Preparation, characterization, and properties of a novel triple-modified derivative of the Ca2+-dependent protein modulator

1978 ◽  
Vol 56 (6) ◽  
pp. 420-429 ◽  
Author(s):  
Michael Walsh ◽  
Frits C. Stevens
Keyword(s):  
Troponin I ◽  
Side Reaction ◽  
Troponin C ◽  
Stable Complex ◽  
Native Protein ◽  
Chemically Modified ◽  
Dependent Change ◽  
Arginine Residues ◽  
Dependent Protein ◽  
A Minor

A novel, chemically modified derivative of the Ca2+-dependent protein modulator of cyclic nucleotide phosphodiesterase was prepared by the sequential treatment of the native protein with tetranitromethane, 1,2-cyclohexanedione, and diethylpyrocarbonate. Three derivatives were isolated during the synthesis, i.e., a single-modified derivative containing two modified tyrosine residues per mole; a double-modified derivative containing two modified tyrosines plus five modified arginine residues per mole; and a triple-modified derivative containing two modified tyrosines, five modified arginines, and one modified histidine residue per mole. Intermolecular cross-linking was observed to occur as a minor side reaction during nitration of the native protein with tetranitromethane. All the derivatives were examined for phosphodiesterase-stimulating activity and troponin C like activities. All derivatives were found to retain the capacity of the native protein to stimulate modulator-deficient phosphodiesterase; furthermore, in each case, the stimulation of phosphodiesterase was a Ca2+-dependent process. On the other hand, both the double- and triple-modified modulators lost the troponin C like activities, i.e., the Ca2+-dependent change in mobility in urea–polyacrylamide gels and the ability to interact with troponin I in the presence of Ca2+ to form a urea-stable complex, while the nitrotyrosyl modulator retained these properties of the native protein.

Circular dichroism studies of native and chemically modified Ca2+-dependent protein modulator

1979 ◽  
Vol 57 (3) ◽  
pp. 267-278 ◽  
Author(s):  
Michael Walsh ◽  
Frits C. Stevens ◽  
Kimio Oikawa ◽  
Cyril M. Kay
Keyword(s):  
Circular Dichroism ◽  
Conformational Change ◽  
Troponin I ◽  
Benzyl Bromide ◽  
Structural Features ◽  
Native Protein ◽  
Chemically Modified ◽  
Helical Content ◽  
Dependent Protein ◽  
Circular Dichroïsm

The structural features of the native Ca2+-dependent protein modulator and two chemically modified derivatives, namely, nitrotyrosyl modulator and alkylated modulator, were examined by circular dichroism. The binding of Ca2+ to the native molecule was accompanied by an increase in helical content from 40 to 49%, with little effect on the local environments of aromatic residues in the modulator. The Mg2+ and Mn2+ do not elicit the conformational change induced by the binding of Ca2+, which also stabilizes the modulator against urea denaturation.The overall secondary structure of nitrotyrosyl modulator is indistinguishable from that of the native protein and undergoes a similar conformational change upon binding Ca2+. These observations are in agreement with the fact that nitration has no effect on modulator functions. Futhermore, nitrotyrosyl modulator interacts with troponin I only in the presence of Ca2+, as detected by circular dichroism (cd). On the other hand, alkylation of five methionine residues on the modulator with benzyl bromide affects protein conformation, as evidenced by a reduced helical content of only 34%. Alkylated modulator retains the ability of the native protein to bind Ca2+ although the affinity of this derivative for Ca2+ is reduced some three orders of magnitude relative to the native protein, with Kd = 3.2 × 10−4 M.The results with the alkylated modulator, in conjunction with previous cd studies on N-chlorosuccinimide oxidized modulator are utilized to advance a model for the Ca2+ activation of modulator protein, based on three conformational states of the molecule.

scholarly journals Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor.

1982 ◽  
Vol 93 (3) ◽  
pp. 719-726 ◽  
Author(s):  
W H Fletcher ◽  
C V Byus
Keyword(s):  
Protein Kinase ◽  
Dose Range ◽  
Hepatoma Cells ◽  
Affinity Column ◽  
Dependent Protein Kinase ◽  
Cytochemical Localization ◽  
Camp Dependent Protein Kinase ◽  
Arginine Residues ◽  
Dependent Protein ◽  
A Minor

A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP-dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP-dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones.

scholarly journals Phosphorylation of troponin and the effects of interactions between the components of the complex

1974 ◽  
Vol 141 (3) ◽  
pp. 733-743 ◽  
Author(s):  
Samuel V. Perry ◽  
Heather A. Cole
Keyword(s):  
Protein Kinase ◽  
Troponin I ◽  
Troponin T ◽  
Troponin C ◽  
Phosphorylase Kinase ◽  
Significant Extent ◽  
Dependent Protein Kinase ◽  
Prolonged Incubation ◽  
Troponin Complex ◽  
Dependent Protein

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3′:5′-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca2+sensitivity on the adenosine triphosphatase of densensitized actomyosin.

scholarly journals Phosphorylation of skeletal-muscle troponin I and troponin T by phospholipid-sensitive Ca2+-dependent protein kinase and its inhibition by troponin C and tropomyosin

1984 ◽  
Vol 218 (2) ◽  
pp. 361-369 ◽  
Author(s):  
G J Mazzei ◽  
J F Kuo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin T ◽  
Troponin C ◽  
Cardiac Troponin I ◽  
Equimolar Amount ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Skeletal-muscle troponin I and troponin T were found to be rapidly phosphorylated by cardiac phospholipid-sensitive Ca2+-dependent protein kinase, with Km values of 6.66 and 0.13 microM respectively. Stoichiometric phosphorylation of skeletal troponin I (endogenous phosphate content 0.7 mol/mol) indicated that the Ca2+-dependent enzyme and cyclic AMP-dependent protein kinase incorporated 0.9 and 0.8 mol/mol respectively. The same experiments with skeletal troponin T (endogenous phosphate content 1.9 mol/mol) revealed a maximal phosphorylation of 2 mol/mol by the Ca2+-dependent enzyme, whereas the cyclic AMP-dependent enzyme was unable to phosphorylate troponin T. The Ca2+-dependent enzyme phosphorylated both serine and threonine residues in skeletal and cardiac troponin I or troponin T; the cyclic AMP-dependent enzyme, in comparison, phosphorylated only serine in skeletal and cardiac troponin I. Although an equimolar amount of skeletal or cardiac troponin C markedly inhibited (80-90%) phosphorylation of skeletal and cardiac troponin I by the Ca2+-dependent enzyme, these troponin C preparations inhibited only phosphorylation of skeletal troponin I, but not that of cardiac troponin I, by the cyclic AMP-dependent enzyme. Calmodulin and Ca2+-binding protein S-100a could mimic the inhibitory effect of troponin C. A tissue specificity appeared to exist for the skeletal troponin T-skeletal troponin C interaction. Inhibition of troponin T phosphorylation by an equimolar amount of troponin C was lower than that of troponin I phosphorylation; these findings might explain in part why troponin T was the major substrate for the Ca2+-dependent enzyme in the troponin complex. The present studies indicate that skeletal and cardiac troponin I and troponin T were effective substrates for phospholipid-sensitive Ca2+-dependent protein kinase, suggesting a potential involvement of this Ca2+-effector enzyme in the regulation of myofibrillar activity.

scholarly journals Proton-magnetic-resonance studies on the interaction of rabbit skeletal-muscle troponin I with troponin C and actin

1982 ◽  
Vol 203 (1) ◽  
pp. 61-68 ◽  
Author(s):  
R J A Grand ◽  
B A Levine ◽  
S V Perry
Keyword(s):  
Skeletal Muscle ◽  
Troponin I ◽  
Specific Interaction ◽  
Troponin C ◽  
Terminal Region ◽  
Side Chains ◽  
Solution Structures ◽  
Arginine Residues ◽  
Cnbr Cleavage ◽  
Fast Twitch

1. The p.m.r. spectra of the larger CNBr-cleavage peptides of troponin I from rabbit fast-twitch skeletal muscle corresponded largely to those of fairly flexible solution structures. 2. On addition of troponin C to each of the CNBr-cleavage peptides in turn, perturbations of side chains were noted only for peptides CN5 (residues 1-21) and CN4 (residues 96-116). 3. In the presence of Ca2+, troponin C induced perturbations of the side chains of threonine-11, alanine, isoleucine and arginine residues of peptide CN5. 4. In the presence of Ca2+, troponin C induced perturbations of the side chains of phenylalanine, lysine and leucine residues of peptide CN4. 5. Irrespective of the presence or absence of Ca2+, specific interaction with actin was observed only with peptide CN4. In this case the side chains of arginine residues were perturbed. 6. It is concluded that actin interacts with the C-terminal region of peptide CN4, whereas troponin C interacts with the N-terminal region of peptide CN4 and with peptide CN5.

scholarly journals Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin

1981 ◽  
Vol 195 (1) ◽  
pp. 307-316 ◽  
Author(s):  
C M Wall ◽  
R J A Grand ◽  
S V Perry
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Troponin I ◽  
Biological Activities ◽  
Troponin C ◽  
Dependent Protein Kinase ◽  
Electrophoretic Mobilities ◽  
Dependent Protein

1. Troponin C and calmodulin were not digested by thrombin at a significant rate in the presence of Ca2+. 2. In the presence of EGTA, troponin C was digested by thrombin to yield three peptides, TH1 (residues 1--120), TH3 (residues 1--100) and TH2 (residues 121--159). 3. In the presence of EGTA calmodulin was digested by thrombin giving two peptides, TM1 (residues 1--106) and TM2 (residues 107--148). 4. The electrophoretic mobilities of peptides TH1 and TM1 were increased at pH 8.6 by Ca2+ both in the presence and absence of urea. The mobilities of peptides TH2 and TM2 were unaltered under these conditions. 5. Peptides TH1, TH2 and tM1 formed complexes with troponin I on polyacrylamide gels at pH 8.6 in the presence of Ca2+. 6. The phosphorylation of troponin I by cyclic AMP-dependent protein kinase was significantly inhibited by peptides TH1 and TH3 and to a lesser extent by peptide TM1. 7. The calmodulin peptide TM1 activated myosin light-chain kinase when present in large molar excess. Peptide TM2 did not activate the enzyme.

scholarly journals The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase

1977 ◽  
Vol 161 (2) ◽  
pp. 371-382 ◽  
Author(s):  
A J G Moir ◽  
H A Cole ◽  
S V Perry
Keyword(s):  
Troponin T ◽  
Troponin C ◽  
Amino Acid Sequences ◽  
The Other ◽  
Phosphorylase Kinase ◽  
Phosphorylation Sites ◽  
Native Protein ◽  
Dependent Protein Kinase ◽  
Troponin Complex ◽  
Dependent Protein

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3′:5′-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx, SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser, SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.

scholarly journals Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I

1978 ◽  
Vol 173 (2) ◽  
pp. 449-457 ◽  
Author(s):  
R A Weeks ◽  
S V Perry
Keyword(s):  
Skeletal Muscle ◽  
Troponin I ◽  
Gel Filtration ◽  
Troponin C ◽  
Fast Skeletal Muscle ◽  
Dependent Protein Kinase ◽  
Mechanism Of Interaction ◽  
Dependent Protein ◽  
Dependent Interaction

1. The CNBr digest of troponin C from rabbit fast skeletal muscle was shown to possess many of the functional properties of the whole troponin C molecule. 2. A peptide corresponding to residues 83-134 was isolated, which forms a Ca(2+-dependent complex with troponin I and neutralizes the inhibition by troponin I of the Mg(2+-stimulated adenosine triphosphatase of desensitized actomyosin. 3. The peptide inhibits the phosphorylation of fast-skeletal-muscle, but not cardiac-muscle, troponin I, by 3′ :5′-cyclic AMP-dependent protein kinase. In this property it was as effective as whole skeletal-muscle troponin C when compared on a molar basis. 4. Biological activity was also present in other fractions obtained from the CNBr digest. 5. By gel filtration and affinity chromatography of the whole CNBr digest of troponin C, two peptides, one of which was identified as representing residues 83-134, were shown to form Ca(2+-dependent complexes with troponin I. 6. The significance of these findings for the mechanism of interaction of troponin C and troponin I is discussed.

scholarly journals The sites of phosphorylation of rabbit cardiac troponin I by adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Effect of interaction with troponin C

1977 ◽  
Vol 167 (2) ◽  
pp. 333-343 ◽  
Author(s):  
A J G Moir ◽  
S V Perry
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Affinity Chromatography ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin C ◽  
Cardiac Troponin I ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein

1. Troponin I prepared from rabbit hearts contains 1.0-1.5 mol of P/mol when isolated by affinity chromatography. Most of the covalently bound phosphate is located in residues 1-48 of the molecule. 2. 3′:5′-Cyclic AMP-dependent protein kinase catalyses phosphorylation at serine-20 and serine-146. Serine-20 is more rapidly phosphorylated than serine-146. 3. In troponin I prepared from frozen hearts by affinity chromatography about 0.3-0.5 mol of P/mol is associated with serine-20 and 0.8-1.0 mol of P/mol with other site(s) in residues 1-48 of the molecule. 4. Phosphorylation at serine-20 and servine-146 is not significantly inhibited by troponin C. 5. The mechansim of the interaction of troponin C with cardiac troponin I is discussed in the light of these results.

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