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.     

High and intermediate affinity calmodulin binding domains of the alpha and beta subunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha beta gamma delta)4, which contains calmodulin as the integral delta subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D.K. ((1989) J. Biol. Chem. 264, 17156-17163) have identified gamma 302-326 and gamma 342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta 770-794; two of intermediate affinity, beta 5-28 and beta 920-946; and one with marginally low affinity, alpha 1070-1093. Peptide beta 770-794 was of higher calmodulin affinity than either gamma 302-326 or gamma 342-366; it was of higher affinity than the model synthetic peptide IV defined by O'Neil, K.T., and DeGrado, W.F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca(2+)-dependent. The beta 770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta subunit.     

Ca2+-independent interaction of the gamma subunit of phosphorylase kinase with dansyl-calmodulin

A strong Ca2+-independent interaction between the isolated, active gamma subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active gamma subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive gamma subunit against 8 M urea, containing 0.1 mM DTT, 0.1 M Hepes at pH 6.8 or 0.1 M Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the gamma subunit to dansyl-CaM are 25.7 +/- 0.6 and 104 +/- 12 nM at pH 6.8 in the presence and absence of CaCl2. At pH 8.2, these values are 4.9 +/- 0.3 and 29 +/- 8 nM in the presence and absence of CaCl2. As the free Ca2+ decreases to as low as 10(-9) M, the fluorescence intensity and the fluorescence polarization of the gamma subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca2+ concentration. The presence of Mg2+ affects the interaction between dansyl-CaM and the gamma subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the gamma subunit and dansyl-CaM. Free ATP has little effect.     

The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

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.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

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.     

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.     

The effect of media pH and autocatalytic phosphorylation on the interaction of kinase phosphorylase with calmodulin

Using calmodulin covalently labeled with dansyl, the Ca2(+)-dependent interaction of phosphorylase kinase with calmodulin has been studied. It has been shown that at pH 6.8 the (alpha beta gamma delta) protomer of the enzyme binds 2.1 +/- 0.8 mol of calmodulin with Kd = (6.67 +/- 1.77).10(-8) M. The enzyme activation induced by the pH increase up to 8.2 does not affect the enzyme interaction with calmodulin [2.14 +/- 0.58 mol calmodulin per mol of (alpha beta gamma delta)]; Kd = (4.14 +/- 1.22).10(-8) M. However, the enzyme activation during its autocatalytic phosphorylation eliminates this effect practically completely.     

Properties of the gamma subunit of phosphorylase kinase

Enzymatic properties of the isolated, active gamma subunit of phosphorylase kinase were characterized. Kinetic parameters indicated that the gamma subunit binds the substrates MgATP and phosphorylase b as well as the holoenzyme with a Km (MgATP) of 98 microM and a Km (phosphorylase b) of 80 microM at pH 8.2, but maximal velocities are significantly lower than the holoenzyme's. Unlike the gamma-calmodulin complex, the gamma subunit activity is dependent on pH in the range of pH 6.2-9.0, with a ratio of activity at pH 6.8 to activity at pH 8.2 of 0.5-0.6. Calmodulin activates the gamma subunit more at low pH than at high pH. ADP inhibits the gamma subunit in a competitive manner with a Ki of 60 microM. Free Mg2+ stimulates gamma subunit activity 3.5-fold at both pH 6.8 and 8.2. MnATP is equivalent to MgATP as a substrate for the enzyme, but free Mn2+ inhibits gamma subunit activity. Several protein substrates of holophosphorylase kinase were found also to be phosphorylated by the gamma subunit. These included kappa-casein, myelin basic protein, the troponin complex, and troponin T alone. In the troponin complex, the proportion of 32P incorporated by the gamma subunit into troponin I compared with troponin T was not Ca2+ dependent, but with the holoenzyme, this proportion was changed greatly by Ca2+ concentration.     

Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme

Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the alpha-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the alpha-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the alpha-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the beta-subunit, and one came from the gamma-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the gamma-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).     

Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol

Two type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.     

Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene

When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.     

Phosphorylase kinase from chicken gizzard. Partial purification and characterization

Phosphorylase kinase was partially purified (530-970-fold) from chicken gizzard smooth muscle by a procedure involving ammonium sulfate fractionation, chromatography on 8-(6-aminohexyl)adenosine-5'-phosphate--Sepharose 4B and glycerol density gradient ultracentrifugation. The final and most efficient purification step takes advantage of the relatively high molecular mass of gizzard phosphorylase kinase, which was found to be similar to that of rabbit skeletal muscle enzyme. The gizzard kinase, further purified to near homogeneity by calmodulin-Sepharose 4 B affinity chromatography, showed one main protein band of 61 kDa, upon dodecyl sulfate acrylamide gel electrophoresis. Four minor protein bands of higher molecular mass were also present but no protein stain was seen at the position of the gamma subunit. The gizzard phosphorylase kinase showed a high pH 6.8/8.2 activity ratio of 0.53, it was stimulated by Ca2+, inhibited up to 80% by EGTA and it was activated about 1.9-fold by calmodulin. The km value for ATP was 0.45 mM, while the K0.5 for rabbit muscle phosphorylase b was extremely low, more than 200-fold lower than the Km of nonactivated skeletal muscle phosphorylase kinase for its protein substrate. High concentrations of phosphorylase b were found to be inhibitory. At 10 mg/ml phosphorylase b, the maximum activity of the kinase was inhibited fivefold. No evidence has been obtained indicating autophosphorylation or the existence of active and inactive forms of gizzard phosphorylase kinase. Limited proteolysis of the smooth muscle kinase with trypsin was accompanied by a twofold activation at pH 6.8.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A.

Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle.