Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Catalytic unit-independent phosphorylation and dephosphorylation of type II regulatory subunit of cyclic AMP-dependent protein kinase in rat liver plasma membranes.

Rat liver plasma membranes contain a 55 kDa protein which proved to be identical with type II regulatory subunit (RII) of the cyclic AMP-dependent protein kinase (kinase A) by several criteria (gel electrophoretic behaviour, peptide map, position of the autophosphorylated site). Analysis of phosphopeptide maps revealed that the membrane-bound RII was phosphorylated by a kinase which is unrelated to the catalytic unit (C) of kinase A. Dephosphorylation of the membrane-bound RII by an endogenous phosphatase was stimulated by both cyclic AMP and fluoride. Addition of C did not stimulate dephosphorylation even in the presence of ADP; moreover, protein inhibitor of C did not modify the effects of cyclic AMP or fluoride. The effects of both cyclic AMP and fluoride were, however, inhibited by C. Results indicate that rat liver plasma membranes contain a phosphorylation-dephosphorylation system for which RII is a relatively specific substrate.     

Adenosine 3',5'-monophosphate receptor proteins in HeLa cells

The adenosine 3',5'-monophosphate receptor proteins of HeLa cells have been characterized. Using the Millipore filter assay, in the presence of 5'AMP and a phosphodiesterase inhibitor, specific [3H]cyclic AMP binding was detected in cytosol and in a nuclear-free particulate fraction, but not in nuclei. Both preparations exhibited biphasic Scatchard plots. 8-Azido[32P]cyclic AMP was used as a photoaffinity probe to covalently link ligand with receptor proteins. Proteins were then separated on denaturing gels and analyzed by autoradiography. The cytosol exhibited four specific binding proteins, with molecular weights of 46 000, 50 000, 52 000 and approx. 120 000. The 50 000/52 000 doublet could not be interconverted by phosphorylation-dephosphorylation reactions. On DEAE-cellulose, the 50 000-dalton protein eluted with peak II cyclic AMP-dependent protein kinase. The other proteins eluted with Peak I and with a binding peak not associated with kinase activity. Only the 50 000 protein was precipitated by type II protein kinase antibody from bovine heart. In the particulate fraction, the 120 000 protein was not detectable, but 8-azido[32P]cyclic AMP treatment revealed the other three proteins, with a relative increase in the 50 000-dalton protein. The results suggest that HeLa cells have four binding proteins which can associate with catalytic subunit and that the Peak I enzyme is heterogeneous, consisting of several distinct regulatory subunits.     

Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart.

1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

Antiserum against the catalytic subunit of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases.

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.     

Inhibitory effect of a lethal toxic fragment of staphylococcal alpha-toxin on cyclic AMP-dependent protein kinase activity.

The effect of a lethal toxic fragment of staphylococcal alpha-toxin on the activity of adenosine 3',5'-monophosphate(cyclic AMP)-dependent protein kinase was examined. 1. The lethal toxic fragment produced a dose-dependent decrease in both the binding of cyclic AMP to the regulatory subunit and phosphorylation activity of cyclic AMP-dependent protein kinase obtained from rabbit skeletal muscles up to a plateau at a 50% inhibitory effect. The decrease in the activity of protein kinase observed with low doses of the lethal toxic fragment (0.1 microM) resulted from a competitive inhibition, probably by its interaction with the cyclic AMP-binding site in the regulatory subunit molecule. 2. The effects of a lethal toxic fragment and epinephrine on the cyclic AMP level and protein kinase activity were investigated in the perfused rabbit heart slices. The lethal toxic fragment attenuated the stimulation of cyclic AMP-dependent protein kinase activity ratio by epinephrine. 3. It is suggested that the specific action of a lethal toxic fragment on the cellular membrane enzymes may be attributable to the inhibition of the cyclic AMP-dependent protein kinase activity.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Cyclic AMP-dependent protein kinase from Ustilago maydis

Summary Ustilago maydis was surveyed for cyclic AMP-dependent protein kinase activity. Using a combination of ion-exchange and molecular filtration techniques, we demonstrate that there is only one form of cyclic AMP-dependent protein kinase in the cytosolic fraction of the fungus. The kinase activity is specifically activated by cyclic AMP and utilizes protamine and kemptide as substrates. Most, if not all, of the cyclic AMP binding detected in the soluble fraction is associated with the protein kinase activity. Cyclic AMP-dependent protein kinase is completely dissociated by cyclic AMP into catalytic and regulatory subunits having an apparent molecular weight of 35 000 daltons as judged by sucrose gradient centrifugation.Post graduate fellow from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).Career investigator from CONICET.     

Evidence against lung galaptin being important to the synthesis or organization of the elastic fibril.

1. The fluctuations in rat hepatocyte volume and protein content in response to dietary perturbations (starvation, protein restriction, refeeding) were accompanied by corresponding fluctuations in the amount of the regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase. Thus the intracellular concentration of this key enzyme was adjusted to be near constant. 2. The adjustment of cellular R was accomplished almost exclusively by regulating cytosolic RI (R subunit of type I kinase). The preferential down-regulation of cytosolic RI in response to starvation/protein restriction indicates that particulate RI and cytosolic as well as particulate RII are more resistant to breakdown during general catabolism in the hepatocyte. 3. The diet-induced fluctuations of kinase subunits were uniformly distributed in all populations of parenchymatous hepatocytes, regardless of their size and density. It is thus possible to isolate hepatocytes with uniformly altered RI/RII ratio from livers of rats with different feeding regimens. 4. The binding of endogenous cyclic AMP to RI and RII was similar in livers with high RI/RII ratio (fed rats) and low RI/RII ratio (fasted rats) as well as in hepatocytes isolated from fasted rats. Under the conditions of the experiment (short-term stimulation by glucagon), therefore, neither the dietary state nor the RI/RII ratio seemed to affect the apparent affinity of the isoreceptors for cyclic AMP. However, RI appeared to show a slightly higher co-operativity of intracellular cyclic AMP binding than did RII in all states.     

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

Altered regulation of cyclic AMP-dependent protein kinase in a mouse lymphoma cell line.

The ability of cyclic AMP to inhibit growth, cause cytolysis and induce synthesis of cyclic AMP-phosphodiesterase in S49.1 mouse lymphoma cells is deficient in cells selected on the basis of their resistance to killing by 2 mM dibutyryl cyclic AMP. The properties of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in the cyclic AMP-sensitive (S) and cyclic AMP-resistant (R) lymphoma cells were comparatively studied. The cyclic AMP-dependent protein kinase activity or R cells cytosol exhibits an apparent Ka for activation by cyclic AMP 100-fold greater than that of the enzyme from the parental S cells. The free regulatory and catalytic subunits from both S and R kinase are thermolabile, when associated in the holoenzyme the two subunits are more stable to heat inactivation in R kinase than in S kinase. The increased heat stability of R kinase is observed however only for the enzyme in which the catalytic and cyclic AMP-binding activities are expressed at high cyclic AMP concentrations (10(-5)--10(-4) M), the activities expressed at low cyclic AMP concentrations (10(-9)--10(-6) M) being thermolabile. The regulatory subunit of S kinase can be stabilized against heat inactivation by cyclic AMP binding both at 2-10(-7) and 10(-5) M cyclic AMP concentrations. In contrast, the regulatory subunit-cyclic AMP complex from R kinase is stable to heat inactivation only when formed in the presence of high cyclic AMP concentrations (10(-5)M). The findings indicate that the transition from a cyclic AMP-sensitive to a cyclic AMP-resistant lymphoma cell phenotype is related to a structural alteration in the regulatory subunit of the cyclic AMP-dependent protein kinase which has affected the protein's affinity for cyclic AMP and its interaction with the catalytic subunit.     

Selective regulation of the amount of catalytic subunit of cyclic AMP-dependent protein kinases during isoprenaline-induced growth of the rat parotid gland.

Stimulation of growth of the rat parotid gland by repeated injection of the beta-agonist isoprenaline led to a significant decrease in the activity of cyclic AMP-dependent protein kinases. Immunochemical quantification of the catalytic (C) and regulatory (RI and RII) subunits of the cyclic AMP-dependent protein kinases type I and type II revealed a loss of 65% of the immunochemically measurable amount of catalytic subunit C. The amount of the regulatory subunits, however, remained constant. The observed decrease in C-subunit was not due to a translocation of the molecule to cellular membranes or to an inhibiting effect of the heat-stable inhibitor of cyclic AMP-dependent protein kinases. A selective decrease in only the C-subunit was also observed after a brief exposure to isoprenaline leading to the stimulation of DNA synthesis. Under these conditions, the decrease was observed at the onset of DNA synthesis (17 h after injection), but not at the the time of an earlier small cyclic AMP peak (13 h after injection) or at the time of maximal DNA synthesis (24 h after injection). The results indicate that the amount of the catalytic subunit of cyclic AMP-dependent protein kinases can be regulated independently from that of the regulatory subunits. The time-limited occurrence of the specific change in the amount of the C-subunit suggests that such a regulation is of physiological significance and that it may participate in cyclic AMP-mediated events involved in the control of cellular proliferation.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Adenosine 3'':5''-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.     

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.     

Serum-activated T51B rat liver cells transiently accumulate cyclic AMP-dependent protein kinases on their surfaces during the G1 phase

Confluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP-binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP-binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP-binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP-dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP-dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP-dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.     

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.