This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project as Los Alamos National Laboratory (LANL). Protein phosphorylation is well established as one of the most important mechanisms of signal transduction and cellular regulation. Two of the key enzymes that catalyze these phosphorylation reactions are the cAMP- (PKA) and cGMP- (PKG) dependent protein kinases. PKA has served as the prototypic model of this class of enzymes that now comprises in excess of 300 phylogenetically related proteins. A large number of these protein kinases are critical for the regulation of cell function and a full analysis of their similarities and differences is essential to understand their diverse physiological roles. The cAMP-dependent protein kinase has the subunit structure R2C2, in which C and R refer to the catalytic and regulatory subunits, respectively. The cGMP-dependent protein kinase (PKG) is highly homologous to PKA but is distinguished from it by having the regulatory and catalytic domains on a contiguous polypeptide. The studies described here use small-angle scattering and Fourier Transform InfraRed (FTIR) spectroscopy to study domain movements and conformational changes in these enzymes in different functional states in order to elucidate the molecular bases for the regulation of their activities.

The high-affinity interaction between protein kinase inhibitor (PKI) (6-22)amide (Thr(6)-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile(22)-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr(6) to Ile(11) sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg(15) to Ile(22). Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the-Ala(12)-Ser-Gly-Arg(15)-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26: 7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des 12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala(12)-Ser-Gly(14)-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The d-alanine(14) PKI analog was as potent as the parent peptide, whereas the beta-alanine(14) and the sarcosine(14) analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).

The phosphorylation of substrate peptides derived from PKI, the heat-stable inhibitor protein of the cAMP-dependent protein kinase (PKA), has been studied with both PKA and the cGMP-dependent protein kinase (PKG) using a variety of substitution and deletion analogs. On the basis of Km, kcat and kcat/Km values, (Ser21)PKI alpha(14-22) amide (numbering based upon native PKI alpha) is the most effective peptide substrate yet discovered for either kinase, although other peptides, while phosphorylated considerably less efficiently by PKG, are more specific. Although the inhibitory peptide corresponding to this sequence (i.e., with an Ala at position 21) is a much more potent inhibitor of PKA than of PKG (approximately 250-fold), PKG actually exhibits a 60% higher kcat than does PKA with the (Ser21)PKI alpha(14-22) amide substrate peptide, with only a 20-fold higher Km value. The two key PKI residues within this peptide which were found to be essential for substrate activity with both kinases were Arg18 (P-3) and Ile22 (P+1). The Arg19 (P-2) residue, which contributes significantly to both PKI-based peptide inhibitors and substrates of PKA, was only a more minor contributor to PKG substrate efficacy. Of particular note, the Phe10 (P-11) residue, which contributes very substantially to high affinity binding of both PKI and longer PKI peptide inhibitors, neither positively nor negatively affects the kinetics of either PKA or PKG with PKI-based substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.

The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

A form of spontaneous Ca2+ release from purified light sarcoplasmic reticulum has been recently described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107). It is characterized by rapid Ca2+ efflux (1-10 mumol/min X mg protein) which begins only after a delay following preloading and depletion of extravesicular Ca2+. In the present study, the influences of a number of ionic factors modulating the spontaneous calcium release phenomenon are described. The divalent metal ions Mn2+ and Sr2+ and higher external [Mg2+] inhibit release. Mn2+ appears to inhibit from outside, whereas Sr2+ must be taken up to inhibit. Decreasing the phosphate concentration during preloading eliminates spontaneous release, but it can be partially restored with salts of other nonprecipitating anions. No such release was obtained with other Ca2+-precipitating anions, i.e. pyrophosphate, oxalate, and fluoride. The release characteristics are strongly dependent on the monovalent cation present. Spontaneous Ca2+ release is optimal at pH 6.8-7.0 and decreases sharply at higher and lower pH. Ca2+ release can be enhanced by addition, during the lag period, of concentrated salt solutions consisting of more permeant cations than anions, which may indicate that an inside positive membrane potential enhances the rate of calcium release. Spontaneous Ca2+ release can take place in the presence of sucrose, and Ca2+ efflux is not accompanied by a corresponding efflux of preloaded sucrose, demonstrating that the vesicles remain sealed during spontaneous release. The spontaneous Ca2+ release process is distinct from several other forms of Ca2+ release from sarcoplasmic reticulum.

A form of spontaneous calcium release from purified sarcoplasmic reticulum isolated from rabbit skeletal muscle is described. The conditions utilized for eliciting spontaneous release rely on preloading the vesicles with calcium in the presence of phosphate. Under the conditions of assay, spontaneous release begins only after a time delay following depletion of calcium ions from the extravesicular space. Release rates as high as 10-20 mumol/mg . min have been observed, but only a portion of the calcium accumulated is released. Released calcium is reaccumulated, and successive spontaneous releases of smaller amounts of calcium are observed under some conditions. Release occurs as a consequence primarily of an increase in unidirectional Ca2+ efflux and, secondarily, a decrease in unidirectional Ca2+ influx. Unidirectional efflux is enhanced by calcium preloading, enhanced by low (0.01-0.1 microM) and reduced by moderate (1-10 microM) extravesicular free calcium levels. Spontaneous Ca2+ release is favored by much lower free calcium concentrations than Ca2+-induced Ca2+ release. The inhibition of unidirectional efflux by calcium appears to involve active calcium uptake. Release is not mediated by a reversal of the calcium pump. The temperature dependence of the release process is steep, comparable with that of energized Ca2+ uptake. This may reflect a process involved in the gating of a hypothetical calcium channel in the sarcoplasmic reticulum membrane.

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.

Spontaneous calcium release from purified light sarcoplasmic reticulum has been previously described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107) and found to be distinct from several other forms of Ca2+ release. Ca2+ release occurs after a lag period following active Ca2+ preloading and depletion of extravesicular Ca2+. In the present study, we find that local anesthetics inhibit spontaneous Ca2+ release, in a time-dependent manner, varying considerably in the preincubation time required to exert maximal effect. At pH 7.0, hydrophilic and mostly charged local anesthetics, such as procaine, procainamide, and N-(2,6-dimethylphenyl carbamoyl methyl)triethyl ammonium bromide, inhibit Ca2+ release only after long preincubations (hours), whereas more hydrophobic local anesthetics are effective after only a short incubation (minutes) with sarcoplasmic reticulum. The more hydrophobic anesthetics take somewhat longer to reach equilibrium, as studied by inhibition of unidirectional Ca2+ efflux, and there is a direct relationship between hydrophobic partition coefficient and half-time to reach equilibrium. Agents known to inhibit permeability pathways for monovalent cations i.e. K+ channel blockers (decamethonium and n-dodecane-1, 12-N,N,N,N',N',N'-hexamethyl-bis-ammonium) or the anion blocker (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), do not inhibit spontaneous Ca2+ release. Carbonyl cyanide m-fluorophenylhydrazone, a protonophore, and gramicidin D, a monovalent cation ionophore, have no effect on Ca2+ release whether local anesthetics are present or not, while the Ca2+ ionophore A23187 relieves inhibition of Ca2+ release by local anesthetics. Ruthenium red does not inhibit spontaneous Ca2+ release. These findings suggest that the binding site(s) for local anesthetics is located on the inner face of the sarcoplasmic reticulum membrane and that local anesthetics interact directly with a Ca2+ channel rather than with other permeability pathways which might indirectly influence Ca2+ channel gating.

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.

The release of Ca2+ from the terminal cisternae of sarcoplasmic reticulum in muscle fiber triggers muscle contraction. The signal for Ca2+ release is mediated via the triad junction, i.e. the junctional association of terminal cisternae and transverse tubule. Recently, highly purified morphologically intact triads were isolated from rabbit skeletal muscle (Mitchell, R. D., Palade, P., and Fleischer, S. (1983) J. Cell Biol. 96, 1008-1016). In this study, biochemical characterization of two variants of purified triad preparations (Pyrophosphate and Standard) is provided. Terminal cisternae of triads sequester Ca2+ at rates comparable to those of purified heavy sarcoplasmic reticulum which is referable to terminal cisternae (Meissner, G. (1975) Biochim. Biophys. Acta 389, 51-68). The permeability for calcium ions, as reflected by a 2-3-fold stimulation of (Ca2+, Mg2+)-ATPase activity in the presence of the Ca2+ ionophore A23187, and by the Ca2+ leak rate, is comparable in triads and heavy sarcoplasmic reticulum. Several transverse tubule characteristics are present in triads. Four of them, i.e. cholesterol content, ouabain binding, dihydroalprenolol binding (beta-adrenergic receptor), and ouabain-sensitive (Na+, K+)-ATPase activity, are comparably enriched in the Pyrophosphate triads and therefore appear to be quantitative indices of the amount of transverse tubule. Adenylate cyclase and basal ATPase are unreliable in this regard. Methodology for analyzing membrane integrity and sidedness was applied (adenylate cyclase activity) and modified (ouabain-sensitive (Na+, K+)-ATPase activity) to characterize the transverse tubule of the triad. In addition, a new method was developed making use of ouabain binding to study sidedness. These studies show that the transverse tubule is largely sealed and inside out in orientation, i.e. with the cytoplasmic face exposed. This report indicates that the t-tubule and sarcoplasmic reticulum components of the triads possess transport capability and retain permeability barriers for ions. Therefore, the isolated triads appear to be suitable for studying the physiological Ca2+ release process in vitro.

A simple method is described for obtaining a representative field of subcellular fractions for thin section electron microscopy. The prefixed sample is admixed together with high molecular weight dextran, filtered onto a Millipore filter, and embedded in polymer. Filtration serves to uniformly distribute structures of different sizes and densities. The dextran serves as a nonosmophilic spacer, increasing the space between individual structures and preventing sample compression during filtration onto Millipore filters. Sample aggregation can also readily be assessed by the procedure.