membrane possessed 21–22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched α 1 → 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunction with water turgor, may contribute to the rigidity of the membrane and the cation binding. 相似文献