共查询到20条相似文献,搜索用时 31 毫秒
1.
Microcystin-LR (MCYST-LR), a cyclic peptide hepatotoxin, associates with high-molecular-weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 +/- 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high-performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time- and temperature-dependent and did not require endogenous molecules less than 6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST-LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration-dependent inhibition of [3H]MCYST-LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A. 相似文献
2.
3.
Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively. 相似文献
4.
Antibodies against a microcystin (MCYST) leucine-arginine variant (MCYST-LR) were demonstrated 4 weeks after immunization of rabbits with either MCYST-LR-polylysine- or MCYST-LR-ethylenediamine-modified bovine serum albumin. A radioimmunoassay (RIA), a direct competitive enzyme-linked immunosorbent assay (ELISA), and an indirect competitive ELISA were developed for characterization of the antibodies. Indirect ELISA and RIA revealed that MCYST-LR-ethylenediamine-bovine serum albumin was a better immunogen. Competitive RIA and direct ELISA revealed that the antibodies had good cross-reactivities with an MCYST-arginine-arginine variant (MCYST-RR), MCYST-LR, an MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN); but they had lower reactivities with variants MCYST-leucine-tyrosine (MCYST-LY) and MCYST-leucine-alanine (MCYST-LA). The antibodies did not cross-react with ozonolyzed MCYST-LR. The concentrations causing 50% inhibition of binding of reduced MCYST-LR to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LA, and MCYST-LY in the RIA were 43, 105, 112, 503, 671, and 1,920 ng/ml, respectively. The concentrations causing 50% inhibition of binding of MCYST-LR-horseradish peroxidase to the antibodies by MCYST-RR, MCYST-LR, MCYST-YR, NODLN, MCYST-LY, and MCYST-LA in the ELISA were 1.75, 2.2, 3.4, 4.6, 50, and 114 ng/ml, respectively. 相似文献
5.
Michael Bachmann August Bernd Heinz C. Schröder Rudolf K. Zahn Werner E.G. Müller 《生物化学与生物物理学报:生物膜》1984,773(2):308-316
The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol Pi/min per 108 ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the ‘carrier structure’ for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase. 相似文献
6.
Philomena Alapatt Fangjian Guo Susan M. Komanetsky Shuping Wang Jinjin Cai Ashot Sargsyan Eduardo Rodríguez Díaz Brandon T. Bacon Pratik Aryal Timothy E. Graham 《The Journal of biological chemistry》2013,288(2):1250-1265
Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues. 相似文献
7.
J L McDonel 《Biochemistry》1980,19(21):4801-4807
125I-Labeled enterotoxin from Clostridium perfringens was utilized to characterize the association of the enterotoxin with cells isolated from rabbit intestine and tissue homogenates from liver, kidney, and brain. The enterotoxin was found to bind in a specific and saturable manner to cells from intestine and to tissue homogenates from liver and kidney but not the brain. Detailed studies of the binding were carried out with the ileal epithelial intestinal cells. The rate and amount of binding of enterotoxin to cells appeared to be temperature dependent. Apparent affinity and association and dissociation rate constants were calculated for what appeared to be two classes of saturable binding sites. The amount of enterotoxin molecules that bound per milligram of cell protein was similar in tissue of intestinal, liver, and kidney origin (approximately 10(13) molecules/mg of cell protein). Spontaneous dissociation into the supernatant medium was observed to be much slower than expected from calculations based on the rate of association. Chaotropic ions did not enhance dissociation of the enterotoxin from cells. Enterotoxin binding was demonstrated to be heat labile (binding ability was lost after the enterotoxin was heated for 10 min at 60 degrees C). A mechanism is described whereby the enterotoxin binds and then is inserted into the membrane where it becomes trapped. 相似文献
8.
Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+ /calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca2+ , the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved. 相似文献
9.
—The major toxin of black widow spider venom, α-latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non-specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre-incubation with 2%β-mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre-treatment with 10m M-EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non-cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M?1 s?1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10?3 and 5.2 × 10?5s?1 corresponding to half-lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α-latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma. 相似文献
10.
C A Colville M K Bansal J H Phillips S van Heyningen 《Biochimica et biophysica acta》1992,1137(3):264-273
Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin. 相似文献
11.
A potential therapy for antibiotic-associated pseudomembranous colitis is to bind Clostridium difficile toxins A and B using cholestyramine, a hydrophobic anion exchange medium. Frontal analysis in isotonic phosphate buffer was studied using post-column derivatization with o-phthalaldehyde, which gave a highly sensitive (> or =30 ng) flow-through analysis. Following load (1.5-3.0 microg toxin/3.6 mg), toxin A was bound at a slightly higher capacity than B, due to slower kinetics. A salt gradient eluted roughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, hence toxin A showed weaker electrostatic affinity. The remainder of toxin A (65%) and some of toxin B (10% out of 50%) were eluted using a subsequent gradient to 60% acetonitrile in normal saline, which measured predominantly hydrophobic binding. Low and high affinity populations of both toxins were observed. Glycocholic acid or amino acids were competitive binders, although these components had little effect on the toxin A population bound primarily through ionic interactions. Competitive protein constituents in hamster cecal contents were also profiled. These results help to explain the variable clinical response in using cholestyramine to treat colitis. Using quaternary amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a trend for increased binding at higher pH was observed, especially for toxin A. Binding to strong cation exchange resins (sulfonate-PHM) was not observed. A range of reversed phase media retained both toxins, although recovery was very poor relative to protein standards. Size exclusion chromatography with light scattering detection showed that toxin B exists in different aggregation states, while toxin A remains monomeric. 相似文献
12.
When free hydroxocobalamin (vitamin B12) is added in vitro to a suspension of intact rat liver mitochondria in the presence of a source of both reducing equivalents and ATP, adenosylcobalamin synthesis is observed. This synthetic process is not dependent on electron transport or oxidative phosphorylation and is not detected when cyanocobalamin is substituted for hydroxocobalamin. Adenosylcobalamin synthesis is linear with time for at least 10 min and with hydroxocobalamin concentration up to 37 nm. At the latter concentration of hydroxocobalamin, the rate of synthesis at 37 °C is 0.26 pmol/min/mg of protein. Only part (<30%) of the newly synthesized adenosylcobalamin is bound to the mitochondrial cobalamin binding protein, whereas most (90%) of the concurrently accumulated hydroxocobalamin is bound. On the other hand, when adenosylcobalamin is added to a suspension of intact mitochondria, it is accumulated at a rate similar to that for hydroxocobalamin, and is bound to the mitochondrial binding protein to a similar extent. These findings indicate that rat liver mitochondria contain all of the enzymatic components necessary to convert hydroxocobalamin to adenosylcobalamin, the coenzyme for the mitochondrial enzyme methylmalonyl CoA mutase. 相似文献
13.
The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes. 相似文献
14.
Yee-Hsiung Chen Ruey-Fen Liou ChienTsung Hu Chung-Ching Juan Jen Tsi Yang 《Molecular and cellular biochemistry》1987,73(1):69-76
The action of 7.2 µM cardiotoxin on 0.25% human erythrocytes in a plasma extender solution was studied by the interaction of toxin with intact red blood cells and subsequent hemolysis of the cells. The binding of toxin to cells was completed within 10 min, whereas the membrane rigidity was weakened in a non-lytic period for about 25 min. The toxin molecules bound almost exclusively to the membrane. The bound toxin could not be liberated with either 0.5% Triton X-100 or 0.1 N NaOH. The degree of binding was slightly reduced in the presence of 10 mM mono- and divalent inorganic salts. The action of toxin might weaken the in situ association of several proteins that are linked with band 3 protein of the membrane, thus making the cells fragile and altering the shape of the cell to a smooth sphere. 相似文献
15.
C. Steven McDaniel Taghi Manshouri M. Zouhair Atassi 《Journal of Protein Chemistry》1987,6(5):455-461
A peptide corresponding to residues 26–41 of α-bungarotoxin, and closed by a disulfide bond between two cysteine residues
at the amino and C terminal ends of the peptide, was synthesized and the monomeric form was purified. The peptide, which represents
the exposed part of the long central loop of the toxin molecule, was examined for binding to acetylcholine receptor. The peptide
was shown by radiometric titrations to bind radiolabeled receptor, and radiolabeled peptide was bound by receptor. The specificity
of the binding was confirmed by inhibition with the parent toxin. A synthetic analog of the peptide in which Trp-28 was replaced
by glycine had very little (10%) of the original activity. Succinylation of the amino groups of the peptide resulted in virtually
complete (98%) loss of the binding activity. These results indicate that a shortened loop peptide corresponding to the region
26–41 of α-bungarotoxin exhibits binding activities mimicking those of the parent molecule. In this region, Trp-28, and one
or both of Lys-26 and Lys-38, are essential contact residues in the binding to receptor. 相似文献
16.
Seungwoo Kang Mingxuan Xu Edward C. Cooper Naoto Hoshi 《The Journal of biological chemistry》2014,289(16):11536-11544
M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex. 相似文献
17.
The binding, mobility, and mode of cell entry of the plant toxin ricin (or RCAII) were investigated on susceptible and partially resistant murine cell lines. When susceptible cells (SV40-transformed 3T3 fibroblast cells and BW5147 lymphoma cells) were examined, ricin bound rapidly, induced endocytosis, and entered the cell cytoplasm via broken endocytotic vesicles to inhibit cell protein synthesis, as found previously (1). Addition of lactose within 15 min after initial ricin binding prevented toxicity. After this time lactose addition no longer blocked the inhibition of protein synthesis. In a partially resistant lymphoma (BW5147/RCA3) that shows only a slight reduction in the total number of ricin-binding sites, ricin bound rapidly to the cell surface, but was endocytosed significantly less at low ricin doses compared to its parental line, indicating a possible difference in cell surface behavior. The exposed surface proteins on the BW5147 parental and BW5147/RCA3 resistant lines were examined by 125I-labeling utilizing lactoperoxidase-catalyzed iodination. The radiolabeled components were solubilized and separated by slab gel electrophoresis in sodium dodecyl sulfate. Autoradiograms of the slab gels indicated that two surface components of approximately 80,000 and 35,000 mol wt were much less exposed or were missing on the resistant line. 相似文献
18.
J. Lindemans E.J.M. de Jongh F.C.M. Brand M. Schoester J. Van Kapel J. Abels 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(2):203-210
The uptake of R-type cobalamin-binding protein from human granulocytes and plasma by isolated parenchymal rat liver cells has been studied. When [57Co]cyanocobalamin-saturated granulocyte-binding protein or transcobalamin III was incubated with the liver cells in a concentration of 500 pM, more than 80% of the vitamin was taken up in 1 h. Vitamin B-12 bound to plasma transcobalamin I, however, was not taken up unless the protein was desialylated by neuraminidase from Vibrio cholerae. The uptake of iodinated pure granulocyte-binding protein, saturated with cobalamin, reached 100% and was accompanied by increasing intracellular proteolytic degradation of the binding protein. EGTA and asialo-orosomucoid completely inhibited this process of uptake and degradation, whereas partial inhibition was caused by chloroquine and colchicine. These observations provide evidence that these (asialo)-R-type cobalamin-binding proteins are taken up by the cell through the plasma membrane receptor for asialoglycoproteins by means of endocytosis followed by proteolysis of the binding protein in the lysosomes. 相似文献
19.
Gélinas M Juneau P Gagné F 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2012,161(3):261-267
Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout. 相似文献
20.
Mikio Kato Robert Blanton Guang-Rong Wang Timothy J. Judson Yuich Abe Masafumi Myoishi Richard H. Karas Michael E. Mendelsohn 《The Journal of biological chemistry》2012,287(49):41342-41351
Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway. 相似文献