Alterations in activities of cyclic nucleotide systems and in beta-adrenergic receptor-mediated activation of cyclic AMP-dependent protein kinase during progression and regression of isoproterenol-induced cardiac hypertrophy.

Initial and transient increases in the basal levels of cyclic GMP in the heart were noted prior to cardiac hypertrophy in rats administered isoproterenol. Increased levels of cyclic AMP-phosphodiesterase (in both the soluble and particulate fractions) and stimulatory modulator of cyclic GMP-dependent protein kinase, however, were associated with the progression, or the state, of cardiomegaly, with their levels returning to the control values upon regression of the hypertrophy. The levels of cyclic GMP phosphodiesterase in the soluble fraction were lower, whereas those in the particulate fraction were higher, in the hypertrophied heart than the control. In cardiac hypertrophy, the maximal activity ratio(--cyclic AMP/+cyclic AMP) of cyclic AMP-dependent protein kinase in the incubated minced heart caused by isoproterenol was lower, whereas the concentration of isoproterenol required to increase the activity ratio half-maximally was higher than controls; the reduced responsiveness to the drug, however, was reversed when the hypertrophy regressed. These observations, taken collectively, appear to suggest that the desensitization of the beta-adrenergic mechanism seen in the cardiac hypertrophy produced by repeated administration of isoproterenol is associated with adaptive modifications in certain parameters of the cyclic nucleotide systems.     

Alterations in activities of cyclic nucleotide systems and in β-adrenergic receptor-mediated activation of cyclic amp-dependent protein kinase during progression and regression of isoproterenol-induced cardiac hypertrophy

Initial and transient increases in the basal levels of cyclic GMP in the heart were noted prior to cardiac hypertrophy in rats administered isoprotenol. Increased levels of cyclic AMP-phosphodiesterase (in both the soluble and particulate fractions) and stimulatory modulator of cyclic GMP-dependent protein kinase, however, were associated with the progression, or the state, of cardiomegaly, with their levels returning to the control values upon regression of the hypertrophy. The levels of cyclic GMP phosphodiesterase in the soluble fraction were lower, whereas those in the particulate fraction were higher, in the hypertrophied heart than the control. In cardiac hypertrophy, the maximal activity ratio (?cyclic AMP/+cyclic AMP) of cyclic AMP-dependent protein kinase in the incubated minced heart caused by isoproterenol was lower, whereas the concentration of isoproterenol required to increase the activit ratio half-maximally was higher than controls; The reduced responsiveness to the drug, however, was reversed when the hypertrophy regressed. These observations, taken collectively, appear to suggest that the desensitization of the β-adrenergic mechanism seen in the cardiac hypertrophy produced by repeated administration of isoproterenol is associated with adaptive modifications in certain parameters of the cyclic nucleotide systems.     

Isoproterenol injection alters protein kinase DEAE-cellulose profiles in selected rat tissues.

Isoproterenol injection produces cardiac hypertrophy and salivary gland enlargement in rats. After subcutaneous injection (5 mg/kg) twice daily for 10 days, the soluble extracts from these and other tissues were subjected to DEAE-cellulose chromatography and the activity of isozymes I and II of cyclic AMP-dependent protein kinase was measured. The activity of isozyme I is decreased 80% in salivary gland and 40% in liver. No significant changes were observed in kidney, brain or skeletal muscle. In contrast to a previous report, no change was observed in the amount of isozyme I in hypertrophied heart. Furthermore, no changes were observed in the activity of isozyme II in response to isoproterenol injection in any of these tissues.     

Independent expression of cardiac type I and II cyclic AMP-dependent protein kinase during murine embryogenesis and postnatal development.

The amount of total cyclic AMP-dependent protein kinase and of the protein kinase isozymes present in mouse heart changes during development. During embryogenesis, the total cardiac protein kinase activity increases most markedly during the 6 days prior to birth. A maximum kinase level is achieved in the 7 day-old neonate, and then activity progressively declines to an adult level approximating that of the mid-embryo. The type II kinase exhibits a moderate increase during late embryogenesis which declines by the time of birth. The type I isozyme increases throughout embryogenesis and the first neonatal week to a maximum specific activity five-fold higher than the mid-embryogenesis level. The isozyme level then falls to an adult activity similar to the mid-embryonic. These changes in isozyme profile are reflected in a changing type I to type II kinase ratio of 1.1 at 13--14 days embryogenesis, 2.4 at birth, 3.0 in the 7 day-old neonates, and 1 in the adult heart. Thus, the two protein kinase isozymes change in association with the developmental process in an independent fashion.     

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.     

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.     

The effects of thyroparathyroidectomy and 1,25 dihydroxyvitamin D3 on changes in the activities of some cytoplasmic and nuclear protein kinases during liver regeneration

Partial hepatectomy (HPX), which proliferatively activates the remaining liver cells, triggered two transient prereplicative surges in the total activities of cytoplasmic types I and II cyclic AMP-dependent protein kinase holoenzymes, and of nuclear catalytic subunits from cyclic AMP-dependent protein kinases. It also induced a transient prereplicative increase in the activities of a nuclear Ca2+-calmodulin-stimulable, protamine-phosphorylating protein kinase, and a nuclear poly(L-lysine)-phosphorylating, 105 kDa protein kinase. Thyroparathyroidectomy (TPTX) delayed and reduced the first surge and completely eliminated the second surge of both of the cytoplasmic cyclic AMP-dependent protein kinases, reduced the rises in the activity of nuclear catalytic subunits, and completely eliminated the surge of the Ca2+-calmodulin-stimulable protein kinase, but did not affect the surge of the nuclear 105 kDa protein kinase. The impairment of the responses of the two cyclic AMP-dependent protein kinases to HPX in TPTX rats was not accompanied by a rise in the level of heat-stable inhibitor of cyclic AMP-dependent protein kinase activity. One intraperitoneal injection of 1,25-dihydroxyvitamin D1 into TPTX rats immediately after HPX completely restored the post-HPX surges in the activity of type I cyclic AMP-dependent protein kinase, but the hormone, even in high doses, had little or not effect on the type II isoenzyme or the nuclear Ca2+-calmodulin-stimulable, protamine-phosphorylating enzyme.     

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium.

Embryonic chick (7-9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionic strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7--9 day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio of newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanied by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.     

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.     

The effect of turpentine-induced inflammation on rat liver glycosyltransferases and Golgi complex ultrastructure

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.     

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.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium

Embryonic chick (7–9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionicf strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7–9-day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio in newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanined by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

Lymphocyte protein kinase activity in cells from young and elderly men and women

Protein kinase activity of lymphocytes isolated from human subjects was assayed using histone as substrate. The activity was stimulated about twofold by cyclic AMP and total enzyme activity, determined in the presence of cyclic AMP, was inhibited by 65% by the specific heat-stable inhibitor of cyclic AMP-dependent protein kinase. Histone phosphorylation was not stimulated by cyclic GMP in the presence of the inhibitor. Cyclic AMP-dependent protein kinase could be activated in vitro by incubating intact cells with isoproterenol or with forskolin and was reflected by a significant (P less than 0.05) increase in the protein kinase activity ratio. In contrast to these well-characterized adenylate cyclase activators, incubating cells for up to 2 hr in vitro in the presence of the specific beta-blocker propranolol had no significant effect on the amount of cyclic AMP-dependent protein kinase that was in the activated state. When compared in subjects between the ages of 21 and 74 years, lymphocyte protein kinase activity was unaltered by age or gender. These results indicate that cyclic nucleotide-dependent protein kinase is of the cyclic AMP-dependent variety in the human lymphocyte. A low amount of the cyclic AMP-dependent activity (about 15%) is in the already activated state in freshly isolated cells, and this is not further reduced by incubation in vitro or by beta-blockade. In contrast to previously reported changes in the capacity to synthesize cyclic AMP, lymphocyte protein kinase is unaltered by gender or age in human subjects.     

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3'':5''-phosphate-dependent protein kinase in vivo.

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.     

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

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.