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1.
cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.  相似文献   

2.
Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.  相似文献   

3.
Direct activation of adrenal Sterol Ester Hydrolase (EC 1.1.1.13) by dibutyryl cAMP, ATP and Mg+2 has been demonstrated in adrenal homogenates from three species. Variability in the degree of activation was minimized by preincubation of the tissue homogenate for two hours prior to addition of the cofactors and subsequent enzyme assay. Although baseline sterol ester hydrolytic activity, independent of cofactors, was present in all subcellular fractions, the cAMP-dependent enzyme was primarily associated with the 105,000 × g soluble fraction of the cell. A requirement for protein kinase in the system was demonstrated with a partially-purified enzyme from bovine adrenal.  相似文献   

4.
Liver post-mitochondrial supernatant from diabetic rats showed a decrease in the [3H] cAMP binding activity which was associated with a decrease in the number of cAMP binding sites. On the other hand, the cAMP binding activity of nuclear fractions from diabetic rat liver was not significantly different than that of control. The cAMP binding activity of post-mitochondrial supernatant was further analyzed by using 8-azido-[32P] cAMP, a photoaffinity probe for cAMP binding sites. The diabetic supernatants showed a selective reduction in the photolabeling of a protein band representing the regulatory subunit of type I cAMP-dependent protein kinase without any appreciable change in the photolabeling of regulatory subunit of type II cAMP-dependent protein kinase.  相似文献   

5.
Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.  相似文献   

6.
The distribution of the cAMP-dependent protein kinase activity in bull ejaculated sperm has been investigated. This activity proved to be mainly present in a soluble form inside the cell. Sperm fractionation into heads and flagellar fragments, using differential centrifugation or centrifugal elutriation, has shown that the particulate cAMP-dependent protein kinase activity was mainly associated with the flagellar structures. A much activity was shown to be associated with the head fraction. Some activity could also be detected in the purified plasma membrane fraction.  相似文献   

7.
In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.  相似文献   

8.
Protein kinase activity was detected and assayed directly on polyacrylamide gels after disc electrophoresis of the 100,000 X g supernatant fraction of brown adipose tissue of infant rats. Nine major bands of activity were detected, eight of which could be stimulated by cAMP or inhibited by the cAMP-dependent protein kinase inhibitor protein. This electrophoretic technique revealed heterogeneity in the cAMP-dependent protein kinase activity eluted from DEAE-cellulose by high concentrations of salt, but not in the peak of activity eluted by low concentrations of salt. The catalytic properties and substrate specificities of the kinases in the various bands were studied while the enzymes were still in the gels. The activity in each band differed from each of the others in at least one of these properties. The activities of the protein kinases in brown fat changed as the animals grew, and each band exhibited a distinct and unique developmental pattern. The major changes in kinase activities occurred in the immediate post-parturition period, then at 15 days after birth and at weaning. These developmental stages coincide with the periods during which the tissue undergoes changes in the rate of its proliferation, differentiation, and functional activity.  相似文献   

9.
The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.  相似文献   

10.
Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.  相似文献   

11.
Protein phosphokinase activity from the cytosol (105,000 X g soluble fraction) of testes from sexually mature rats has been resolved be DEAE-cellulose chromatography in three forms of protein kinase, cAMP-dependent protein kinases I and II and cAMP-independent protein kinase III. Adenosine 3':5'-monophosphate-binding activity (cAMP-binding activity) was associated with protein kinases I and II but not with protein kinase III. Protein kinases I, II, and III exhibited different pH optima, cyclic nucleotide dependency, and relative substrate specificity. Protein kinases I and II were inhibited by a heat-stable protein inhibitor from rat skeletal muscle, whereas protein kinase III was not inhibited. According to previously established criteria (Traugh, J. A., Ashby, C.D., and Walsh D. A. (1974) Methods Enzymol. 38, 290-299) protein kinases I and II can be classified as cAMP-dependent holoenzymes consisting of regulatory and catalytic subunits. Protein kinase III is a cAMP-independent protein kinase.  相似文献   

12.
A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type I alpha regulatory subunit (RI alpha). The full-length RI alpha from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RI alpha, the [3H]cAMP exchange rate was comparable to that of the B domain of full-length RI alpha containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.  相似文献   

13.
Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.  相似文献   

14.
Abstract: Increased intracellular adenosine 3':5'-monophosphate (cAMP) levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM-6 N.O 2-dibutyryl 3':5'-monophosphate (Bt2cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ-32P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.  相似文献   

15.
Differentiation of human peripheral blood monocytes into macrophages was accompanied by induction of the regulatory subunit of cAMP-dependent protein kinase I as determined by photoaffinity labeling of cytosol proteins with 8-N3-[32P]cAMP and DEAE-Sephacel chromatography. The appearance of cAMP-dependent protein kinase I in macrophages was not due to translocation from the particulate fraction of monocytes. The regulatory subunit of cAMP-dependent protein kinase II was present in both monocytes and in vitro-differentiated macrophages. Protein kinase I in macrophages demonstrated higher affinity for 8-N3-cAMP (KD = 0.7 nM) than did protein kinase II from either monocytes (KD = 14.5 nM) or macrophages (KD = 4.9 nM). These studies demonstrate induction of the regulatory subunit of cAMP-dependent protein kinase I during the differentiation of a normal human cell and support the hypothesis that cAMP may regulate some stages of differentiation.  相似文献   

16.
The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.  相似文献   

17.
In contrast to other tissues (e.g. brain, heart), no cAMP dependent protein kinase activity and little cAMP-binding activity could be detected in crude homogenates of purified human PMN leucocytes. This was due to the presence of an inhibitor of cAMP binding and protein kinase activity in PMN leucocytes. Since the inhibitor was entirely segregated in PMN lysosomes (rich in β-glucuronidase and acid phosphatase), lysosomefree supernatants yielded cAMP-dependent protein kinase (> 5-fold stimulation with 5 μM cAMP) and considerable cAMP binding activity. The inhibitor was not dialyzable, and unlike the usual protein kinase modulators, was heat-labile. Preparations of beef-heart protein kinase, treated with the PMN inhibitor, lost cAMP-binding and protein kinase activities simultaneously. The presence of this lysosomal inhibitor may invalidate studies of cAMP binding and protein kinase activities in crude homogenates prepared from lysosome-rich tissues.  相似文献   

18.
Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.  相似文献   

19.
In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP-dependent protein kinase (A kinase), Ca2+/calmodulin-dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid-dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise.  相似文献   

20.
J Bubis  S S Taylor 《Biochemistry》1987,26(19):5997-6004
Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.  相似文献   

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