J. F. Kuo

Cyclic nucleotide-dependent protein kinases. V. Preparation and properties of adenosine 3′,5′-monophosphate-dependent protein kinase from various bovine tissues

Abstract Adenosine 3′,5′-monophosphate (cyclic AMP)_dependent protein kinases, which catalyze the phosphorylation of proteins by ATP, have been found in each of fifteen bovine tissues examined. The enzymes were partially purified from these tissues, and their properties studied. Cyclic AMP increased by 5–15-fold the ability of each of these enzymes to phosphorylate histones. The concentration of cyclic AMP required to give half-maximal activation ranged from 30 to 160 nM. The 3′,5′-monophosphate derivatives of inosine, guanosine, uridine, and cytidine also activated the enzymes, but only at concentrations considerably higher than those required for cyclic AMP. 2′-Deoxythymidine 3′,5′-monophosphate was incapable of stimulating enzyme activity. Of the proteins tested as substrates, histone was the most effective for all of the enzymes studied. Protamine and casein were also phosphorylated by the enzymes. All of the enzymes had an absolute requirement for a divalent metal. Cyclic AMP stimulated enzyme activity in the presence of Mg2+, Mn2+, or Co2+, whereas it inhibited enzyme activity in the presence of Ca2+. The concentration of ATP required to give half-maximal velocity was determined for several of the enzyme preparations in the absence and presence of cyclic AMP; in each case, the apparent K m for ATP was significantly lower in the presence than in the absence of the activator. It was found that adenine, adenosine, AMP, ADP, GDP, riboflavin, FMN, and FAD inhibited enzyme activity to varying degrees. The fifteen enzyme preparations were found to be generally similar, but some important differences were observed.

Regulation of myocardial cyclic AMP by isoproterenol, glucagon and acetylcholine☆

Abstract A method for preparing rat and calf heart slices which retain their responsiveness to various hormones is described. It is shown that isoproterenol and glucagon independently and maximally increased myocardial cyclic AMP, up to about 6-fold, that the stimulatory action of these two hormones was not additive, and that propranolol abolished the action of isoproterenol but not that of glucagon. These data suggest that isoproterenol and glucagon interact with functionally distinguishable receptors on myocardial adenyl cyclase. Acetylcholine antagonized the action of isoproterenol, of glucagon, or of both, greatly reducing the elevation of myocardial cyclic AMP. Acetylcholine also lowered the basal level of cyclic AMP slightly.

Stimulation of adenosine 3′,5′-monophosphate-dependent and guanosine 3′,5′-monophosphate-dependent protein kinases by some analogs of adenosine 3′,5′-monophosphate

Abstract The effect of some analogs of adenosine 3′,5′-monophosphate (cyclic AMP) on the activity of protein kinases activated specifically either by cyclic AMP or by guanosine 3′,5′-monophosphate (cyclic GMP) has been examined. Tubercidin 3′,5′-monophosphate (cyclic TuMP) stimulated the activity of cyclic AMP-dependent protein kinases as effectively as cyclic AMP. The ability of cyclic TuMP to stimulate cyclic GMP-dependent protein kinases was intermediate between that of cyclic GMP and cyclic AMP. The 5′-methylene cyclic phosphonate analog of cyclic AMP caused varying degrees of activation of different cyclic AMP-dependent protein kinases, but was inactive with cyclic GMP-dependent enzymes. The 3′-methylene cyclic phosphonate analog of cyclic AMP was inactive with enzymes of either class.

Activation and dissociation of adenosine 3′,5′-monophosphate-dependent and guanosine 3′,5′-monophosphate-dependent protein kinases by various cyclic nucleotide analogs

Abstract The effects of various analogs of adenosine 3′,5′-monophosphate (cyclic AMP), guanosine 3′,5′-monophosphate (cyclic GMP), and inosine 3′,5′-monophosphate (cyclic IMP) in stimulating and dissociating cyclic AMP-dependent and cyclic GMP-dependent classes of protein kinases were examined. With the possible exceptions of the dibutyryl derivative of cyclic GMP and the cyclothiophosphate analog of cyclic AMP, all compounds tested were capable of maximally stimulating both classes of protein kinases, and some analogs were more reactive than the parent compounds. The effectiveness of these compounds in activating these protein kinases paralleled their ability to cause the dissociation of the enzymes into subunits. Some compounds, notably the 8-thio and 8-methylthio analogs of cyclic AMP, which were at least as reactive as cyclic AMP in dissociating and stimulating the cyclic AMP-dependent enzyme, did not compete, but unexpectedly cooperatively enhanced the binding of the radioactive cyclic AMP to this enzyme, suggesting that these sulfur-containing analogs may interact with specific sites on the enzyme different from cyclic AMP. Many of the cyclic AMP analogs, like cyclic GMP, supported the stimulatory action of the protein kinase modulator on arginine-rich histone phosphorylation catalyzed by cyclic GMP-dependent protein kinase. The modulator, however, inhibited the cyclic AMP-dependent class of protein kinase activated by any of the cyclic nucleotides and analogs under the same assay conditions.

Differential effects of Ca2+, EDTA and adrenergic blocking agents on the actions of some hormones on adenosine 3′,5′-monophosphate levels in isolated adipose cells as determined by prior labelling with [8−14C]adenine☆

The effects of several hormones on the adenyl cyclase-adenosine 3′,5′-monophosphate (cyclic AMP) system in isolated adipose cells were studied by measuring the formation and accumulation of radioactive cyclic AMP by cells which have been prelabeled with [8−14C]adenine. Based on the differential effects of Ca2+, EDTA and adrenergic blocking agents, the lipolytic hormones that activate the adenyl cyclase in adipose cells can be divided into the following categories: (1) norepinephrine, (2) glucagon, and (3) general one which includes corticotropin, thyroid-stimulating hormone and probably luteinizing hormone. It is suggested that in adipose cells there may exist at least three functionally distinguishable receptor sites on the cyclase, one responsive to each category of these hormones.

Cyclic nucleotide−dependent protein kinases. VII. Comparison of various histones as substrates for adenosine 3′,5′−monophosphate−dependent and guanosine 3′,5′−monophosphate−dependent protein kinases☆

Abstract A study was made of the ability of purified histones Ib, IIb and IV to serve as phosphate acceptors in reactions catalyzed by adenosine 3′,5′-monophosphate (cyclic AMP)-dependent and guanosine 3′,5′-monophosphate (cyclic GMP)-dependent protein kinases. All three histones were able to serve as phosphate acceptors for all of the protein kinases tested. The relative reactivity of the histones was similar in the case of each of twenty-six cyclic AMP-dependent protein kinases obtained from deverse tissues; for all of these cyclic AMP-dependent enzymes, the reactivity of the histones decreased in the order IV>IIb>Ib . The relative reactivity of the histones exhibited a much different and more complex pattern when a cyclic GMP-dependent protein kinase was used. It is suggested that cyclic GMP-dependent protein kinases possess specificityk for endogenous proteins different from that of cyclic AMP-dependent protein kinases. This different may account for the separate and independent physiological roles believed to be mediated by cyclic AMP and cyclic GMP.

Studies on the mechanism of action of cyclic AMP in nervous and other tissues

Summary Thermodynamic studies, involving equilibrium and calorimetric measurements, indicate that cyclic AMP is a high-energy compound. The hydrolysis of the 3′bond of cyclic AMP to 5′AMP releases about 12,000 cal/mole of free-energy. The free-energy change results from a large decrease in enthalpy of the cyclic nucleotide upon its conversion to 5′AMP. Cyclic AMP-dependent protein kinases and cyclic GMP-dependent protein kinases have been shown to be distributed widely in nature. The results support a hypothesis that all of the diverse effects of cyclic AMP and cyclic GMP are mediated through regulation of the activity of protein kinases. The two classes of protein kinase differ in their substrate specificity. Enzymological, cytochemical and electrophysiological evidence is presented in support of the concept that cyclic AMP is intimately associated with the molecular events underlying the process of synaptic transmission in the nervous system.

[47] Purification and characterization of cyclic GMP-dependent protein kinases

Publisher Summary Many arthropod tissues are rich in cGMP-dependent protein kinases and provide favorable material for study of the properties and function of enzymes. In contrast, it has not yet been possible to detect cGMP-dependent protein kinase activity in most vertebrate tissues examined. A correlation of the relative tissue levels of cAMP and cGMP with the apparent relative tissue levels of cAMP-dependent and cGMP-dependent protein kinases would appear to be lacking. For instance, cecropia silkmoth larval fat body, a tissue in which only cyclic GMP-dependent protein kinase activity has been detected, has a ratio of cGMP to cAMP of about 0.4; in contrast, rat cerebellum and lung, tissues in which only cAMP-dependent protein kinase has been detected, have a cGMP to cAMP ratio of about 0.7, highest of any of the mammalian or arthropod tissues examined. This lack of correlation is not entirely surprising, when one considers the great difficulty of estimating levels of enzyme activity in vivo from studies of broken cell preparations.

[12] Assay of cyclic GMP by activation of cyclic GMP-dependent protein kinase

Publisher Summary The assay method is based upon the ability of low concentrations of cyclic GMP (eGMP) to activate cyclic GMP-dependent protein kinases, which catalyze the phosphorylation of substrate proteins (e.g., histone, protamine) by ATP. Under the standard assay conditions, the extent of histone phosphorylation is directly proportional to the amount of cGMP present in the incubation tube, thus providing a direct, sensitive and specific method for the measurement of the cyclic nucleotide in tissue and body fluid samples. The limit of sensitivity of the procedure for assaying cGMP with cGMP-dependent protein kinase is about 0.5 pmole. The sensitivity can be improved to about 0.3 pmole of cGMP if the incubation volume is reduced to about 0.1 ml. The preliminary purification of tissue cGMP is found to effectively separate this cyclic nucleotide from cAMP (which is usually present in amounts 5–100 times that of cGMP in most tissues; the ratios are even higher if the tissues have been exposed to certain agents) and remove any substances, such as ATP, ADP, originally present in the crude extracts, which might interfere with the assay for cGMP based upon its ability to activate the enzyme.