The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Phosphorylation of the myosin light chains and satellite proteins in detergent-skinned arterial smooth muscle

Isoelectric focusing of extracts prepared from detergent-skinned porcine carotid artery showed that contraction was associated with phosphorylation of the regulatory myosin light chains and two additional proteins of the same apparent molecular weight (20,000). These two proteins, previously described as satellites, did not appear to be artifactually derived from the phosphorylated light chains during electrophoresis. That is, each of the phosphorylated proteins migrated as separate and distinct proteins when subjected to a second cycle of isoelectric focusing. Moreover, relaxation of skinned fibers was associated with dephosphorylation of the light chains and both satellites. These findings suggest that the satellites may represent varients of the light chains per se, or another regulatory protein which is reversibly phosphorylated and dephosphorylated during contraction and relaxation of vascular smooth muscle.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

The ATPMg-dependent phosphatase is present in mammalian vascular smooth muscle

An ATPMg-dependent phosphorylase phosphatase was identified in vascular smooth muscle from bovine aorta. The smooth muscle enzyme, like the corresponding enzyme from striated muscle, exists as an inactive phosphatase (F_C-enzyme) which can be activated by a protein, F_A, in the presence of ATP and Mg²⁺. Moreover, smooth muscle F_C is activatable by skeletal muscle F_A and skeletal muscle F_C can be activated by smooth muscle F_A. The mode of activation of aortic F_C by aortic F_A is similar to that reported for the skeletal muscle proteins. In accord with earlier findings obtained with the skeletal muscle system, the activity of the aortic phosphatase is inhibited by a specific heat-stable modulator protein (previously called phosphatase inhibitor-2). Thus, the fundamental properties of arterial ATPMg-dependent phosphatase appear to be identical to those of its skeletal muscle counterpart which purportedly represents the major phosphorylase phosphatase in that tissue. Since glycogen phosphorylase is activated when vascular smooth muscle contracts, ATPMg-dependent protein phosphatase may participate in coordinating arterial metabolism and contractility.     

Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase.

The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate ³²P from [γ-³²P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca²⁺ ATPase and Mg²⁺ ATPase activities without any changes in Na⁺K⁺ ATPase activity. The observed increase in $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity was found to be associated with an increase in V_max value of the reaction whereas K_a value for $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Evidence for dysfunction in the regulation of cytosolic Ca2+ in excitation-contraction uncoupled dysgenic muscle

In noncontracting, dysgenic murine muscle, excitation is uncoupled from contraction. To test whether the gene lesion is expressed as a defect in the regulation of the intracellular free Ca2+ levels, cultured normal and dysgenic muscle at various stages of development (proliferative myoblasts, early, late, and mature myotubes) were exposed to increasing increments (0.5-mM steps) of extracellular Ca2+ in ionophore A23187-Ca2+-EGTA-buffered media. Normal and dysgenic muscle at all stages (except myoblast) displayed contractures at approximately 500 microM free Ca2+ and higher. Experiments using finer increments of Ca2+ and different ionophore concentrations indicated an external Ca2+ threshold for contracture at 265 microM Ca2+ for early and late myotubes and 47-78 microM for mature normal and dysgenic myotubes. Low extracellular concentrations of calcium (14 microM and 0.76 nM) caused elongation of both normal and dysgenic myotubes. Mature cells were depolarized by exposure to increasing extracellular K+ and monitored by intracellular recording; normal and dysgenic myotubes showed similar reductions in membrane potentials. Depolarization to -35 mV elicited contractures in normal myotubes, but even depolarization to -9 mV in dysgenic cells elicited no response. Thus steady-state depolarization of dysgenic muscle does not cause contractures, which can, however, be elicited by increasing the intracellular free Ca2+. These results offer new evidence for a possible defect in the regulation of Ca2+ levels in dysgenic muscle.     

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: comparison between Mg2+- and Mn2+-supported enzyme activities in particulate. detergent-solubilized and detergent-insoluble fractions

Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg²⁺ or Mn²⁺ as sole cation in the presence and absence of Triton X-100. With Mg²⁺, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg²⁺-supported enzyme activity. With Mn²⁺, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg²⁺ or Mn²⁺, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg²⁺ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg²⁺-dependent activity; Mn²⁺-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn²⁺ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg²⁺.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

Comparative changes of levels of nitrendipine Ca2+ channels, of tetrodotoxin-sensitive Na+ channels and of ouabain-sensitive (Na+ + K+)-ATPase following denervation of rat and chick skeletal muscle

Three major ion transport systems, the nitrendipine-sensitive Ca2+ channels, the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na+ channel and the (Na+ + K+)-ATPase and that regulation of the nitrendipine-sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase which show a quite similar evolution.     

Stimulation of ion release from liposomes by the polyene antibiotic, filipin.

The effect of the polyene antibiotic, filipin, upon release of the ions Ca²⁺, Sr²⁺, SO₄^2? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.     

Ca2+ regulated modulator protein interacting agents: inhibition of Ca2+-Mg2+-ATPase of human erythrocyte ghost.

Agents such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and its derivatives, chlorpromazine and amitriptyline that interact with calcium-regulated modulator protein were found to inhibit not only Ca²⁺ dependent cyclic nucleotide phosphodiesterase but also Ca²⁺-Mg²⁺-ATPase of human erythrocyte ghosts. I₅₀ values of modulator interacting agents for testis modulator-activated, brain modulator-activated and erythrocyte modulator-activated-ATPase are indistinguishable. However, I₅₀ of W-7 for troponin C-activated-ATPase is lower than that for modulator-activated ATPase. The specificity of these agents toward modulator-related enzyme reaction is also shown by the negative effect on modulator-unrelated enzyme system such as erythrocyte ghost protein kinase and Mg²⁺-ATPase. These agents provide a useful tool for elucidating the physiological role of modulator.     

Phosphorylation of calf uterus 17 beta-estradiol receptor by endogenous Ca2+-stimulated kinase activating the hormone binding of the receptor

Direct evidence is presented that uterus 17_β-estradiol receptor is phosphorylated $\text{in}\text{,}\text{vitro}$ by an endogenous kinase. Nuclear phosphatase, cytosol Ca²⁺-stimulated kinase (the former inactivating and the latter reactivating the hormone binding of the 17_β-estradiol receptor) and receptor were purified from calf uterus. 17_β-estradiol binding was inactivated by phosphatase, then reactivated by kinase in the presence of [_γ-³²P] ATP, Ca²⁺ and calmodulin, and the receptor was examined by various methods. The results of gel electrophoresis in non denaturating and denaturating conditions, and of centrifugation through sucrose gradients of receptor preincubated with monoclonal antibodies showed that the receptor is phosphorylated.     

Activation of ribulose-1, 5-bisphosphate oxygenase, The role of Mg2+, CO2, and pH.

Ribulose-1,5-bisphosphate oxygenase was activated by incubation with CO₂ and Mg²⁺ and inactivated upon removal of CO₂ and Mg²⁺ by gel filtration. The activity of the enzyme was dependent upon the preincubation concentrations of CO₂ and Mg²⁺ and upon the preincubation pH. This indicated that activation involved the reversible formation of an equilibrium complex of enzyme-CO₂-Mg. The kinetics of the activation process were the same as those described by G. H. Lorimer et al. ((1976) Biochemistry15, 529–536), for ribulose bisphosphate carboxylase and are consistent with the ordered reversible reaction sequence:

The activity of the enzyme, after preincubation at constant concentrations of CO₂ and Mg²⁺, increased as the pH was raised, suggesting that CO₂ reacted with an enzyme group having an alkaline pK. Since CO₂ and O₂ interact competitively at the catalytic site, the activation of ribulose bisphosphate oxygenase by CO₂ and Mg²⁺ indicates that the CO₂ molecule which takes part in the activation process is not the same as that which becomes fixed during the carboxylase reaction. These results also indicate that the oxygenase and carboxylase functions of the catalytic site are tightly coupled rather than independent of one another.     

Properties of the calcium-sensitive components of bovine arterial actomyosin.

A calcium-sensitive actomyosin was prepared from bovine aortic muscularis. Calcium binding to this arterial actomyosin was measured using a centrifugation method. The amount of bound calcium necessary for full activation of the arterial actomyosin adenosine triphosphatase was approximately 36 μmol of Ca²⁺/kg of aorta. Calcium stimulation of the actomyosin ATPase could be prevented by lowering the free magnesium from 7 to 1 mm. However, calcium binding to the actomyosin increased slightly with a reduction of free magnesium levels. Positive cooperativity was evident in the sequence of reactions beginning with the binding of calcium and ending with the hydrolysis of ATP. However, there was no evidence for the cooperativity occurring at the initial calcium-binding step.     

Equimolar interaction between calmodulin and the Ca2+ ATPase from human erythrocyte membranes

The interaction between calmodulin and the pure, solubilized Ca²⁺ ATPase from human erythrocyte membranes was examined by kinetic titration. The data indicated that the two proteins interacted in a molar ratio of 1:1 with a K_d of 4.2 nm. The dependence of enzyme activity on calmodulin concentration agreed quantitatively with that predicted by kinetic theory.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

RNA synthesis in Escherichia coli during K + -depletion

During K⁺ depletion of a mutant of $\text{Escherichia}$$\text{coli}$ which cannot concentrate this cation, protein synthesis is inhibited but RNA formation continues. The RNA produced during K⁺ depletion was analyzed by gel electrophoresis. It was found that 4S, 5S and 23S RNA were synthesized by K⁺-depleted cells whether uninfected or infected with phage T4. In addition, an RNA species moving close to 16S (presumably 17S) and material of about 6–10S were made during K⁺ depletion. These species of RNA were not evident in growing cells. Methylation of RNA is severely inhibited during K⁺ depletion.