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• W2085950174 abstract "The C terminus of the catalytic γ-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the γ·γ complex. Antibodies raised against the very C terminus of the γ-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the γ·γ complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the γ·γ complex 2.5-fold. Activation of the holoenzyme and the γ·γ complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the γ·γ complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the γ-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity. The C terminus of the catalytic γ-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the γ·γ complex. Antibodies raised against the very C terminus of the γ-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the γ·γ complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the γ·γ complex 2.5-fold. Activation of the holoenzyme and the γ·γ complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the γ·γ complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the γ-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity. Phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC) catalyzes the conversion of phosphorylase b to a and plays a key role in regulating glycogen breakdown in response to adrenergic and neuronal stimuli. The skeletal muscle isozyme is a multimeric enzyme with the subunit composition (αβγγ)4 and a molecular weight of 1.3 × 106 (reviewed in Ref. 1Pickett-Gies C.A. Walsh D.A. Boyer P.D. Krebs E.G. The Enzymes. Academic Press, Inc., Orlando, FL1986: 395Google Scholar). The α-, β-, and γ-subunits are regulatory subunits, whereas the γ-subunit is the catalytic subunit (2Reimann E.M. Titani K. Ericsson L.H. Wade R.D. Fischer E.H. Walsh K.A. Biochemistry. 1984; 23: 4185-4192Google Scholar). Interactions of the regulatory subunits with the γ-subunit serve to modulate the activity of the enzyme in response to the second messengers, cAMP and Ca2+. Phosphorylation of the α- and β-subunits by the cAMP-dependent protein kinase and by autophosphorylation activates the enzyme at pH 6.8. The unphosphorylated α- and β-subunits are thought to inhibit the catalytic site from exhibiting maximum catalytic potential, since inhibition can be relieved by increasing pH to 8.2, by phosphorylation, by proteolysis, or by dissociation of these subunits from the holoenzyme complex. Ca2+ dependence is conferred upon the enzyme by the γ-subunit, which is identical to calmodulin (3Cohen P. Burchell A. Foulkes J.G. Cohen P.T.W. Vanaman T.C. Nairn A. FEBS Lett. 1978; 92: 287-293Google Scholar). Residues 19-276 of the γ-subunit represent the catalytic domain of the enzyme based on sequence homology with other protein kinases (2Reimann E.M. Titani K. Ericsson L.H. Wade R.D. Fischer E.H. Walsh K.A. Biochemistry. 1984; 23: 4185-4192Google Scholar). Two crystal structures of a constitutively active catalytic core of the γ-subunit (residues 1-298) have recently been solved to a resolution of 3.0 Å or better (4Owen D.J. Noble M.E.M. Garman E.F. Papageorgiou A.C. Johnson L.N. Structure. 1995; 3: 467-482Google Scholar). Overall, these two γ-subunit structures are very similar to the catalytic cores of other protein kinases. The C-terminal 110 amino acids of the γ-subunit (277-386) are thought to contain pseudosubstrate/autoinhibitory domains and subunit interaction domains based on this region's lack of sequence similarity to other protein kinases (2Reimann E.M. Titani K. Ericsson L.H. Wade R.D. Fischer E.H. Walsh K.A. Biochemistry. 1984; 23: 4185-4192Google Scholar) and studies involving limited proteolysis (5Harris W.R. Malencik D.A. Johnson C.M. Carr S.A. Roberts G.D. Byles C.A. Anderson S.R. Heilmeyer Jr., L.M.G. Fischer E.H. Crabb J.W. J. Biol. Chem. 1990; 265: 11740-11745Google Scholar) and site-directed mutagenesis (6Huang C.-Y.F. Yuan C.-J. Blumenthal D.K. Graves D.J. J. Biol. Chem. 1995; 270: 7183-7188Google Scholar, 7Huang C.-Y.F. Yuan C.-J. Livanova N.B. Graves D.J. Mol. Cell. Biochem. 1993; 127/128: 7-18Google Scholar, 8Cox S. Johnson L.N. Protein Eng. 1992; 5: 811-819Google Scholar). By using a library of overlapping synthetic γ-subunit peptides, the regions corresponding to γ302-326 and γ342-366 have been identified as being regulatory subdomains that act in concert to mediate interactions between the γ-subunit and the catalytic domain of the γ-subunit (6Huang C.-Y.F. Yuan C.-J. Blumenthal D.K. Graves D.J. J. Biol. Chem. 1995; 270: 7183-7188Google Scholar, 9Dasgupta M. Honeycutt T. Blumenthal D.K. J. Biol. Chem. 1989; 264: 17156-17163Google Scholar, 10Dasgupta M. Blumenthal D.K. J. Biol. Chem. 1995; 270: 22283-22289Google Scholar). Peptides corresponding to these two regions (termed PhK13 and PhK5, respectively) bind calmodulin with high affinity (9Dasgupta M. Honeycutt T. Blumenthal D.K. J. Biol. Chem. 1989; 264: 17156-17163Google Scholar) and competitively inhibit phosphorylase kinase catalytic activity (6Huang C.-Y.F. Yuan C.-J. Blumenthal D.K. Graves D.J. J. Biol. Chem. 1995; 270: 7183-7188Google Scholar, 10Dasgupta M. Blumenthal D.K. J. Biol. Chem. 1995; 270: 22283-22289Google Scholar). Site-directed mutagenesis experiments (6Huang C.-Y.F. Yuan C.-J. Blumenthal D.K. Graves D.J. J. Biol. Chem. 1995; 270: 7183-7188Google Scholar) and small-angle scattering studies (11Trewhella J. Blumenthal D.K. Rokop S.E. Seeger P.A. Biochemistry. 1990; 29: 9316-9324Google Scholar) have provided limited structural information regarding the interactions of PhK5 and PhK13 with the γ-subunit catalytic domain (6Huang C.-Y.F. Yuan C.-J. Blumenthal D.K. Graves D.J. J. Biol. Chem. 1995; 270: 7183-7188Google Scholar) and the γ-subunit (11Trewhella J. Blumenthal D.K. Rokop S.E. Seeger P.A. Biochemistry. 1990; 29: 9316-9324Google Scholar). However, detailed information regarding the structure of the γ-subunit regulatory domain is not presently available. It has also been proposed that the regulatory domain of the γ-subunit might interact directly with the α- and β-subunits (2Reimann E.M. Titani K. Ericsson L.H. Wade R.D. Fischer E.H. Walsh K.A. Biochemistry. 1984; 23: 4185-4192Google Scholar), but the sites of interaction on the γ-subunit for the α- and β-subunits have yet to be firmly established. The present investigation was undertaken to better define the potential regulatory functions of specific regions within the regulatory domain of the γ-subunit of phosphorylase kinase. Antibodies were raised against each of four peptides, PhK1 (γ362-386), PhK5 (γ342-366), PhK9 (γ322-346), and PhK13 (γ302-326), that together span the C-terminal 85 amino acids of the γ-subunit (Fig. 1). These monospecific antibodies were then affinity-purified and assayed for their ability to activate or inhibit the catalytic activity of several different forms of phosphorylase kinase. The approach of using antipeptide antibodies as probes to assess the functional properties of putative regulatory domains within protein kinases has previously been used to characterize the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (12Nunnally M.H. Blumenthal D.K. Krebs E.G. Stull J.T. Biochemistry. 1987; 26: 5885-5890Google Scholar), the pseudosubstrate domain of protein kinase C (13Makowske M. Rosen O.M. J. Biol. Chem. 1989; 264: 16155-16159Google Scholar), and potential regulatory domains in casein kinase II (14Charlton L.A. Sanghera J.S. Clark-Lewis I. Pelech S.L. J. Biol. Chem. 1992; 267: 8840-8845Google Scholar), the insulin-like growth factor-I receptor kinase (15Herrera R. Petruzzelli L.M. Rosen O.M. J. Biol. Chem. 1986; 261: 2489-2491Google Scholar, 16Kaliman P. Baron V. Gautier N. Van Obberghen E. J. Biol. Chem. 1992; 267: 10645-10651Google Scholar), and rhodopsin kinase (17Palczewski K. Buczyłko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Google Scholar). The results of the studies presented here indicate that binding of monospecific antibodies to the regulatory domain of the γ-subunit can have profound effects on catalytic activity and provide important insights into structural features involved in regulatory interactions in the phosphorylase kinase holoenzyme complex. Phosphorylase kinase holoenzyme was prepared by the procedure of Cohen (18Cohen P. Eur. J. Biochem. 1973; 34: 1-14Google Scholar), and phosphorylase b was prepared using the procedure of Fischer and Krebs (19Fischer E.H. Krebs E.G. Methods Enzymol. 1962; 55: 369-373Google Scholar). Protein concentrations were determined spectrophotometrically using values of E1%280 nm of 12.4 (18Cohen P. Eur. J. Biochem. 1973; 34: 1-14Google Scholar) and 13.2 (20Kastenschmidt L.L. Kastenschmidt J. Helmreich E. Biochemistry. 1968; 17: 3590-3608Google Scholar) for phosphorylase kinase and phosphorylase b, respectively. The γ-subunit of phosphorylase kinase was purified using reversed-phase HPLC 1The abbreviations used are: HPLChigh performance liquid chromatographyMOPS4-morpholinepropanesulfonic acidELISAenzyme-linked immunosorbent assay. as described by Crabb and Heilmeyer (21Crabb J.W. Heilmeyer Jr., L.M.G. J. Biol. Chem. 1984; 259: 6346-6350Google Scholar), except that a Vydac C-4 analytical column (5 mm, 0.46 × 25 cm) was used instead of a Vydac C-18 column. The subunit elution pattern obtained using the C-4 column was similar to that reported for the C-18 column. The active γ·γ complex was prepared from HPLC-purified γ-subunit using the reactivation procedure described by Kee and Graves (22Kee S.M. Graves D.J. J. Biol. Chem. 1986; 261: 4732-4737Google Scholar). The reactivation buffer contained 50 m Tris, 50 m β-glycerophosphate, pH 8.0, 2 m dithiothreitol, 0.1 m CaCl2, 3 m calmodulin, 1 mg/ml bovine serum albumin, and HPLC-purified γ-subunit (diluted 10-fold into the reactivation buffer). Reactivation was carried out at 0°C for 18 h. high performance liquid chromatography 4-morpholinepropanesulfonic acid enzyme-linked immunosorbent assay. Synthesis of peptides was done by standard solid-phase techniques using t-butoxycarbonyl chemistry as described previously (9Dasgupta M. Honeycutt T. Blumenthal D.K. J. Biol. Chem. 1989; 264: 17156-17163Google Scholar). Peptide purification was performed using reversed-phase HPLC, and each sequence was confirmed by amino acid analysis and protein sequence analysis. Monospecific polyclonal antibodies were raised in rabbits against the synthetic peptides PhK1 (γ362-386), PhK5 (γ342-366), PhK9 (γ322-346), and PhK13 (γ302-326), which span the C-terminal 85 residues of the γ-subunit (see Fig. 1). In the case of PhK1, PhK5, and PhK9, the peptides were coupled to keyhole limpet hemocyanin using glutaraldehyde, and then each peptide was used to immunize two rabbits using the Ribi Adjuvant System (Hamilton, MT). Booster injections were given on days 14 and 28 and every month thereafter. Rabbits were bled each week (3 times a month) between immunizations. In the case of PhK13, the peptide was coupled to Imject maleimide-activated keyhole limpet hemocyanin (Pierce) and used to immunize two rabbits. These rabbits were immunized and bled by Bethyl Laboratories (Montgomery, TX) according to their recommended schedule. Initial screening using an enzyme-linked immunosorbent assay (ELISA) was performed to select the rabbits giving the best responses and the bleeds with the highest titer against phosphorylase kinase holoenzyme. These antisera were used for the subsequent workup described below. Each antiserum was subjected to the following prepurification procedure to remove contaminating lipids and lipoproteins. To each milliliter of antiserum, 0.05 ml 5% (w:v) sodium dextran sulfate (Pharmacia Biotech Inc.) was added, stirred, then allowed to sit on ice for 1 h. To this solution was added 0.09 ml of 11.1% (w/v) CaCl2/ml of antiserum. The solution was allowed to settle on ice for 1-2 h, and the precipitate was removed by centrifugation at 5000 × g for 20 min. To further purify the antipeptide antibodies, the supernatant was subjected to ammonium sulfate fractionation by slowly adding, while stirring, 0.667 ml of saturated ammonium sulfate (pH 7.3)/ml of antibody solution. The solution was allowed to stir for 1 h on ice and then was centrifuged at 5000 × g for 25 min. The precipitated pellet was resuspended in a minimal volume of Milli-Q water and then dialyzed overnight against Milli-Q water at 4°C. The dialysate was then subjected to affinity purification using phosphorylase kinase immobilized to Sepharose 4B. Phosphorylase kinase holoenzyme was immobilized on CNBr-activated Sepharose 4B at a density of 5 mg of protein/ml of gel to produce affinity columns for the isolation of each of the antipeptide antibodies. Dry CNBr-activated Sepharose (1.14 g; Sigma) was suspended in 1 m HCl to dissolve stabilizers and hydrate the gel. After about 1 min, the suspension was filtered, and to the moist gel was added 20 mg of phosphorylase kinase dissolved in 0.1 borate, 0.5 NaCl, pH 8.3. The gel and phosphorylase kinase were gently shaken for 24 h at 4°C, after which the suspension was mixed with 1 ethanolamine and shaken at room temperature for another 2 h to block unreacted sites on the gel. The gel was washed with 0.1 borate, 0.5 NaCl, pH 8.3, followed by 0.1 acetic acid, 0.5 NaCl, pH 4.0, and was then aliquoted into four 1-ml columns. The columns were stored in 50 m MOPS, 2 m EDTA, pH 7.0, at 4°C. Before use, the columns used for purification of anti-PhK1 and anti-PhK5 were equilibrated with buffer containing 50 m MOPS, pH 7.0, 1 m dithiothreitol, and 1 µ leupeptin. The columns used for anti-PhK9 and anti-PhK13 were preequilibrated with the above buffer containing 200 µ CaCl2. Antipeptide antibodies were applied to the columns in the presence (anti-PhK9 and anti-PhK13) or absence (anti-PhK1 and anti-PhK5) of 200 µ Ca2+. The columns were then washed with equilibration buffer until the A280 of the column effluent was less than 0.05. The anti-PhK1 and anti-PhK5 antibodies were eluted with 500 m MgCl2, and the anti-PhK9 and anti-PhK13 antibodies were eluted with 50 m MOPS, pH 7.0, 1 m dithiothreitol, 1 µ leupeptin, and 2 m EDTA. Eluates were desalted using Bio-Gel P6 DG (Bio-Rad) desalting columns and stored in phosphate-buffered saline at 4°C. Antibody concentrations were determined spectrophotometrically using a E1%280 nm value of 15 for IgG (23Fasman G.D. Practical Handbook of Biochemistry and Molecular Biology. CRC Press Inc., Boca Raton, FL1989Google Scholar). Fab fragments were prepared from affinity-purified anti-PhK1 antibody according to the procedure of Gibson et al. (24Gibson A.L. Herron J.N. He X.-M. Patrick V.A. Mason M.L. Lin J.-N. Kranz D.M. Voss E.W.J. Edmundson A.B. Proteins Struct. Funct. Genet. 1988; 3: 155-160Google Scholar) as briefly described here. A sample of affinity-purified antibody (0.5 mg) was dialyzed overnight at pH 7.5 against 50 m Tris-HCl, 0.15 NaCl, and 2 m EDTA. After dialysis, dithioerythritol was added to a final concentration of 1 m. A suspension of mercuripapain (Sigma) was prepared in the above buffer in the presence of 10 m dithioerythritol. The solutions of protease and antibody were mixed in a ratio of 1:33 (w:w) and incubated at 37°C for 30 min. The reaction was stopped by the addition of dehydroascorbic acid to a final concentration of 1 m, and the solution was allowed to sit for 15 min. The solution was filtered (0.22-µm filter, Pharmacia), and the Fab fragments were separated by fast protein liquid chromatography using a Superdex 200 prep grade (HiLoad 16/60) column. A sandwich ELISA method was used to determine the ability of anti-PhK1 Fab fragments to bind phosphorylase kinase and the γ·γ complex. The procedure used the reagents and protocol provided in the ELISAmate kit (Kirkegaard and Perry Laboratories). Polystyrene microtiter plates (96-well flat-bottom, Corning) were first coated with the indicated concentrations of anti-PhK1 or anti-PhK1 Fab fragment for 1 h at room temperature. Either phosphorylase kinase holoenzyme (0.9 µg/ml) or γ·γ complex in 1% (w/w) bovine serum albumin was then added and allowed to bind for 1 h before the plate was emptied and rinsed with 1% bovine serum albumin. Antibody raised against a synthetic multiple antigen peptide corresponding to the N terminus of the γ-subunit, GKSHSGPLAADRT, was then added (0.7 µg/ml in 1% bovine serum albumin) and allowed to bind for 1 h. The plate was then emptied and washed three times with 0.02% Tween 20 (v/v). Peroxidase-labeled goat anti-rabbit IgG (0.3 µg/ml in 1% bovine serum albumin) was added and incubated for 1 h. The plate was then washed three times (0.02% Tween 20) before adding the color development reagents, 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. The plate was read in kinetic mode on a UVmax microplate reader (Molecular Devices) at 650 nm using SOFTmax (version 2.01) for data acquisition and analysis. A radioactive protein kinase assay was used to monitor antipeptide antibody interactions with phosphorylase kinase holoenzyme and the γ·γ complex. Enzymatic activities of the holoenzyme and the γ·γ complex were determined by measuring the rate of 32P incorporation from [γ-32P]ATP into phosphorylase b or a synthetic peptide substrate (SDQEKRKQISVRGLG). The reaction mixture (50 µl) contained 50 m HEPES, 42 m Tris, 1 m dithiothreitol, pH 8.2 or 6.8 (as indicated), 10 m magnesium acetate, 200 µ CaCl2, 25 µ phosphorylase b or 250 µ peptide substrate (as indicated), 100 ng of phosphorylase kinase, and 1 m [γ-32P]ATP (300 cpm/pmol; DuPont NEN). The reaction mixtures were preincubated for 5 min at 30°C before the reactions were started by the addition of ATP. Aliquots of 10 or 20 µl of each reaction were removed after 5 and 15 min and spotted on Whatman 3MM or P81 filter paper squares. Whatman 3MM paper squares were used for the protein substrate, and P81 filter paper squares were used for the synthetic peptide substrate. The paper squares were immediately placed in 10% trichloroacetic acid, 4% sodium pyrophosphate (3MM paper) or 75 m H3PO4 (P81 paper). The papers were washed at least three times, rinsed in 95% ethanol, dried, and counted in a scintillation counter following the addition of Opti-fluor (Packard Instrument Co.) scintillation mixture. Assays containing antibodies or Fab fragments were performed as described above except that the enzyme was preincubated on ice (in a volume of 10 µl) with the indicated concentrations of antibody or Fab fragment for the indicated times before addition to the protein kinase reaction mixture. Phosphorylase kinase (2 mg/ml) was activated by incubation with 0.02 milliunits of cAMP-dependent protein kinase catalytic subunit (Promega; units of activity are as defined by Promega) and 1 m ATP for 30 min in a volume of 100 µl in the presence of the pH 6.8 kinase assay buffer described above. The protein kinase-activated phosphorylase kinase was then preincubated with antibody and assayed as described above. Error bars associated with each data set represent standard errors of the mean calculated from duplicate or triplicate assay tubes, each of which was sampled at two time points. In some cases, the data represent means and standard errors from more than one experiment. Nonlinear least squares curve-fitting was performed using MacCurveFit (version 1.03) to estimate Km and Vmax values and to determine partial inhibition constants. The ability of each affinity-purified antipeptide antibody to alter the enzymatic activity of phosphorylase kinase holoenzyme (Fig. 2) and γ·γ complex (Fig. 3) at pH 8.2 was examined. Two of the antibodies, anti-PhK1 and anti-PhK5, induced activation of both phosphorylase kinase and the γ·γ complex in the presence and absence of Ca2+. In the presence of Ca2+, anti-PhK1 antibody increased holoenzyme activity by approximately 1150% and the γ·γ complex activity by 250%. The degree of holoenzyme activation induced by anti-PhK5 antibody was approximately 70% of that seen with anti-PhK1 antibody and somewhat less (50%) for the γ·γ complex. The extent of holoenzyme and γ·γ complex activation induced by anti-PhK1 in the presence of 3 m EGTA was greater than that seen in the presence of Ca2+ with no antibody present. The extent of stimulation of holoenzyme and γ·γ complex activity by anti-PhK5 in the presence of 3 m EGTA was nearly equal to the Ca2+-stimulated activity in the absence of antibody. The concentration of anti-PhK1 required for activation was comparable for the holoenzyme and the γ·γ complex, in the presence or absence of Ca2+, with maximal activation occurring at an antibody concentration of 0.3 mg/ml (data not shown).Fig. 3Effect of affinity-purified antipeptide antibodies on γ·γ complex activity in the presence of Ca2+ or EGTA. The γ·γ complex was incubated in the presence of antipeptide antibodies and assayed at pH 8.2 as described under “Experimental Procedures.” The horizontal dashed line represents the Ca2+-stimulated activity of the γ·γ complex in the absence of antibody. Error bars represent the standard error of the mean.View Large Image Figure ViewerDownload (PPT) To determine whether the activation of phosphorylase kinase by anti-PhK1 required antibody with intact Fc regions, Fab fragments of anti-PhK1 were tested for their ability to activate the holoenzyme and the γ·γ complex. Other antipeptide antibodies were not investigated due to difficulties in purifying these other antibodies in sufficient quantities to conduct such studies. The anti-PhK1 Fab fragments were unable to activate either the holoenzyme or the γ·γ complex (Fig. 4). A sandwich ELISA was used to determine whether the inability of the Fab fragments to activate was due to the loss of binding capability. It was found that the anti-PhK1 Fab fragments were still able to bind holoenzyme and the γ·γ complex as tightly as the intact antibody (Fig. 5).Fig. 5Binding of anti-PhK1 antibody (filled symbols) and anti-PhK1 Fab fragments (open symbols) to phosphorylase kinase holoenzyme and the γ·γ complex as determined by sandwich ELISA. Data obtained using anti-PhK1 antibody are represented by filled symbols, and anti-PhK1 Fab data are represented by open symbols. Data obtained using holoenzyme are depicted by square symbols, while those obtained using the γ·γ complex are represented by circles. Details of the sandwich ELISA are described under “Experimental Procedures.” Each point represents the mean of three independent assays (each with quadruplicate wells) ± S.D.View Large Image Figure ViewerDownload (PPT) The time dependence of antibody activation was assessed by measuring holoenzyme and γ·γ complex activity after overnight and 1-h incubations. Antibody-induced activation by anti-PhK5 occurred in a time-dependent manner with holoenzyme but not with the γ·γ complex (data not shown). Anti-PhK1 exhibited no difference between the overnight and 1-h incubations in the extent of activation of either holoenzyme or the γ·γ complex. To examine the possible mechanisms for antibody-induced activation, kinetic analyses were performed with anti-PhK1 and anti-PhK5 antibody using both the holoenzyme and the γ·γ complex in the presence and absence of Ca2+ at pH 6.8. The Km and Vmax values determined from these analyses are shown in Table I. In the presence of Ca2+, the effects of both anti-PhK1 and anti-PhK5 on holoenzyme activity were primarily attributable to changes in Vmax, with only modest or minimal effects on Km values for ATP or phosphorylase b. Because of the intrinsic difficulty in accurately determining Km values, it is difficult to know whether the 2-fold decrease in Km value for ATP seen with anti-PhK1 and the comparable effect of anti-PhK5 on the Km value for phosphorylase b are due to normal assay variability or whether these lower Km values reflect real but modest effects of these antibodies on enzyme-substrate interactions. The effects of anti-PhK1 and anti-PhK5 on holoenzyme Vmax values were much more robust, with anti-PhK1 increasing Vmax values 10-13-fold and anti-PhK5 increasing Vmax values 7-9-fold.TABLE ISummary of kinetic data for antibody activation and inhibition of holoenzyme and γ·γ complex activityPhosphorylase bATPKmVmaxKmVmaxµmµmol/min/mgµmµmol/min/mgPhosphorylase kinase holoenzyme in the presence of Ca2+ (1 m)Control64.0 ± 3.16 (53.2)2.23 ± 0.05 (2.01)0.25 ± 0.03 (0.33)1.37 ± 0.06 (1.55)Anti-PhK155.8 ± 8.11 (55.9)28.8 ± 1.96 (28.9)0.13 ± 0.01 (0.21)13.9 ± 0.04 (15.7)Anti-PhK532.7 ± 7.51 (38.6)14.8 ± 1.26 (15.9)0.26 ± 0.01 (0.25)12.7 ± 0.24 (12.6)Anti-PhK1365.9 ± 13.8 (53.3)1.34 ± 0.14 (1.20)0.31 ± 0.05 (0.48)0.79 ± 0.05 (0.96)Phosphorylase kinase holoenzyme in the absence of Ca2+ (3 m EGTA)Control69.0 ± 11.2 (49.7)0.49 ± 0.04 (0.61)0.19 ± 0.06 (0.17)0.39 ± 0.04 (0.38)Anti-PhK117.5 ± 10.1 (23.1)4.56 ± 0.77 (5.04)0.45 ± 0.05 (0.73)8.79 ± 0.46 (11.4)Anti-PhK528.9 ± 10.2 (24.2)1.57 ± 0.20 (1.50)0.42 ± 0.06 (0.50)2.70 ± 0.15 (2.94)γ·γ complex in the presence of Ca2+ (1 m)Control47.9 ± 9.57 (31.5)1.76 ± 0.15 (1.46)0.13 ± 0.02 (0.15)1.02 ± 0.03 (1.06)Anti-PhK112.5 ± 3.00 (17.0)3.81 ± 0.22 (4.13)0.10 ± 0.01 (0.09)3.00 ± 0.06 (2.94)Anti-PhK531.2 ± 11.2 (38.5)2.94 ± 0.38 (3.17)0.27 ± 0.05 (0.27)1.71 ± 0.11 (1.72)γ·γ complex in the absence of Ca2+ (3 m EGTA)Control59.5 ± 9.37 (72.4)0.41 ± 0.03 (0.45)0.21 ± 0.04 (0.27)0.37 ± 0.02 (0.41)Anti-PhK116.7 ± 5.25 (26.2)1.43 ± 0.12 (1.65)0.14 ± 0.01 (0.15)1.49 ± 0.03 (1.52)Anti-PhK585.2 ± 26.9 (96.9)2.06 ± 0.36 (2.16)0.11 ± 0.02 (0.12)0.75 ± 0.03 (0.78) Open table in a new tab Slightly different kinetic patterns were observed for holoenzyme activation when the Ca2+ concentration was lowered using EGTA, although the effect of antibody was still predominantly a Vmax effect. Assays of the holoenzyme with varying concentrations of phosphorylase b in the presence of 3 m EGTA resulted in a 9.3-fold increase in Vmax with anti-PhK1 antibody but an increase of only 3.2-fold with anti-PhK5 antibody (Table I). There were also 4-fold and 2.4-fold decreases in the Km value for phosphorylase b with anti-PhK1 antibody and anti-PhK5 antibody, respectively. Kinetic analyses using ATP as the varied substrate indicated there were no significant differences in Km values for ATP, but there were 22.5- and 7-fold increases in Vmax values with anti-PhK1 and anti-PhK5 antibodies, respectively. These observations indicate that in the absence of Ca2+, anti-PhK1 and anti-PhK5 both activate the holoenzyme through a mixed type mechanism, which is mostly due to an effect on Vmax, but with some rate-enhancing effects on the Km for phosphorylase b and no effect on the Km for ATP. The kinetic patterns observed for activation of the γ·γ complex by anti-PhK1 and anti-PhK5 were similar overall to those seen with the holoenzyme, with activation being predominantly a Vmax effect (Table I). Differences between holoenzyme activation and γ·γ complex activation were primarily seen in the extent of activation and the somewhat more complex pattern of γ·γ complex activation observed with anti-PhK1 in the presence of Ca2+. A 4-fold decrease in the Km value for phosphorylase b and a 2-fold increase in the Vmax was seen when the γ·γ complex was assayed with anti-PhK1 antibody in the presence of Ca2+, indicating a mixed type activation, which differs from the pure Vmax effect seen with the holoenzyme. A 1.7-fold increase in Vmax was seen with the anti-PhK5 antibody, with no significant change in Km for phosphorylase b. With regard to ATP, there was no significant change in Km value observed with anti-PhK1 and a 2-fold increase with anti-PhK5, whereas the Vmax value increased 3- and 1.7-fold with anti-PhK1 and anti-PhK5, respectively. The effects of anti-PhK1 and anti-PhK5 on the Km and Vmax values of the γ·γ complex in the presence of EGTA were qualitatively the same as those seen in the presence of Ca2+, with Vmax effects always being observed and Km effects being seen to a variable degree. Phosphorylase kinase is activated by cAMP-dependent protein kinase-catalyzed phosphorylation of its α- and" @default.
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• W2085950174 title "Activation and Inhibition of Phosphorylase Kinase by Monospecific Antibodies Raised against Peptides from the Regulatory Domain of the γ-Subunit" @default.
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