Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.     

The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin

Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.     

Direct Visualization of the Calmodulin Subunit of Phosphorylase Kinase via Electron Microscopy Following Subunit Exchange

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.     

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.     

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase. Probes for studies of subunit function

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase were produced by the conventional hybridoma cell technique. 90 out of 600 hybridomas were found to produce phosphorylase kinase binding antibodies from which only five secreted also phosphorylase kinase activity affecting antibodies. Three of them were cloned; two hybridomas resisted all cloning efforts. Employing immunoblot technique all monoclonal antibodies show cross-reactivity with the alpha, beta, and gamma subunits of phosphorylase kinase indicating that similar, if not identical, epitopes are present on these three subunits. No cross-reactivity with delta is observed. Monoclonal antibodies secreted by two clones which bind to the alpha subunit stimulate the Ca2+-independent A0 activity of phosphorylase kinase more than 30-fold, whereas all other monoclonal antibodies obtained are ineffective in this respect. Monoclonal antibodies binding to the beta subunit inhibit the Ca2+-dependent activities significantly. Antibody produced by one hybridoma binds to the alpha, beta, and gamma subunits with approximately the same affinity. Based on the dual function of calmodulin in phosphorylase kinase (Hessová, Z., Varsányi, M., and Heilmeyer, L.M.G., Jr. (1985) Eur. J. Biochem. 146, 107-115) we conclude that binding of anti-alpha monoclonal antibodies to a regulatory domain in the alpha subunit results in an uncoupling of the inhibitory function of the Ca2+-free delta from the holoenzyme which leads to a concomitant increase in A0 activity. Furthermore, binding of anti-beta monoclonal antibodies to the beta subunit prevents a signal transfer from the Ca2+-saturated delta to the catalytic site of the holoenzyme which inhibits the Ca2+-dependent activities.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

Molecular mechanisms of the regulation of phosphorylase kinase from skeletal muscles of mammals and birds

Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses of beta- and gamma-subunits of phosphorylase kinase from rabbit, chicken and pigeon muscles are not identical. Electron microscopy data suggest that the quaternary structure of chicken and pigeon phosphorylase kinase is of the same type. The alpha'-isozyme of chicken and pigeon phosphorylase kinase is strongly activated by calmodulin and troponin C. Avian phosphorylase kinase is activated 2--3-fold by phosphorylation with cAMP-dependent protein kinase and by autophosphorylation. This activation is associated with the phosphorylation of both alpha'- and beta-subunits. The affinity of pigeon phosphorylase kinase a for Ca2+ is 20 times as high as that of phosphorylase kinase b.     

Activators of phosphorylase kinase alter the cross-linking of its catalytic subunit to the C-terminal one-sixth of its regulatory alpha subunit

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.     

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).     

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.     

Dual function of calmodulin (delta) in phosphorylase kinase

The Ca2+-independent activity of fast skeletal muscle phosphorylase kinase, A0, can be reversibly stimulated by heparin more than 20-fold; concomitantly the Ca2+-dependent A2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less delta and show an about fivefold higher A0 activity than the tetrameric form containing delta stoichiometrically. We interpret this to mean that delta has two functions in the phosphorylase kinase: an inhibitory one with respect to A0 and an activating one with respect to A2. The inhibition of A0 by Ca2+-free delta is released, i.e. A0 increases when this subunit dissociates from the holoenzyme. The maximally heparin-stimulated A0 activity, A0,hep, is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A2. The phosphorylase kinase is not eluted from DEAE-cellulose as a symmetrical bell-shaped protein peak. The peak fraction contains the activities A2 and A0,hep superimposed and yields a nearly homogeneous sedimentation boundary with an S20,w value of 25.5 S. The A0 yields a much broader eluation profile showing a distinct maximum from the A2 activity which contains slower sedimenting species of 12.1 S, some tetrameric enzyme of 22.7 S and higher aggregated material. Over the whole profile the activity ratio A2/A0 decreases about sevenfold whereas the ratio A2/A0,hep is constant on average. This shows that A0 is an intrinsic activity of phosphorylase kinase. The heparin-activated A0 activity or A0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca2+-independent flash activation of phosphorylase in a protein-glycogen complex. Thus, A0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca2+ in resting, hormone-stimulated, muscle.     

Influence of the alpha-, beta- and gamma-subunits of the energy-transducing adenosine triphosphates from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method.

We developed a sensitive and specific radioimmunoassay of the energy-transducing adenosine triphosphatase (F1-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and extended the assay to the alpha-, beta- and gamma-subunits of the enzyme. We isolated these subunits and studied cross-reactions. We found the immunochemical properties of alpha- and beta-subunits to differ, and gamma-subunits showed an intermediate behaviour between that of alpha- and beta-subunits. Our findings indicate that each subunit of M. lysodeikticus F1-ATPase has its own identity and that conformational antigenic determinants and/or co-operative antigenic sites-arise from subunit assembly. Equimolecular amounts of alpha- and beta-subunits (up to three copies of each) reconstituted partially the immunochemical properties of the ATPase molecule, and addition of 2 mol of gamma-subunit per mol of alpha 3 beta 3 complex improved reconstitution. Our findings describe the first reconstitution of biological activity of this ATPase by assembly of the isolated subunits, and provide support for earlier proposals on the stoicheiometry of the alpha 3 beta 3 gamma 2 type for M. lysodeikticus F1-ATPase. The radioimmunoassay method affords opportunities to study the homologies between different energy-transducing ATPases and their constituent polypeptides before the primary structure of these complex proteins has been determined.     

Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.     

Molecular mechanisms of G-proteins coupling with membrane receptors and second messenger systems

G-proteins transmit the signals from hormone receptors onto intracellular effector systems which take part in production of the second messengers such as cAMP, IP3, DAG and Ca2+. Molecular mechanisms of G-protein participation in the coupling of the seven-domain receptors to adenylate cyclase, phospholipase C and channels for Ca2+ and K+ ions are discussed in this paper. G-protein is a heterotrimers built of alpha-, beta- and gamma-subunits, which dissociate onto alpha- and beta gamma-subunits during interaction with hormone-receptor complex. alpha-subunit as well as beta gamma-dimmer may interact with effector system that leads to acceleration or slowing down of second messengers formation. Molecular mechanisms of such regulatory signal diversification are described. Seven-domain receptors possess very high recognition specificity of G-proteins. It is defined by combination of both alpha- and beta gamma-subunits in the G-protein structure. There is well-defined interaction specificity of G-protein alpha-subunit with effector systems. Combinations of different beta- and gamma-subunits involved in complex formation define interaction specificity of G-protein beta gamma-complex with effector systems. The highest interaction specificity of receptors with G-proteins and G-proteins with effector systems is found during triple complex formations: receptor--G-protein--effector. Such specificity is stronger in living cells than in membrane preparations. It can be an evidence of intracellular factors influence on the processes of interaction of the proteins involved in transmembrane regulatory signal transduction.     

Inhibition of the phosphorylase kinase activity by ATP analogs and their binding to the enzyme subunits

The interaction between phosphorylase kinase (EC 2.7.1.38), isolated from rabbit skeletal muscles, and the ATP analogs with the modified triphosphate fragment: adenosine-5'-chloromethane pyrophosphonate (1), adenosine-5'-chloroethyl phosphate (2), adenosine-5'-bromethane pyrophosphonate (3), adenosine-5'-bromoethane phosphonate (4), adenosine-5'-chloroacetylaminomethane phosphonate (5), adenosine-5'-chloroacetylaminomethane pyrophosphonate (6) and adenosine-5'-chloromethane phosphonate (7), was studied. The compounds 1, 2 and 3 irreversibly inhibit the enzyme activity. In the presence of ATP the rate of inactivation is decreased. The radioactive compounds 1, 2 and 3 are stoicheometrically incorporated into the beta- and gamma-subunits of phosphorylase kinase. A correlation is shown to exist between the degree of the beta-subunit modification by compound 1 and the enzyme inactivation. The compounds 4, 5 and 6 inhibit the enzyme reversibly: in the presence of ATP complete protection of the enzyme activity is observed. The compound 7 does not affect the kinase activity; however, it binds itself to the beta-subunit of the enzyme. The binding of analogs 1 and 7 to the beta-subunit occurs at different sites. The data obtained are indicative of the catalytic role of the beta-subunit of phosphorylase kinase.     

Rabbit skeletal muscle protein kinase. Conversion from cAMP dependent to independent form by chemical perturbations.

Protein kinase isolated from rabbit skeletal muscle can be reversibly converted from the cAMP dependent form to the indepent form by chaotropic salts and urea. A similar but irreversible conversion can also be induced by trypsin digestion of the holoenzyme. The dissociation of cAMP dependent protein kinase by low concentrations of thiocyanate raises the possibility of isolating both native regulatory and catalytic subunits. From various changes in enzymatic activity caused by urea and trypsin perturbation, it is proposed that the conversion of protein kinase from the cAMP dependent to the independent form is due primarily to preferential modification of the regulatory subunit of the holoenzyme.     

The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle

In the I/Lyn mouse strain a mutation on the X chromosome results in a deficiency of the major calmodulin-regulated enzyme in skeletal muscle, phosphorylase kinase. Calmodulin has been identified as the delta-subunit of phosphorylase kinase, and it is estimated that approximately 40% of the total calmodulin in rabbit skeletal muscle is associated with the phosphorylase kinase hexadecamer (alpha, beta, gamma, delta)4. The absence of phosphorylase kinase in I/Lyn skeletal muscle results in a reduction in the total amount of calmodulin. The mechanisms affecting this reduction were investigated by comparing the abundance and heterogeneities in calmodulin mRNAs between normal and phosphorylase kinase-deficient skeletal muscles. The results demonstrate that in normal tissue there are four species of calmodulin mRNA distinguished by their molecular weight. All four of these species are present in the deficient tissue, and none of them are preferentially reduced. However, there is a 54% reduction in all four mRNAs as well as in calmodulin in the deficient skeletal muscle relative to normal skeletal muscle. These results indicate that the expression of calmodulin mRNAs is coordinated with the expression of its major enzyme target in skeletal muscle.