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1.
Javier Ortego Julio Escribano John Crabb Miguel Coca-Prados 《Journal of neurochemistry》1996,66(2):787-796
Abstract: The ocular ciliary epithelium, the site of aqueous humor secretion in the mammalian eye, is believed to play a key function in signaling mechanisms that regulate the rate of secretion, and thus intraocular pressure. One possible way of mediating these signaling functions is through neuropeptides and hormones secreted into the aqueous humor and acting on target tissues. We recently identified a cDNA clone sharing 100% identity with carboxypeptidase E (CPE), a neuropeptide-processing enzyme. Utilizing polymerase chain reaction, we further identified and characterized another processing enzyme, the peptidylglycine α-amidating monooxygenase (PAM), and the neuropeptide secretogranin II, a molecular marker restricted to neuroendocrine tissues. Using specific probes, we found that the nonpigmented ciliary epithelial cells express CPE, PAM, and secretogranin II mRNA, and protein. We also found that CPE and secretogranin II are abundant in aqueous humor. Treatment of cultured ciliary epithelial cells with veratridine and phorbol ester up-regulates CPE and PAM. Secretogranin II was found to be induced by veratridine, whereas phorbol ester had little effect, suggesting different mechanisms for secretion. The results demonstrate that secretogranin II, CPE, and PAM represent a specialized group of neuropeptide and neuropeptide-processing enzymes secreted by the ciliary epithelial cells which may confer to them neuroendocrine functions in cell-cell communication or cell signaling. 相似文献
2.
Frederick R. Derubertis Patricia A. Craven 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(4):499-511
Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation. 相似文献
3.
M. M. Abou-Donia S. P. Wilson T. P. Zimmerman† C. A. Nichol O. H. Viveros 《Journal of neurochemistry》1986,46(4):1190-1199
Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation. 相似文献
4.
5.
Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase. 相似文献
6.
NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH. 相似文献
7.
Chick embryos were injected on the fifth day of incubation with 75 ng cis-diamminedichloroplatinum II (cisplatin) and killed at daily intervals. Bilateral microphthalmia appeared in 88% of the surviving embryos; the decrease in eye size was noticeable 2 or 3 days after injection. Coinciding with this, macroscopic, histological, and ultrastructural changes started to appear in the ciliary body: ciliary processes failed to form and the cells in the inner layer of the ciliary epithelium underwent degenerative changes. Changes in the retina appeared somewhat later. Despite the decreased growth rate of the whole eye the neural layer of the retina continued to grow rapidly; as a result, it formed numerous folds and acquired a glandular appearance. In the most severe cases the rapidly growing retina would invade the ciliary region and replace completely the degenerated inner layer of the ciliary epithelium. It has been shown by previous authors that intraocular pressure is a determinant of eye expansion and also that the secretion of water and ions by the ciliary epithelium is important for the maintenance of that intraocular pressure. On this basis, our results are interpreted as indicating that the primary lesion induced by cisplatin was in the ciliary epithelium and that microphthalmia was the consequence of decreased pressure. It is also concluded that the retinal changes were due to the fact that the retina continued to grow despite the lack of expansion of the eye as a whole. 相似文献
8.
BMP signaling is required for development of the ciliary body 总被引:4,自引:0,他引:4
The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye. 相似文献
9.
Kinetics of the inhibition by naphtholsulfonate compounds of AMP deaminase from chicken erythrocytes
The effect of a variety of naphthalene sulfonate compounds on the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction was analyzed kinetically. Of the naphthalene sulfonate derivatives tested, the compounds with hydroxyl, sulfonate and nitrogen groups such as amino, anilino or azo groups showed an inhibitory effect. The cooperative effect of AMP, analyzed in terms of Hill coefficient, was increased from about 2 to 4 and the maximal velocity was unchanged with the addition of these compounds, suggesting the ligands as an allosteric inhibitor of the enzyme. The inhibition of AMP deaminase by naphtholsulfonate compounds can be qualitatively and quantitatively accounted for by the Monod-Wyman-Changeux model. Theoretical curves yield a satisfactory fit of all experimental saturation and inhibition curves, assuming four binding sites for AMP and the inhibitor, and various KT(I) values. The structure-activity analysis of the interaction of the naphtholsulfonate compounds with AMP deaminase has demonstrated that the affinity of the enzyme for naphtholsulfonates as the inhibitors is correlated with electronic properties of the nitrogen atoms attached to naphthalene moiety: the delocalization of lone electron pair on nitrogen through naphtholsulfonate group makes the compound less basic, resulting in more tight binding of the ligand to the enzyme. Introduction of hydrophobic group to naphtholsulfonate moiety increases the binding affinity for the enzyme, and of the inhibition. These results suggest the location of hydrophobic regions as the allosteric inhibitory sites of the enzyme for the binding of naphtholsulfonate compounds. 相似文献
10.
A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes. 总被引:13,自引:11,他引:2
1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics. 相似文献
11.
An adenosine 3'5'-cyclic-monophosphate (Cyclic AMP)-dependent protein kinase has been identified and partially purified from the rat prostate tumor induced by 20-methylcholanthrene. This enzyme is stimulated 2- to 3-fold by the nucleotide. Equilibrium studies at pH 5.0 suggest the presence of a major class of binding site for cyclic AMP with an association constant of approximately 10(8) M-1. The concentration of binding site is about 1 pmol/mg of protein of the enzyme preparation. The enzyme is stimulated by other cyclic nucleotides as well, but only by higher concentrations. In comparing the ability of different histone subfractions, casein and protamine, to serve as substrate for this particular protein kinase, maximal cyclic-AMP-dependent enzyme activity was observed with histones. The results suggest that factors contributing to the malignant growth of the prostatic tissue do not directly involve changes in the characteristics of a cyclic-AMP-dependent protein kinase. 相似文献
12.
At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding. 相似文献
13.
The rate of glucose and fructose 6-phosphate phosphorylation in islet homogenates is reduced by prior fasting of the donor rats. In fed rats, the velocity of glucose phosphorylation at increasing glucose concentrations (0.1 to 100 mM) is compatible with the presence of two enzyme activities. A preferential effect of fasting upon the high Km enzyme activity can be documented either at low ATP concentration which enhances the fractional contribution of the high Km enzyme activity, or in the presence of glucose 6-phosphate, which suppresses the low Km enzyme activity. Islet phosphofructokinase activity was characterized by inhibition by citrate or high ATP concentrations, and relief from ATP inhibition by AMP. Fasting reduces the activity of phosphofructokinase without altering its sensitivity to ATP and AMP. Cyclic AMP fails to overcome the effect of fasting upon phosphofructokinase. The activity of phosphoglucoisomerase is unaffected by fasting. The fasting-induced adaptation of key glycolytic enzymes could account, in part at least, for reduced metabolism of glucose in islets from fasted rats. 相似文献
14.
A direct, stimulating effect of cyclic GMP on purified phosphoribosyl pyrophosphate synthetase and its antagonism by cyclic AMP 总被引:1,自引:0,他引:1
The activity of phosphoribosyl pyrophosphate synthetase, purified from a line of rat hepatoma cells in continuous culture, is maximally stimulated (2–4 fold) by less than 10?7M cyclic GMP. Half maximal stimulation occurs at 2 × 10?9M. Cyclic GMP stimulates phosphoribosyl pyrophosphate synthetase by decreasing the Km of the enzyme for ATP from 50 μM to 10 μM without affecting the Vmax; it has no effect on the Km for ribose 5-phosphate, the other substrate. Cyclic AMP alone has no effect on the enzyme activity, but at micromolar concentrations it antagonizes the stimulation by cyclic GMP. GMP, GDP, and GTP do not stimulate enzyme activity; and AMP and ADP at micromolar concentrations do not antagonize the effect of cyclic GMP.There is no detectable cyclic nucleotide-activated protein kinase in the enzyme preparation. Cyclic GMP significantly stabilizes the enzyme to heat inactivation. We conclude that cyclic GMP binds directly to the enzyme in an allosteric fashion, causing it to have an increased affinity for one of its substrates, and that cyclic AMP directly antagonizes this effect. 相似文献
15.
Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was partially purified from cotton seeds. The enzyme shows normal kinetics toward phosphoenol-pyruvate, ADP, and magnesium or manganese. Of nearly 50 metabolites tested, the enzyme is inhibited only by ATP, UTP, citrate, and malate, and activated by AMP, GMP, and fumarate. The inhibition by citrate and ATP is not due to metal chelation; both compounds appear to directly affect the enzyme. The kinetics of the activations by AMP and by fumarate suggest the existence of separate activator sites for the two compounds.It is suggested that cotton seed pyruvate kinase is a regulatory enzyme, although it differs markedly from the regulatory pyruvate kinases which have been described in animals and in microorganisms. This is the first instance in which regulatory properties have been reported for a pyruvate kinase from a higher plant. 相似文献
16.
Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell. 相似文献
17.
Adenosine 3':5' -monophosphate (cyclic AMP) -dependent protein kinase from bovine heart muscle catalyzes the phosphorylation of its regulatory, cyclic AMP-binding subunit. Phosphorylation enhances net dissociation of the enzyme by cyclic AMP. Chromatography on omega-aminohexyl-agarose was used to study the effects of phosphorylation on cyclic AMP binding and subunit dissociation and reassociation. This method permitted rapid separation of the catalytic subunit from the cyclic AMP -binding protein and holoenzyme. Phospho- and dephosphoprotein kinases were found to dissociate to the same extent at any given concentration of cyclic AMP and completely at saturation. At equilibrium, the amount of cyclic AMP bound was the same for both forms of enzyme and was directly proportional to the degree of dissociation of the holoenzyme. In the absence of cyclic AMP, phospho- and dephospho-cyclic AMP-binding proteins reassociated completely with the catalytic subunit. However, the rate of reassociation of the dephospho-cyclic AMP-binding protein was at least 5 times greater than the phospho-cyclic AMP-binding protein. Retardation of reassociation was directly proportional to the extent of phosphorylation. We conclude that the degree to which the cyclic AMP-binding protein is phosphorylated markedly affects its intrinsic ability to combine with the catalytic subunit to regenerate the inactive cyclic nucleotide-dependent kinase and that the state of phosphorylation of this subunit may be important in detemining the proportion of dissociated (active) and reassociated (inactive) protein kinase at any given time. 相似文献
18.
Christakis Panayiotou Nicola Solaroli Magnus Johansson Anna Karlsson 《The international journal of biochemistry & cell biology》2010,42(1):62-69
Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import. 相似文献
19.
E S Severin M V Nesterova L P Sashchenko V V Rasumova 《Biochimica et biophysica acta》1975,384(2):413-422
2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding. 相似文献
20.
The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data. 相似文献