Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Role of cyclic AMP-dependent protein kinase activation in regulating rat submandibular mucin secretion

The extent of activation of rat submandibular gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was determined in vitro using dispersed cells to assess the involvement of this enzyme in submandibular mucin secretion. cAMP-dependent protein kinase activation, as determined by activity ratio method, was markedly increased following β-adrenergic receptor activation. 0.5 M NaCl was required in the homogenization buffer for stabilization of the hormonally activated cAMP-dependent protein kinase. A role for cAMP-dependent protein kinase activation in regulating mucin secretion was strongly suggested by the following: (1) the kinase activity ratio increased rapidly after β-adrenergic receptor stimulation; (2) dose-response relationship of the kinase activation following β-adrenergic receptor activation correlated with isoproterenol induced mucin release; (3) termination of β-adrenergic mediated mucin secretion caused a rapid decrease in the kinase activity ratio; (4) dibutyryl cyclic AMP stimulation caused an increase in the kinase ratio; whereas (5) pure cholinergic and pure α-adrenergic receptor stimulation had no effect on endogenous kinase activity. Although cAMP-dependent protein kinase activation may not be the only regulator of mucin secretion, these data suggest an important regulatory role for this kinase activation during rat submandibular mucin release.     

Modulation of lyso-platelet activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of cyclic AMP-dependent protein kinase

Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of Mg-ATP stimulated 2-3-fold lyso-platelet-activating factor:acetyltransferase activity. This activation was due to an increase in the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction, whereas the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for acetyl-CoA was not affected. The ATP derivative, AMPPNP, could not replace ATP and preincubation of the microsomes with the heat-stable inhibitor of protein kinase prevented the activation by Mg-ATP obtained in the presence of the protein kinase. Activation of the acetylation reaction by the protein kinase was reversible. Evidence is provided that the reversal of activation is due to dephosphorylation of the enzyme. These data provide evidence that in vitro lyso-platelet-activating factor:acetyltransferase from splenic microsomes is regulated by phosphorylation.     

Activation of type I and type II cyclic AMP-dependent protein kinases by 2,8-disubstituted derivatives of cyclic AMP

Derivatives of cyclic AMP with substituents in both the 2-position (methyl or butyl) and the 8-position (bromo, benzylthio, p-chlorophenylthio or azido) and their singly modified parent compounds were examined for their abilities to activate type I isozymes of cyclic AMP-dependent protein kinases from rabbit and porcine muscle and type II isozymes of cyclic AMP-dependent protein kinases from bovine brain and heart. The specificity of 2-n-butyl-cyclic AMP for type II was substantially reduced or eliminated by the addition of 8-substituents. The lack of specificity of 2-methyl-cyclic AMP for either type I or II was not changed by the addition of 8-substituents.     

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3–0.4%/min) for at least 30 min incubation at 37°C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cell preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (K_i=5·10^?7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H₂-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.     

Differential effects of histamine, vasoactive intestinal polypeptide, prostaglandin E2 and somatostatin on cyclic AMP-dependent protein kinase activation in gastric glands isolated from the guinea pig fundus and antrum

Histamine, vasoactive intestinal polypeptide (VIP), secretin and prostaglandin E2 (PGE2) stimulate cyclic AMP-dependent protein kinase activity in gastric glands isolated from the guinea pig fundus and antrum. The effects are observed in the absence of any cyclic AMP phosphodiesterase inhibitor and maximal stimulation of the protein kinases occurs within 0.5 min of incubation at 20 degrees C. As shown by dose-response studies, VIP is equally potent in the antrum as in the fundus (identical values of the activation constant are found in both types of gland, Ka = 2.5 . 10(-9) M); a similar situation occurs for PGE2 action (but with Ka = 2.0 . 10(-8) M), whereas the potency of histamine is higher in the fundus (ka = 8.0 . 10(-6)M) than in the antrum (Ka = 5.0 . 10(-5) M). Secretin also increases the protein kinase activity ratio but with a 1000 times lower potency than VIP. In fundic glands, histamine (10(-3) M) is the activator of by far the greatest efficacy (increasing protein kinase activity at 4 times of the basal value) as compared with the effect obtained with 10(-6) M PGE2 (2.7 times) and 10(-7) M VIP (1.4 times). In contrast, VIP has greater efficacy (2.3 times) than histamine (2.1 times) in antral glands, whereas PGE2 is equally active in the two parts of the gastric mucosa. In addition, somatostatin (10(-6) M) inhibits partially (30%) and specifically the protein kinase activation stimulated by histamine, whereas it has no effect on VIP- and PGE2-induced activation. The results are consistent with increased cyclic AMP levels in response to these effectors in this system. A physiological role of histamine on acid-secreting parietal cells, of VIP on nonparietal cells and of PGE2 on both cell types, mediated by the cyclic AMP/protein kinase system is proposed.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis.Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function,such as adhesion,aggregation,and secretion.Elevation of intracellular cAMP,which induces the activation of PKA,results in the inhibition of platelet function.Thus,tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy.In this review,we summarize the PKA substrates and their contributions to platelet function,especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology.In addition,we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function, such as adhesion, aggregation, and secretion. Elevation of intracellular cAMP, which induces the activation of PKA, results in the inhibition of platelet function. Thus, tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy. In this review, we summarize the PKA substrates and their contributions to platelet function, especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology. In addition, we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

Glycogen synthase activity in Chinese hamster ovary cells: Studies with wild-type and mutant cells defective in cyclic AMP-dependent protein kinase

Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous hisone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation)_or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. Since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

Activation of cyclic AMP-dependent protein kinase A common step in vasopressin- and hypertonicity-induced water flow

Water flow across the amphibian urinary bladder can be induced by either vasopressin or serosal hypertonicity. In an effort to determine the common intracellular steps mediating both responses, we determined the in situ activation of cyclic AMP-dependent protein kinase in bladders stimulated by vasopressin or hypertonicity. Treatment of bladders with vasopressin (1 mU/ml) caused in situ activation of cytosolic cyclic AMP-dependent protein kinase of epithelial cells, with a rise in the kinase ratio and cyclic AMP content. Similarly, hyperonicity increased the kinase ratio, but this occured without a measurable increase in cyclic AMP content per mg protein. Because of the hypertonicity-induced cell shrinkage, epithelial cell water decreased by 20%, which may result in a proportionate increase in cyclic AMP concentration (per ml cell water). Furthermore, cell shrinkage also increases intracellular electrolyte concentration, which, in turn, should delay reassociation and consequent inactivation of the predominant Type II cyclic AMP-dependent protein kinase of the epithelial cells. Thus activation of cyclic AMP-dependent protein kinase during hypetonicity may be the result of cell shrinkage, with an associated increase in cyclic AMP and electrolyte concentrations. Studies with prostaglandin synthesis inhibitors and colchicine, a microtubule disrupting agent, also indicated common pathways for vasopressin and hypertonicity. Both naproxen and meclofenamate significantly enhanced the hypertonicity response. Colchicine pretreatment, on the other hand, caused a small (18%) but significant inhibition of the hypertnicity response, similar to its effect on the vasopressine response (25% inhibition). Thus, the increased water permeability of the toad bladder in response to both vasopressin and hypertonicity follows a similar pathway. Activation of cyclic AMP-dependent protein kinase represents the first common step yet identified.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats

In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical sympathectomy and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats.Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP content of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but not phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue.Thus we have established the responsiveness of the cyclic GMP content of brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the α-adrenergic stimulation and in the regulation of proliferative processes in the tissue.     

Endogenous substrates for cyclic AMP-dependent and calcium-dependent protein phosphorylation in rabbit peritoneal neutrophils

As in other cells, cAMP-dependent (protein kinase A) and calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil. The major substrates for protein kinase A in the cytosol of rabbit peritoneal neutrophil is a 43 kDa protein which appears to be actin (pI 5.7). The other substrates for protein kinase A in the cytosol are very acidic proteins with molecular weights of 135 000 (pI 4.6) and 130 000 (pI 4.8). Two classes of calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil: one is calcium, calmodulin-dependent, the other is calcium, phosphatidylserine-dependent. Phosphatidylserine appears to be much more effective than calmodulin in stimulting calcium-dependent protein kinase activity. The phospolipid-sensitive, calcium-dependent protein kinase (protein kinase C), present only in the cytosol fraction, exhibits much higher activity than the cAMP-dependent protein kinase from the same source. At least four substrates (M_r 130 000 (pI 4.6) 43 000 (pI 4.8), 41 000 (pI 6.3) and 34 000) of the protein kinase C in the cytosol were identified. Trifluoperazine, a compound which inhibits the degranulation, aggregation and stimulated oxygen consumption of rabbit peritoneal neutrophils. (Alobaidi, T., Naccache, P.H. and Sha'afi, R.I. (1981) Biochim. Biophys. Acta 675, 316–321), also inhibits the activity of protein kinase C. The possible role of cAMP-dependent and calcium-dependent phosphorylation system in neutrophil function is discussed.     

Lipoprotein lipase activity in cultured macrophage cell line J7742 and its increase in variants deficient in adenylate cyclase and cyclic AMP-dependent protein kinase

Three macrophage cell lines, J774₂, CT₂ and J7H₁ were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCl. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT₂ line which is adenylate cyclase deficient while that in the J7H₁ line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30–40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50–70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity use was made of dibutyryl cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J774₂ line but only dibutyryl cAMP was active in the CT₂ line (adenylate cyclase deficient). In the J7H₁ (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein lipase.     

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protien kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.     

Activation of cyclic AMP-dependent protein kinase by VIP in blood mononuclear cells

Vasoactive intestinal peptide stimulated cyclic AMP-dependent protein kinase activity in human blood mononuclear cells. The simultaneous presence of a phosphodiesterase inhibitor was required to elicit maximal activation. The apprent K_a value of half the maximal stimulation was about 60 pmol. Secretin exhibited a 170-times lower potency. Other peptides such as glucagon or insulin had no effect event at 1 μM.     

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na⁺-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. ³²P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the ³²P from [γ-³²P]ATP is incorporated into protein phosphomonoester linkages.     

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

Summary The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R₂C₂ of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behaviour towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.Abbreviations C catalytic subunit monomer of protein kinase - R regulatory subunit monomer of protein kinase - 8-N₃-cyclic AMP 8-azido-cylic AMP - SDS sodium dodecyl sulfate - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) See AcknowledgementsCareer Investigators from the CONICET