Properties of epinephrine-induced activation of cardiac adenosine 3',5'-monophosphate-dependent protein kinase.

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (-cyclic AMP/"cyclic AMP) of 12 000 X g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

高温胁迫下葡萄叶片蛋白激酶的诱导形成与活性变化

以"京秀"葡萄(Vitis vinifera L.cv.Jingxiu)幼苗为试材,研究了高温胁迫激活的蛋白激酶的类型和活性.结果表明,高温胁迫10～60min明显地激活了一个分子量约为52 kD的蛋白激酶,该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白(MBP)磷酸化,在放射自显影中表现出很高的放射活性,而对凝胶中的组蛋白-Ⅲ(histone-Ⅲ)则没有这样的作用.在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性,而对histone-Ⅲ却无作用.Ca2 对其活性变化无显著影响.酪氨酸特异性蛋白磷酸酶(YOP)对该激酶的活性有显著的钝化作用.结果表明该52 kD蛋白激酶是MAPK家族中的一种.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Proteolysis of a 34 kDa Phosphoprotein Coincident with a Decrease in Protein Kinase Activity During the Erythrocytic Schizont Stage of the Malaria Parasite

ABSTRACT. Protein phosphorylation events may play important roles in the replication and differentiation of the malarial parasite. Investigations into the lability of a Plasmodium protein kinase revealed that a 34 kDa parasite phosphoprotein is rapidly converted into a 19 kDa fragment. Coincident with this conversion is a nearly total loss of a protein kinase activity, as determined from the phosphorylation of endogenous protein substrates. Both the conversion of the 34 kDa protein to the 19 kDa protein and the loss of protein kinase activity are inhibited by thio-protease inhibitors. The presence of low levels of the intact 34 kDa protein restores the protein kinase activity to almost maximum levels. However, it was not possible to demonstrate protein kinase activity associated with the 34 kDa protein, thus suggesting that the 34 kDa protein is probably an activator or regulator of the protein kinase activity and not a protein kinase. The conversion to the 19 kDa fragment also occurs in vivo and only during the schizont stage prior to the appearance of ring forms. During this same period the protein kinase activity decreases suggesting that the proteolytic processing of the 34 kDa protein may be a physiological regulator of the protein kinase.     

Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe protein modification in Rhodospirillum rubrum.

A procedure for the immunoprecipitation of Fe protein from cell extracts was developed and used to monitor the modification of Fe protein in vivo. The subunit pattern of the isolated Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Coomassie brilliant blue protein staining and autoradiographic 32P detection of the modifying group. Whole-cell nitrogenase activity was also monitored during Fe protein modification. The addition of ammonia, darkness, oxygen, carbonyl cyanide m-chlorophenylhydrazone, and phenazine methosulfate each resulted in a loss of whole-cell nitrogenase activity and the in vivo modification of Fe protein. For ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for Fe protein modification. The reillumination of a culture incubated in the dark brought about a rapid recovery of nitrogenase activity and the demodification of Fe protein. Cyclic dark-light treatments resulted in matching cycles of nitrogenase activity and Fe protein modification. Carbonyl cyanide m-chlorophenylhydrazone and phenazine methosulfate treatments caused an immediate loss of nitrogenase activity, whereas Fe protein modification occurred at a slower rate. Oxygen treatment resulted in a rapid loss of activity but only an incomplete modification of the Fe protein.     

Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.     

Differential regulation of phenylalanine ammonia lyase activity and protein level by light in tomato seedlings

Red light, acting via phytochrome, stimulates phenylalanine ammonia lyase (PAL) activity in cotyledons and hypocotyls of tomato seedlings. The time course of photoinduction of PAL activity has a peak level at 4 h after which activity declines significantly. In tomato seedlings PAL activity comprised of three isoforms and light stimulated activity of all three isoforms. A polyclonal antibody raised against PAL purified from tomato leaves recognized PAL protein belonging to PAL-II and PAL-III isoforms. The mode of increase in PAL activity was investigated by immunochemical techniques. The photostimulated increase in PAL activity appeared to be dependent on de novo synthesis of protein and nucleic acid. However, inhibition of protein phosphatase activity blocked increase in PAL activity without affecting the increase in PAL protein levels. The results indicate that in addition to de novo synthesis, the photostimulation of PAL activity likely requires dephosphorylation by a type 2C protein phosphatase.     

Studies on Protein Metabolism in Higher Plants

Changes in ribulose diphosphate (RuDP) carboxylase activity and the content of fraction 1 protein, which had been elucidated to be identical with protein of RuDP carboxylase, in tobacco leaves were examined with age, comparing with change in total protein content. Fraction 1 protein was determined by an immunological precipitin method developed in this experiment. Fraction 1 protein decreased with age and the rate was similar or a little larger than those of total protein and total chlorophyll. The carboxylase activity decreased more rapidly than fraction 1 protein during the senescent process. In a plant, upper leaves showed higher values of the carboxylase activity and fraction 1 protein content than lower leaves. The specific activity, RuDP carboxylase activity per unit fraction 1 protein, in upper leaves was higher than that in lower leaves.     

Protein kinase in human plasma analogous to that present in control and interferon-treated HeLa cells

A protein kinase activity analogous to that found in interferon-treated HeLa cells is detectable in human plasma. This kinase activity is manifested by the phosphorylation of an endogenous 72,000 molecular weight protein which is stimulated markedly by Mn²⁺. The protein kinase activity in human plasma can phosphorylate calf thymus histones and is independent of cyclic AMP. The phosphate is linked to the phosphorylated 72,000 molecular weight protein by its serine and threonine residues. The level of protein kinase activity in 50 different normal human plasma that we analysed varied from one individual to the other. Treatment of two patients with fibroblast (β) interferon resulted in an enhanced level of the protein kinase activity in the plasma. These results suggest that such protein kinase activity in human plasma may reflect the presence and action of circulating interferon.     

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.     

Stimulation of ascites tumor RNA polymerase II by protein kinase.

The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.     

Activation of adenosine 3',5'-monophosphate-dependent protein kinase and its relationship to cyclic AMP and lipolysis in hamster adipose tissue

The interrelationships among cAMP-dependent protein kinase activity, lipolysis, and cellular concentrations of cAMP were investigated in hamster epididymal adipose tissue. Isoproterenol, norepinephrine, and theophylline increased the protein kinase activity assayed in tissue extracts with no added cAMP, but not in the presence of added cyclic nucleotide. The maximum rate of lipolysis was associated with a nearly three-fold increase in cAMP levels and a protein kinase activity ratio of 0.8 (the ratio of activity assayed without cAMP to that assayed with cAMP). Rates of lipolysis less than maximum were associated with lesser degrees of protein kinase activity and lower levels of cAMP. The relatively pure alpha-adrenergic agent phenylephrine partially suppressed the isoproterenol-stimulated protein kinase activity, lipolysis, and cAMP levels. Conversely, the alpha-adrenergic blocking agent phentolamine increased the activity of protein kinase and cAMP levels in adipose tissues exposed to norepinephrine. These data are consistent with the primary role for cAMP and its dependent protein kinase in control of lipolysis in adipose tissue. Moreover, our data are consistent with the view that the antilipolytic action of alpha-adrenergic agents is mediated by a decrease in activity of protein kinase, caused by a decrease in cellular cAMP concentrations.     

Distribution of protein kinase activities in subcellular fractions of rat brain

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.     

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Demonstration of protein kinase activities in the coronary artery of cattle]

Protein kinase activities were identified in a soluble and a particulate fraction from the A. coronaria of cattle. For both protein kinase activities Mg++ is essential. Protamine was used as a substrate of the protein kinase activity of the soluble fraction. The pH optimum of the protein kinase activity of the soluble fraction is around 6.5. The Km-value of the protein kinase for ATP is 1.9 +/- 0.4 - 10(-5) M. cAMP stimulates the protein kinase activity more effectively than cGMP. Ca++ cannot replace Mg++; monovalent cations (Na+ and K+) show no influence. The protein kinase activity of the fraction was determined via endogenous phosphorylation. By means of the cAMP-dependent particulate protein kinase 72 to 80 percent of the serine residues are phosphorylated. The pH optimum of the protein kinase activity of the particulate fraction lies around 7.0. The Km-value of the enzyme for ATP is 6.6 +/- 0.8 - 10(-5) M. cGMP stimulates the protein kinase of the particulate fraction better than cAMP. For the protein kinase activity of this fraction Ca++ replaces Mg++ in the endogenous phosphorylation but not in the exogenous phosphorylation (protamine). In the presence of Mg++ and in the additional presence of Na+ or K+, the protein kinase activity is suppressed in the endogenous phosphorylation whereas it is stimulated in the exogenous phosphorylation.     

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.     

On the Relationship between Food Composition and Serine Dehydrase Activity in Rat Liver

Relationship between serine dehydrase activity in rat liver and dietary protein and carbohydrate levels, using casein and wheat starch, was investigated. Between 10% to 50% of dietary protein levels the enzyme activity sharply increased with increase of protein level. The enzyme activity was also increased by the restriction of carbohydrate intake, even when protein intake was equal in amount. Apparently enzyme activity is controlled by dietary protein level and also by carbohydrate level. In the case of high protein or low carbohydrate diet these changes may be mainly acting for utilization of protein as caloric source.     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.