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1.
The substrate specificity of cGMP-dependent protein kinase has been investigated by examining the ability of the enzyme to phosphorylate a series of synthetic peptides that correspond to the amino acid sequence at its site of autophosphorylation. The undecapeptide Ile53-Gly-Pro-Arg-Thr-Thr58-Arg-Ala-Gln-Gly-Ile63 which corresponds to the sequence around threonine-58 in cGMP-dependent protein kinase (Takio, K., Smith, S.B., Walsh, K.A., Krebs, E.G., and Titani, K. (1983) J. Biol. Chem. 258, 5531-5536) was synthesized and tested as a substrate for that enzyme. It was phosphorylated to the extent of 1.0 mol of phosphate/mol of peptide. Analysis of the products of Edman degradation of the phosphopeptide indicated that only threonine-58 was phosphorylated, as is the case for the autophosphorylation reaction in the native enzyme. The peptide was phosphorylated by cGMP-dependent protein kinase with a Km value of 578 +/- 25 microM and a Vmax of 0.069 +/- 0.003 mumol/min/mg of enzyme. This low Vmax value is consistent with the relatively slow rate of the autophosphorylation reaction. An analog peptide that contained serine in place of threonine-58 was also phosphorylated to 1.0 mol of phosphate/mol of peptide. That phosphopeptide contained only phosphoserine. The serine-containing analog peptide had a Km value similar to that of the parent peptide but was phosphorylated with a 70-fold higher Vmax value. Substitution of arginine-56 in the parent peptide by an alanine residue resulted in a peptide that was essentially not a substrate. Substitution of arginine-59, COOH-terminal to the phosphorylatable threonine, yielded a peptide with a Vmax similar to that of the parent peptide but a Km value of almost 22,000 microM. These results indicate that serine is a better phosphate-accepting residue than is threonine and that both arginine residues around the site of autophosphorylation are important specificity determinants for the cGMP-dependent protein kinase. 相似文献
2.
Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I 总被引:3,自引:0,他引:3
A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated. 相似文献
3.
Phosphorylation of rabbit cardiac-muscle troponin I by phosphorylase kinase. The effect of adrenaline. 总被引:1,自引:1,他引:0
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1. Incubation of rabbit cardiac-muscle troponin I with phosphorylase b kinase leads to the incorporation of .07-1.2 mol of Pi/mol. 2. The major site of phosphorylation is a serine residue at position 72. 3. Lesser amounts of phosphate are incorporated into threonine-138, threonine-162 and serine 20. 4. Serine-20 is the only site that contains a significant amount of phosphate before incubation with phosphorylase b kinase. 5. Unlike the situation with serine-20, the extent of phosphorylation of serine-72 and threonine-138 in the perfused rabbit heart does not change when the heart is exposed to adrenaline (4 microM). 相似文献
4.
Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides. 相似文献
5.
Annikka Linnala-Kakkunen Pekka H. Mäenpää 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,587(3):324-332
A method of steady-state electrophoresis in polyacrylamide gels was used to analyze the presence of cyclic nucleotide binding components in cell extracts. Multiple cyclic AMP and cyclic GMP binding components were detected in soluble cytoplasmic and nuclear extracts derived from avian liver, but only a single cyclic GMP binding protein was found in the 0.3 M NaCl extract of liver nucleoli. In the presence of cyclic GMP, this protein phosphorylated efficiently a calf thymus histone mixture and an endogenous nucleolar protein which migrated identically with histone H4 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the cyclic GMP-binding protein was 4.8. Addition of cyclic GMP did not influence the activity of the endogenous nucleolar RNA polymerase. 相似文献
6.
Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides. 相似文献
7.
Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues. 总被引:3,自引:0,他引:3
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M G Larrabee 《The Biochemical journal》1990,272(1):127-132
Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP. 相似文献
8.
T Yamamoto Y Furuki S Guild J W Kebabian 《Biochemical and biophysical research communications》1987,143(3):1076-1084
The effect of Mg-ATP and cyclic AMP on the secretion of alpha-melanocyto-stimulating hormone (alpha-MSH) from electrically permeabilized cells of rat intermediate lobe (IL) were investigated. Addition of exogenous Ca2+ stimulated alpha-MSH secretion in a concentration- (EC50 = 4.8 microM) and temperature-dependent manner. This Ca2+-evoked secretion was further enhanced by Mg-ATP and cyclic AMP. Mg-ATP was required for the fully secretory response in the electrically permeabilized IL cells and the maximal secretion was reached at 1 mM. Cyclic AMP in the presence of GTP gamma S also potentiated Ca2+-evoked alpha-MSH secretion to the same magnitude as Mg-ATP. In the absence of Ca2+ both the cyclic AMP and Mg-ATP did not stimulate alpha-MSH secretion from IL cells. The data suggest that Mg-ATP and cyclic AMP may modulate directly the secretory components rather than change intracellular concentration of free Ca2+. 相似文献
9.
Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase. 相似文献
10.
Some properties of cyclic AMP-dependent pig brain protein kinase were studied. The holoenzyme was shown to exist in solution in the form of a tetramer complex R2C2 with mol. weight of 180 000. The limited proteolysis of the regulatory subunit caused the formation of a fragment with mol. weight of 35 000, capable of independent binding of 3H-cyclic AMP and containing a site, which can be phosphorylated in the autophosphorylation reaction. Autophosphorylation of the holoenzyme led to an increase in the degree of dissociation of the former into individual subunits under the effect of cyclic AMP. The ability of the phosphoform of the catalytic subunit was demonstrated. The autophosphorylation process and the phosphotransferase reaction involve the same active site of the catalytic subunit. 相似文献
11.
Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site. The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP. Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 4 . 10(-8) M) belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2--5 . 10(-6) M) was demonstrated by the inhibitory effect of 10(-5) M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site. 相似文献
12.
Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.
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Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP. 相似文献
13.
Anna Faragó Péter Hasznos Ferenc Antoni Tibor Romhányi 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,538(3):493-504
Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site.The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP.Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 44 · 10?8 M belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2–5 · 10?6 M) was demonstrated by the inhibitory effect of 10?5 M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site. 相似文献
14.
A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart. 相似文献
15.
When the homogenate of rabbit superior cervical ganglia (SCG) was incubated in the presence of [gamma-32P]ATP and Mg2+, two specific proteins were strongly labeled. Their apparent molecular weights were 90,000 and 54,000, respectively. The phosphorylation of the latter was significantly stimulated by 10-50 nM cyclic GMP but to a lesser extent by cyclic AMP, whereas that of the former was not stimulated significantly by either of the cyclic nucleotides. The purified protein kinase inhibitor from rabbit skeletal muscle did not inhibit the phosphorylation. These results indicated that the observed phosphorylation of 54K protein was dependent on cyclic GMP but not on cyclic AMP. When intact SCG was incubated in the presence of 32Pi, phosphorylation of 90K protein was stimulated by cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP (10 microM), whereas phosphorylation of 54K protein was not significantly stimulated by any of these substances. The present demonstration of endogenous cyclic GMP-dependent protein kinase activity and its endogenous substrate proteins raises a possibility that the physiological actions of cyclic GMP in SCG are mediated by the phosphorylation of these proteins. 相似文献
16.
Buffalo sperm heads contain more than 50% of the total cyclic AMP-phosphodiesterase activity (EC 3.1.4.17) present in spermatozoa. Its distribution in sperm heads revealed no activity in acrosome and other membrane structures present in the head. All the cyclic AMP-phosphodiesterase activity was found firmly bound to sperm chromatin which could not be solubilized. In addition to cyclic AMP, cyclic GMP was also hydrolysed by chromatin preparation. The rate of hydrolysis was 2.5-times more rapid with cyclic AMP than with cyclic GMP at their optimum pH of 7.5 and 8.0, respectively. The pH and heat stability profiles, inhibition studies and the effect of divalent metal ions indicated that the two activities are not associated with the same protein. Mixed substrate analysis showed two sites at which the hydrolysis of cyclic AMP and cyclic GMP is catalysed. Chromatin cyclic nucleotide phosphodiesterases exhibited kinetics typical of one enzyme species both for cyclic AMP (K m = 100 microM; V = 1.0 nmol/min per mg protein) and cyclic GMP (Km = 23 microM; V = 0.4 nmol/min per mg protein). Each cyclic nucleotide was found to be a competitive inhibitor of the hydrolysis of the other with a Ki value of 30.18 microM for cyclic AMP hydrolysis and 256 microM for cyclic GMP hydrolysis. Hill coefficients of 1.0 obtained in the presence of cyclic AMP for cyclic GMP hydrolysis and vice-versa indicated no allosteric interactions. It is suggested that chromatin cyclic nucleotide phosphodiesterase may have a role post fertilization in cell growth and differentiation with no role in sperm motility which is regulated by similar enzymes present in sperm flagella. 相似文献
17.
The insulin- and glucagon-stimulated ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity. 总被引:6,自引:3,他引:3
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The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652]. 相似文献
18.
G. W. DeVries A. I. Cohen I. A. Hall J. A. Ferrendelli 《Journal of neurochemistry》1978,31(6):1345-1351
Abstract— The content of cyclic AMP and cyclic GMP was measured in whole eyes and in normal retinas from C57BL(6)J mice, in receptorless retinas from congenic mice homozygous for the receptor dystrophy gene (rd/rd), and in retinas from mice treated postnatally with monosodium glutamate. Normal retinas contain approx 320 μg of protein: dystrophic (rd/rd) retinas contain approx 110μg of protein, lack rods but possess some surviving cone somata and terminals: glutamate-modified retinas contain approx 200 μg of protein and have both a reduced area and thickness with a marked deficiency of ganglion cells and amacrine cells. In normal mice, more than 90% of the cyclic GMP, but only 607, of the cyclic AMP of the whole eye was in the retina. In normal dark-adapted retinas isolated under dim red light cyclic AMP and cyclic GMP content was 4.1 and 20.2pmol/retina, respectively. The content of both cyclic AMP and cyclic GMP was 40% less, 2.5 and 11.5pmol/retina, respectively, in light-adapted retinas. In dark-adapted retinas isolated under infra-red light, cyclic AMP content was 40%, higher than that in retinas isolated under dim red light; cyclic GMP content was the same under these two conditions. Receptorless retinas contained approx 50% as much cyclic AMP and only 1-2% as much cyclic GMP as normal retinas. Although glutamate-modified retinas also had approx 50% as much cyclic AMP, they contained 60-85%, as much cyclic GMP as normal retinas. Light decreased by 30-50% levels of both cyclic AMP and cyclic GMP in glutamate-modified retinas, but only reduced cyclic nucleotide levels in receptorless retinas by 20%.
These data indicate that 95% or more of the cyclic GMP is in photoreceptor cells, whereas cyclic AMP is more evenly distributed throughout the retina. In addition, both cyclic AMP and cyclic GMP levels are influenced by light- and dark-adaptation. 相似文献
These data indicate that 95% or more of the cyclic GMP is in photoreceptor cells, whereas cyclic AMP is more evenly distributed throughout the retina. In addition, both cyclic AMP and cyclic GMP levels are influenced by light- and dark-adaptation. 相似文献
19.
Moussavi Robabeh S. Kelley Christine A. Adelstein Robert S. 《Molecular and cellular biochemistry》1993,127(1):219-227
In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC
myosin light chain
- MHC
myosin heavy chain
- Tris
tris(hydroxymethyl)aminomethane
- EGTA
[ethylenebis(oxyethylenenitrilo)]tetraacetic acid
- EDTA
ethylenediaminetetraacetate
- TPCK
N-tosyl-L-phenylalanine chloromethyl ketone
- PMA
phorbol 12-myristate 13-acetate 相似文献
20.
Myosin light chain phosphorylation in contraction and relaxation of intact rat thoracic aorta 总被引:4,自引:0,他引:4
Myosin light chain phosphorylation in intact rat thoracic aorta was elevated during contraction induced by 0.3 microM norepinephrine, but was not maintained. Addition of 0.5 microM sodium nitroprusside to norepinephrine treated rat aorta strips led to elevation of cyclic GMP levels, relaxation of tension, and dephosphorylation of myosin light chain. Depletion of extracellular calcium or addition of calmodulin antagonists trifluoperazine and W7 diminished the contraction and phosphorylation of myosin light chain by norepinephrine, but did not prevent dephosphorylation by sodium nitroprusside or the elevated levels of cyclic GMP. Isoproterenol, 8-bromo cyclic GMP, and dibutyryl cyclic AMP all caused dephosphorylation of myosin light chain and induced relaxation during the period of development of tone. Eight other proteins had increased phosphorylation following norepinephrine treatment and one protein had less phosphorylation. The different proteins phosphorylated by norepinephrine showed varying degrees of sensitivity to Ca2+-free solution and to the calmodulin antagonists. The pattern of protein phosphorylation caused by sodium nitroprusside was best mimicked by 8-bromo cyclic GMP, rather than isoproterenol and dibutyryl cyclic AMP. These proteins were, generally, unaffected by Ca2+-free solution and the calmodulin antagonists. The present observations support the hypothesis that vasodilators inhibit tone development through myosin light chain dephosphorylation. Furthermore, the nitrovasodilators act through elevation of cyclic GMP and phosphorylation of proteins by cyclic GMP-dependent protein kinase. 相似文献