Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca(2+) ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca(2+) activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca(2+) have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca(2+) ions using a battery of biophysical probes as a function of temperature. Ca(2+)-induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca(2+) ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca(2+) binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca(2+) rendered the enzyme increasingly labile to thermal perturbation.     

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.     

Effect of molecular crowding on self-association of phosphorylase kinase and its interaction with phosphorylase b and glycogen

Self-association of phosphorylase kinase (PhK) and its interaction with glycogen (M=5500 kDa) and phosphorylase b (Phb) has been studied using analytical ultracentrifugation and turbidimetry under the conditions of molecular crowding arising from the presence of high concentrations of osmolytes. In accordance with the predictions of the molecular crowding theory, trimethylamine N-oxide (TMAO) and betaine greatly favor self-association of PhK induced by Mg2+ and Ca2+ and PhK interaction with glycogen. In contrast, proline suppresses these processes, probably, due to its specific interaction with PhK. All osmolytes tested prevented the complex formation between PhK and its physiological substrate, Phb. The specific interactions of PhK and Phb with glycogen, in the living cell, presumably is a factor allowing the negative effect of crowding on the recognition of Phb by PhK to be overcome.     

Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.     

High-affinity calcium-stimulated, magnesium-dependent adenosine triphosphatase in Trypanosoma cruzi.

1. A high-affinity (Ca2+ + Mg2+)-ATPase and a low-affinity Mg(2+)-ATPase were identified in the 105,000 g fraction from epimastigote forms of Trypanosoma cruzi, the agent of Chagas' disease (Tulahuen strain). 2. Activities were conserved after enzyme solubilization with deoxycholate. 3. The Ca(2+)-stimulated ATPase activity was (a) lower than that of the Mg(2+)-ATPase; (b) inhibited by p-chloromercurobenzoate and orthovanadate and (c) insensitive to oligomycin. 4. Optimal stimulation by Ca2+ was observed at pH 6.5-6.8 in the presence of 1 mM MgCl2 and 0.1 M KCl. 5. The Mg(2+)-ATPase was insensitive to p-chloromercurobenzoate and orthovanadate and did not require KCl for activity. 6. Kinetic analysis of the (Ca2+ + Mg2+)-ATPase yielded a half-maximal stimulating concentration of 1.1 microM for Ca2+ and a Km of 66 microM for ATP. 7. The (Ca2+ + Mg2+)-ATPase clearly differed from the Ca(2+)- or Mg(2+)-ATPases previously characterized in the same strain of T. cruzi (Frasch et al., 1978; Comp. Biochem. Physiol. 60B, 271-275).     

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.     

The labeling with 8-azido-cyclic adenosine monophosphate of proteins in vesicles of sarcoplasmic reticulum from rabbit skeletal muscle

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (NA+, K+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1. 10(2) M-1 while ATP binds with an apparent Ka = 1.1.10(4) M-1 for 1 mM NaCl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCl2, 0.1 mM Tris-HCl (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasielastic light scattering spectrum is 203.7 +/- 15.2 nm. In the presence of ATP a reduction in size is observed.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. II. Dependence on calcium and a cytoplasmic activator.

1. Activity of the (Ca2+ + Mg2+)-ATPase of erythrocyte membrane may be enhanced by a cytoplasmic protein activator. The presence of Ca2+ is necessary for the ionic strength-dependent interaction between the erythrocyte membrane and the activator. This is true no matter the purity of activator (unfractionated hemolysis supernatant or partially purified activator) or the major source of ionic strength (imidazole or NaCl). 2. When the endogenous activator enhances (Ca2+ + Mg2+)-ATPase activity of the erythrocyte membrane, there is a physical association between activator and membrane. This association is not disrupted by a decrease in ionic strength to 0.005 but is reversed by exposure to 5 mM ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 3. Activator binding necessary for enhancement of (Ca2+ + Mg2+)-ATPase activity may occur during preparation of membranes or during incubation for assay of ATPase.     

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.     

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.     

Physicochemical changes in phosphorylase kinase associated with its activation

Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca(2+)-dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as upward arrow pH), but Ca(2+) dependence is retained. Little is known about the structural changes associated with PhK's activation by upward arrow pH and Ca(2+), but activation by both mechanisms is mediated through regulatory subunits of the (alphabetagammadelta)(4) PhK complex. In this study, changes in the structure of PhK induced by upward arrow pH and Ca(2+) were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca(2+) and upward arrow pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH approximately 7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 - Ca(2+)) versus that where it would be most active (pH 8.2 + Ca(2+)), the joint activation by upward arrow pH and Ca(2+) is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of upward arrow pH and Ca(2+) in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.     

Comparison of the structural and dynamical properties of holo and apo bovine alpha-lactalbumin by NMR spectroscopy

In the presence of 0.5 M NaCl at pH 7.1, the Ca(2+)-free apo form of recombinant bovine alpha-lactalbumin (BLA) is sufficiently stabilised in its native state to give well-resolved NMR spectra at 20 degrees C. The (1)H and (15)N NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native holo protein. Large changes observed between the two forms of BLA are mainly limited to the Ca(2+)-binding region of the protein. These data suggest that Na(+) stabilises the native apo state through the screening of repulsive negative charges, at the Ca(2+)-binding site or elsewhere, rather than by a specific interaction at the vacant Ca(2+)-binding site. The hydrogen exchange protection of residues in the Ca(2+)-binding loop and the C-helix is reduced in the apo form compared to that in the holo form. This indicates that the dynamic behaviour of this region of the protein is substantially increased in the absence of the bound Ca(2+). Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the holo to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganisations of the structure in the vicinity of the Ca(2+)-binding site that are coupled to the intrinsic fluctuations in the protein structure.     

Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhances overall vinblastine- or vincristine-induced tubulin self-association. As demonstrated in previous studies, GDP enhances overall self-association more than GTP, although in the presence of salt, GDP enhancement is reduced. For example, in 150 mM NaCl, GDP enhancement is 0.24 kcal/mol for vinblastine and 0.36 kcal/mol for vincristine versus an average enhancement of 0.87 (+/- 0.34) kcal/mol for the same drugs in the absence of salt. Wyman linkage analysis of experiments with vinblastine or vincristine over a range of NaCl concentrations showed a twofold increase in the change in NaCl bound to drug-induced spirals in the presence of GTP compared to GDP. These data indicate that GDP enhancement of Vinca alkaloid-induced tubulin self-association is due in part to electrostatic inhibition in the GTP state. In the absence of NaCl, we found that vinblastine and 1 mM Mn2+ or Ca2+ causes immediate condensation of tubulin. The predominant aggregates observed by electron microscopy are large sheets. This effect was not found with 1 mM Mg2+. At 100 microM cation concentrations (Mn2+, Mg2+, or Ca2+), GDP enhances vinblastine-induced spiral formation by 0.55 (+/- 0.26) kcal/mol. This effect is found only in K2, the association of liganded heterodimers at the ends of growing spirals. There is no GDP enhancement of K1, the binding of drug to heterodimer, although K1 is dependent upon the divalent cation concentration. NaCl diminishes tubulin condensation, probably by inhibiting lateral association, and allows an investigation of higher divalent cation concentrations. In the presence of 150 mM NaCl plus 1 mM divalent cations (Mn2+, Mg2+, or Ca2+) GDP enhances vinblastine-induced spiral formation by 0.35 (+/- 0.21) kcal/mol. Relaxation times determined by stopped-flow light scattering experiments in the presence of 150 mM NaCl and vincristine are severalfold longer than those in the presence of vinblastine, consistent with a mechanism involving the redistribution of longer polymers. Unlike previous results in the absence of NaCl, relaxation times in the presence of NaCl are only weekly protein concentration dependent, suggesting the absence of annealing or an additional rate-limiting step in the mechanism.     

The cell surface glycoprotein of Halobacterium halobium. Physico-chemical characterization in the absence and presence of salt

The cell surface glycoprotein of Halobacterium halobium is soluble in dilute buffer at neutral pH. At low counterion concentrations, the protein is monomeric (Ms,D = 209 kDa) and exhibits the characteristics of a highly charged polyelectrolyte. Evidence obtained from intrinsic fluorescence and far-UV circular dichroism shows that the monomer at low salt loses both its native conformation and its inherent tendency to form high molecular mass assemblies. In 4M NaCl, 25 mM KCl, and in the presence of divalent ions (greater than or equal to 50mM Mg2+ or Ca2+), association to well-defined assemblies of up to approximately 4 X 10(6) Da occurs. At low Mg2+ concentration and in the presence of Ba2+, a wide size-distribution of aggregates is observed. The assembly pattern of the protein may be correlated with salt-dependent alterations in the morphology of the bacterium.     

Studies on interaction of phosphorylase kinase from rabbit skeletal muscle with glycogen in the presence of ATP and ADP.

The influence of ATP on complex formation of phosphorylase kinase (PhK) with glycogen in the presence of Ca(2+) and Mg(2+) has been studied. The initial rate of complex formation decreases with increasing ATP concentration, the dependence of the initial rate on the concentration of ATP having a cooperative character. Formation of the complex of PhK with glycogen in the presence of ATP occurs after a lag period, which increases with increasing ATP concentration. The dependence of the initial rate of complex formation (v) on the concentration of non-hydrolyzed ATP analogue, beta,gamma-methylene-ATP, follows the hyperbolic law. A correlation between PhK-glycogen complex formation and (32)P incorporation catalyzed by PhK itself and by the catalytic subunit of cAMP-dependent protein kinase has been shown. For ADP (the product and allosteric effector of the PhK reaction) the dependence of v on ADP concentration has a complicated form, probably due to the sequential binding of ADP at two allosteric sites on the beta subunit and the active site on the gamma subunit.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Divalent ion binding properties of the timothy grass allergen, Phl p 7

The timothy grass allergen, Phl p 7, was studied by calorimetry, spectroscopy, and analytical ultracentrifugation. As judged by isothermal titration calorimetry (ITC), the protein binds Ca (2+) cooperatively with stepwise macroscopic association constants of 1.73 x 10 (6) and 8.06 x 10 (6) M (-1). By contrast, Mg (2+) binding is sequential with apparent macroscopic association constants of 2.78 x 10 (4) and 170 M (-1). Circular dichroism and ANS fluorescence data suggest that Ca (2+) binding provokes a major conformational change that does not occur upon Mg (2+) binding. Conformational stability was assessed by differential scanning calorimetry (DSC). In phosphate-buffered saline (PBS) containing EDTA, the apoprotein undergoes two-state denaturation with a T m of 78.4 degrees C. In the presence of 0.02 mM Ca (2+), the T m exceeds 120 degrees C. Phl p 7 is known to crystallize as a domain-swapped dimer at low pH. However, analytical ultracentrifugation data indicate that the protein is monomeric in neutral solution at concentrations exceeding 1.0 mM, in both the apo and Ca (2+)-bound states.     

Aggregation and swelling of brain synaptic vesicles in rats

Mg-ATPase (1 mM) induces a decrease in the intensity of light scattering (I1) at 620 nm of rat brain synaptic vesicles (SV) suspended in sucrose, with this decrease being indicative of the swelling of the vesicles. The Mg-ATPase-induced swelling appears to be associated with the function of H+-ATPase of SV membranes, since it is completely abolished by the proton pump blocker dicyclohexylcarbodiimide and the protonophore carbonylcyanide m-chlorophenylhydrazone. The Mg-ATPase-induced swelling was enhanced in the presence of the permeable anion Cl- (in the range of 25-50 mM KCl). Ca2+ (and Mg2+) at high concentrations (0.1-1.0 mM) cause aggregation of the SV as measured by changes in the I1. Colchicine and cytochalasin do not affect SV swelling and aggregation whereas Mg-ATP (1 mM) lowers aggregation caused by Ca2+ (1 mM).     

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

Summary The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK ) as reported by others (2). In the absence of calcium the K_m of PhK for phosphorylase b was 53 + 10 U/ ml with a V_m of 17 = 1 U/gm of tissue. The K_m of PhK for phosphorylase b was 20 + 2 U/ml with a V_m of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A_0.5 0.03 M). In the presence of 0.1 M calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the V_m of both forms, but the effect was greater for PhK b than for PhK . The K_m of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.