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1.
The insulin-like effects of selenate in rat adipocytes 总被引:21,自引:0,他引:21
O Ezaki 《The Journal of biological chemistry》1990,265(2):1124-1128
Selenate was found to have several insulin-like effects in rat adipocytes: stimulation of glucose transport activity by translocation of two types of glucose transporters from intracellular sites to the plasma membrane, stimulation of cAMP phosphodiesterase activity, and stimulation of ribosomal S6 protein phosphorylation. Furthermore, in intact cells addition of 1 mM selenate stimulated tyrosyl phosphorylation of 210-, 170-, 120-, 95-, 70-, and 60-kDa proteins but failed to stimulate insulin receptor kinase activity, suggesting that selenate stimulated other tyrosine kinase. In the presence of insulin, selenate enhances insulin receptor kinase activity and phosphorylations of insulin-stimulated tyrosyl phosphoproteins. These results may provide clues for the elucidation of the role of selenium in animals and the mechanism of insulin action. 相似文献
2.
Exposure of adipocytes of rats to CdCl2 caused acceleration of [3-3H]glucose incorporation into lipid maximally at 500 microM in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 0.2% albumin. T.l.c. of the lipids extracted from adipocytes showed that Cd2+ increased labelling of di- and tri-[14C]acylglycerols predominantly. With increasing concentrations of glucose the apparent Km value was not affected by Cd2+, but the V value was increased, similarly to the effect of insulin. In the presence of insulin, Cd2+ (5 microM) exerted a consistent additive effect with a stimulatory effect of insulin on lipogenesis at all concentrations of insulin tested (5-50 mu units/ml). The stimulation was observed at a high concentration of glucose, suggesting that Cd2+ accelerated intracellular metabolism of glucose, mimicking insulin. However, although Zn2+ and Mn2+ stimulated the transport at a rate similar to that observed with insulin (200 mu units/ml), Cd2+ had no stimulating effect on the membrane transport of 3-O-methylglucose. The biological potency of Cd2+ and the insulin-like effects of Zn2+, both of which metals belong to the same group in the Periodic Table, are similar towards glucose metabolism, but quite different towards glucose transport. 相似文献
3.
E M Wilson 《The Journal of biological chemistry》1985,260(15):8683-8689
The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor. 相似文献
4.
Raman spectroscopic study of the effects of Ca2+, Mg2+, Zn2+, and Cd2+ ions on calf thymus DNA: binding sites and conformational changes 总被引:4,自引:0,他引:4
The interaction of Mg2+, Ca2+, Zn2+, and Cd2+ with calf thymus DNA has been investigated by Raman spectroscopy. These spectra reveal that all of these ions, and particularly Zn2+, bind to phosphate groups of DNA, causing a slight structural change in the polynucleotide at very small metal: DNA (P) concentration ratio (ca. 1:30). This results in increased base-stacking interactions, with negligible change of the B conformation of DNA. Contrary to Zn2+ and Cd2+, which interact extensively with the nucleic bases (particularly at the N7 position of guanine), the alkaline-earth metal ions are bound almost exclusively to the phosphate groups. The affinity of both the Zn2+ and Cd2+ ions for G.C base pairs is comparable, but the Cd2+ ions interact more extensively with A.T pairs than Zn2+ ions. Interstrand cross-linking through the N3 atom of cytosine is suggested in the presence of Zn2+, but not Cd2+. 相似文献
5.
The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin. 相似文献
6.
Gregory S. Makowski Shan -Meei Lin Sean M. Brennan Henry M. Smilowitz Sidney M. Hopfer F. William Sunderman 《Biological trace element research》1991,29(2):93-109
Two Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenile Xenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with 65Zn2+, 63Ni2+, or 109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 micrograms/well by 109Cd(2+)-probing, 0.13 micrograms/well by 65Zn(2+)-probing, and 0.26 mu/well by 63Ni(2+)-probing. Protein p43 was more clearly visualized by probing with 63Ni2+ than with 65Zn2+ or 109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide, 65Zn2+, 109Cd2+, and 63Ni2+ distinctly labeled the 22 kDa middle fragment; 65Zn2+ and 109Cd2+ also labeled the 11 kDa N-terminal fragment, but did not label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with 65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+ = Cu2+ greater than or equal to Hg2+ greater than Cd2+ greater than Co2+ greater than or equal to Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with 65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43. 相似文献
7.
Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase 总被引:1,自引:0,他引:1
J C Lai A Baker K C Carlson J P Blass 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,80(2):291-294
The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested. 相似文献
8.
Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated. 相似文献
9.
A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
10.
Resistance to cadmium, cobalt, zinc, and nickel in microbes. 总被引:23,自引:0,他引:23
The divalent cations of cobalt, zinc, and nickel are essential nutrients for bacteria, required as trace elements at nanomolar concentrations. However, at micro- or millimolar concentrations, Co2+, Zn2+, and Ni2+ (and "bad ions" without nutritional roles such as Cd2+) are toxic. These cations are transported into the cell by constitutively expressed divalent cation uptake systems of broad specificity, i.e., basically Mg2+ transport systems. Therefore, in case of a heavy metal stress, uptake of the toxic ions cannot be reduced by a simple down-regulation of the transport activity. As a response to the resulting metal toxicity, metal resistance determinants evolved which are mostly plasmid-encoded in bacteria. In contrast to that of the cation Hg2+, chemical reduction of Co2+, Zn2+, Ni2+, and Cd2+ by the cell is not possible or sensible. Therefore, other than mutations limiting the ion range of the uptake system, only two basic mechanisms of resistance to these ions are possible (and were developed by evolution): intracellular complexation of the toxic metal ion is mainly used in eucaryotes; the cadmium-binding components are phytochelatins in plant and yeast cells and metallothioneins in animals, plants, and yeasts. In contrast, reduced accumulation based on an active efflux of the cation is the primary mechanism developed in procaryotes and perhaps in Saccharomyces cerevisiae. All bacterial cation efflux systems characterized to date are plasmid-encoded and inducible but differ in energy-coupling and in the number and types of proteins involved in metal transport and in regulation. In the gram-positive multiple-metal-resistant bacterium Staphylococcus aureus, Cd2+ (and probably Zn2+) efflux is catalyzed by the membrane-bound CadA protein, a P-type ATPase. However, a second protein (CadC) is required for full resistance and a third one (CadR) is hypothesized for regulation of the resistance determinant. The czc determinant from the gram-negative multiple-metal-resistant bacterium Alcaligenes eutrophus encodes proteins required for Co2+, Zn2+, and Cd2+ efflux (CzcA, CzcB, and CzcC) and regulation of the czc determinant (CzcD). In the current working model CzcA works as a cation-proton antiporter, CzcB as a cation-binding subunit, and CzcC as a modifier protein required to change the substrate specificity of the system from Zn2+ only to Co2+, Zn2+, and Cd2+. 相似文献
11.
We have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+ and Ni2+ ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3 M urea-induced transformation to the DNA-binding form, and (2) limited trypsin digestion to separate the steroid- and DNA-binding domains. Imidazole (2-200 mM) affinity elution and pH-dependent (pH 7-3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+ and Ni2+ ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+ greater than Cu2+ much greater than Zn2+) at neutral pH. Generally, similar results were obtained with IDA-Cu2+ and IDA-Ni2+ in the absence of urea. Receptors were tightly bound and not eluted before 100 mM imidazole or pH 3.6. Different results were obtained using IDA-Zn2+; at least four receptor isoforms were resolved on IDA-Zn2+. Receptor-metal interaction heterogeneity and affinity for IDA-Zn2+ and IDA-Cu2+, but not IDA-Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA-Zn2+ chromatography were not separable using high-performance size-exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA-affinity chromatography. The affinity of trypsin-generated (mero)receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High-affinity metal-binding sites were mapped to the DNA-binding domain, but at least one of the metal-binding sites is located on the steroid-binding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor-metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal-binding proteins suggests new and potentially powerful receptor immobilization and purification regimes previously unexplored by those in this field. 相似文献
12.
Biosorption of Cu2+, Cd2+, Pb2+, and Zn2+ using dried marine green macroalga Caulerpa lentillifera 总被引:2,自引:0,他引:2
Pavasant P Apiratikul R Sungkhum V Suthiparinyanont P Wattanachira S Marhaba TF 《Bioresource technology》2006,97(18):2321-2329
The sorption of Cu2+, Cd2+, Pb2+, and Zn2+ by a dried green macroalga Caulerpa lentillifera was investigated. The removal efficiency increased with pH. The analysis with FT-IR indicated that possible functional groups involved in metal sorption by this alga were O-H bending, N-H bending, N-H stretching, C-N stretching, C-O, SO stretching, and S-O stretching. The sorption of all metal ions rapidly reached equilibrium within 20min. The sorption kinetics of these metals were governed by external mass transfer and intraparticle diffusion processes. The sorption isotherm followed the Langmuir isotherm where the maximum sorption capacities was Pb2+>Cu2+>Cd2+>Zn2+. 相似文献
13.
The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation. 相似文献
14.
Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation. 相似文献
15.
H Shibata F W Robinson C F Benzing T Kono 《Archives of biochemistry and biophysics》1991,285(1):97-104
Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high. 相似文献
16.
Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite. 相似文献
17.
The effect of Zn2+, Mn2+, Cd2+ and Hg2+ ions on the kinetics of growth was studied with Candida utilis. The inhibition of Candida utilis growth by Zn2+ and Mn2+ ions is described by the equation for noncompetitive inhibition of enzymatic reactions which is not the case with Cd2+ and Hg2+ ions. The inhibition constants (Ki) for these metals have been determined. 相似文献
18.
Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells 总被引:2,自引:0,他引:2
R Saperstein P P Vicario H V Strout E Brady E E Slater W J Greenlee D L Ondeyka A A Patchett D G Hangauer 《Biochemistry》1989,28(13):5694-5701
An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa beta-subunit of the insulin receptor (IC50 = 200 microM). The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 microM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa beta-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. Our data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases. 相似文献
19.
R Regazzi G D Li J Deshusses C B Wollheim 《The Journal of biological chemistry》1990,265(25):15003-15009
The hamster islet B cell line HIT retains the ability to secret insulin in response to glucose and several receptor agonists. We used HIT cells to study the initial signaling events in glucose or receptor agonist-stimulated insulin secretion. Glucose stimulated insulin release from HIT cells in a dose-dependent manner with a half-maximal effect seen already at 1 mM. Insulin release was also stimulated by carbachol in a glucose-dependent manner. Glucose depolarized the HIT cell membrane potential as assessed with the fluorescent probe bisoxonol and raised intracellular Ca2+ as revealed by fura-2 measurements. Using a Mn2+ fura-2 quenching technique, we could show that the rise in intracellular Ca2+ was due to Ca2+ influx following opening of voltage-gated Ca2+ channels. Glucose is thought to increase the diacylglycerol (DAG) content of insulin-secreting cells. However, although HIT cells respond to glucose in terms of insulin secretion, membrane depolarization, and Ca2+ rise, the hexose was unable to increase the proportion of protein kinase C activity associated with membranes. In contrast, the membrane-associated protein kinase C activity increased in HIT cells exposed to the two receptor agonists carbachol and bombesin. Bombesin was shown to generate DAG with the expected fatty acid composition of activators of phospholipase C. Glucose, in contrast, only caused minor increases in DAG containing myristic and palmitic acid without affecting total DAG mass. The failure to detect stimulation of protein kinase C by glucose could be due to both the limited amount and to the different fatty acid composition of the metabolically generated DAG. The latter was in part supported by experiments performed on protein kinase C partially purified from HIT cells. Indeed, 1,2-dipalmitoylglycerol, presumed to be the main DAG species generated by glucose, was only one-third as active as 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonylglycerol in stimulating the isolated enzyme at physiological Ca2+ concentration. It is therefore unlikely that DAG and protein kinase C play a major role in glucose-stimulated insulin secretion. 相似文献