The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

Detection of cGMP dependent protein kinase isozymes by specific antibodies.

Two isozymes of cGMP-dependent protein kinase (cGMP kinase) have been identified. Polyclonal antibodies were developed which recognize both isozymes or specifically the I alpha and I beta isoform. The specificity of these antibodies was verified by using the recombinant or purified I alpha and I beta isozymes. The antibodies cross-reacted with the purified isozymes of cGMP kinase from bovine tracheal smooth muscle. The tissue concentration of cGMP kinase was determined by ELISA. High concentrations (greater than 10 pmol/g wet tissue) were present in bovine lung, rumen, trachea, aorta, uterus and stomach. The tissue distribution of the isozymes I alpha and I beta was investigated by immunoblots using crude extracts of the different tissues. The I beta-specific antibody yielded strong signals with extracts of trachea, aorta, stomach and uterus, whereas heart, cerebellum and lung apparently contain mainly the I alpha isozyme.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini.

cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.     

Comparison of cAMP-dependent protein kinase in normal and Rous sarcoma virus transformed chick embryo fibroblasts

cAMP-dependent protein kinase was compared in normal and Rous Sarcoma Virus transformed chicken embryo fibroblasts. Total cAMP binding activity and cAMP-dependent histone kinase activity were unaltered by RSV transformation. The apparent Km for activation of histone kinase activity by cAMP was 35 nM in both normal and transformed cells. Using 8-N3-cAMP photoaffinity labeling, normal and transformed cells were also found to contain equal quantities of a single 42,000 Mr regulatory sub-unit isoenzyme of A-kinase. This isoenzyme corresponded to the lower molecular weight isoenzyme of the two enzymes found in normal chicken skeletal muscle. Both avian isoenzymes were about 4,000 Mr smaller than the corresponding bovine type I and type II regulatory subunits. Rous Sarcoma Virus transformation does not directly alter the amount or activity of cAMP-dependent protein kinase.     

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog.

Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.     

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.     

Interaction of the alpha beta dimers of the insulin-like growth factor I receptor is required for receptor autophosphorylation.

We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.     

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.     

Cyclic AMP-dependent protein kinase does not phosphorylate cyclic GMP-dependent protein kinase in vitro

The autophosphorylation reaction of purified cGMP-dependent protein kinase has been studied. Apparent initial rates of autophosphorylation in the absence of cyclic nucleotides and in the presence of cGMP and cAMP are 0.006, 0.04, 0.4 mol Pi incorp./min-1. mol cGMP-kinase subunit-1. In the presence of cGMP and cAMP approximately 1 and 2 mol Pi are incorporated/mol enzyme subunit. These values are independent of the enzyme concentration. Stimulation of autophosphorylation by cAMP is not due to activation of a contaminating cAMP-dependent protein kinase since: (a) addition of the heatstable inhibitor protein of cAMP-kinase does not inhibit autophosphorylation; and (b) catalytic subunit of cAMP-kinase added at a 10-fold excess over cGMP-kinase does not phosphorylate cGMP-kinase.     

Studies of functional domains of the regulatory subunit from cAMP-dependent protein kinase isozyme I

Homogenous regulatory subunit from rabbit skeletal muscle cAMP-dependent protein kinase (isozyme I) was partially hydrolyzed with low (1 g/1300 g) or high (1 g/6 g) concentrations of trypsin. After treatment with low trypsin two main peptides (Mr = 35,000 and 12,000) were produced. The cAMP-binding activity (2 mol cAMP/mol of subunit monomer) was recovered in the monomeric Mr = 35,000 peptide. The ability of either fragment to inhibit catalytic subunit activity was lost. Treatment of the regulatory subunit with a high concentration of trypsin yielded three main fragments (Mr = 32,000, 16,000, and 6,000) which could be resolved by Sephadex G-75 and purified further on DEAE-cellulose columns. One of the peptides (Mr = 32,000) bound 2 mol cAMP/mol fragment. The Mr = 16,000 fragment was very labile and bound cAMP with an undetermined stoichiometry. Cyclic AMP dissociation curves for the native regulatory subunit and its Mr = 32,000 component were similar and suggested the presence of two nonidentical binding sites in each monomer. Using the same procedure, the Mr = 16,000 fragment or homogenous cGMP-dependent protein kinase appeared to contain a single type of binding site. Purified Mr = 32,000 fragment was readily converted to the Mr = 16,000 fragment using high trypsin as assessed by protein bands on SDS-disc gels or by following transfer of radioactivity from Mr = 32,000 peptide covalently labeled with 8-N3-[32P] cAMP to radiolabeled Mr = 16,000 fragment. The smallest regulatory subunit fragment (Mr = 6,000) did not bind cAMP, but was dimeric and could be part of the dimerization domain in the native protein. A model is presented to explain the possible structural-functional relationships of the regulatory subunit.     

Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases.

Cyclic AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified, and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit molecular weight of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These data suggest an asymmetric tetrameric (R2C2) structure (Mr approximately equal to 160,000) for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit molecular weight, pI values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 microgram/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but are clearly dissimilar from those of the soluble type II enzyme. Moreover, regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit molecular weight, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the present results suggest that the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

The cDNA of the two isoforms of bovine cGMP-dependent protein kinase

cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.     

Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.     

The cyclic nucleotide-dependent phosphorylation of aortic smooth muscle membrane proteins

Membrane proteins of Mr 240,000, 130,000, and 85,000 (GS-proteins) were rapidly and selectively phosphorylated in particulate fractions of rabbit aortic smooth muscle in the presence of [Mg-32P]ATP and low concentrations of cGMP (Ka = 0.01 microM) or cAMP (Ka = 0.2 microM). The effects of both cyclic nucleotides in this preparation were mediated entirely by an endogenous, membrane-bound form of cGMP-dependent protein kinase (G-kinase). The GS-proteins were also phosphorylated by the soluble form of G-kinase purified from bovine lung; this effect was most evident following removal of endogenous G-kinase from the membranes using Na2CO3 and high salt washes. The membrane-bound and cytosolic forms of G-kinase phosphorylated the Mr 130,000 GS-protein with the same specificity as determined by two-dimensional peptide mapping. Despite this functional homology between the two forms of G-kinase, only the particulate enzyme appears to play a role in phosphorylating the GS-proteins. Although little endogenous cAMP-dependent protein kinase (A-kinase) activity was detected in washed aortic smooth muscle membranes, the GS-proteins could be phosphorylated when purified A-kinase catalytic subunit was added to this preparation. Peptide mapping of the Mr 130,000 GS-protein indicated that A-kinase phosphorylated a subset of the same peptides labeled by the two forms of G-kinase. The endogenous A-kinase of rabbit aortic smooth muscle homogenates was also found to phosphorylate the GS-proteins. Since the intracellular concentrations of cGMP or cAMP can be selectively elevated by different stimuli, these results suggest several possible mechanisms by which the phosphorylation state of the GS-proteins may be regulated by cyclic nucleotides: activation of the membrane-bound G-kinase by cGMP or cAMP; and activation of cytosolic A-kinase by cAMP.