Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using ¹²⁵I-CDR as a tracer molecule. ¹²⁵I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca²⁺ and Mg²⁺ dependence was observed for ¹²⁵I-CDR binding and for brain adenylate cyclase activation by CDR.     

Complete nucleotide sequence of the messenger RNA coding for chicken muscle glyceraldehyde-3-phosphate dehydrogenase

The complete nucleotide sequence for chicken glyceraldehyde-3-phosphate dehydrogenase mRNA has been determined, thereby extending the longest such sequence previously reported (Dugaiczyk et al. Biochemistry, 1983, 22, 1605-1613) by 27 nucleotides. The complete mRNA with the exclusion of poly(A) is 1284 nucleotides long and contains 56 nucleotides of 5' non coding sequence and 229 nucleotides of 3' non coding region. Knowledge of the complete sequence allows us to propose secondary structures models which may be of biological significance.     

Disassembly of microtubules by the action of calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubules assembled by the incubation of GTP at 37 °C were disassembled by the action of calmodulin-dependent protein kinase (Kinase II) which occrs only in the brain tissues. This disassembly required the presence of ATP and physiological concentrations of Ca²⁺ and calmodulin.     

Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Protein phosphorylation in pancreatic islets: evidence for separate Ca2+ and cAMP-enhanced phosphorylation of two 57,000 Mr proteins

There is a phosphopeptide that has an Mr of 53,000 to 60,000 in insulin-secreting tissues and there is general agreement that this peptide can be phosphorylated in a calcium-dependent manner. The present report shows that there are at least two phosphoproteins with Mr's near 57,000 in rat pancreatic islet cytosol. One peptide has an Mr of 57,000, a pl of 7.5 - 8 and is phosphorylated in a Ca2+-enhanced manner, and the other has an Mr of 54,000, a pl of 5 - 5.5 and is phosphorylated in a cAMP-enhanced manner, as judged by two-dimensional polyacrylamide gel electrophoresis. Sepharose 4B chromatography indicated that the former polypeptide resides in a native protein complex that has an Mr of about 500,000 and the latter in a complex that has an Mr of about 180,000. Tritiated azido cyclic AMP binds to an islet polypeptide that has an Mr of 54,000. The results suggest that Ca2+ and cAMP could regulate stimulus-secretion coupling in pancreatic islets via protein phosphorylation.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.     

Hyaluronic acid salt — a mechanoelectrical transducer

Hyaluronic acid transduces a very gentle pressure into an electrical potential. Such pressure, depending on its direction, changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the unpressured salt or changing and increasing the rotation in the opposite direction. These findings have implications for understanding the funtion of the cochlear and vestibular fluids, renal function, and the approximation to frictionless motion of normal joints.     

Cation dependent conformations of brain CA2+-dependent regulator protein detected by nuclear magnetic resonance.

The 250MHz NMR spectrum of the brain Ca²⁺-dependent regulator protein was examined in the absence of cations and in the presence of Ca²⁺ or Mg²⁺. The Ca²⁺-saturated regulator protein and Mg²⁺-saturated regulator protein exhibited several spectral differences in the aromatic and aliphatic regions of their spectra. Certain spectral changes observed to occur upon addition of metal ions are qualitatively similar to those which have been observed in the spectrum of skeletal troponin-C. These results suggest that the large sequence homology between skeletal troponin-C and the regulator protein results in similar conformational changes due to the binding of Ca²⁺ or Mg²⁺.     

Effects of monovalent cations on the activities associated with coupling site III of rat liver mitochondria

The effects of substitution of K⁺ by Li⁺, Na⁺, or Rb⁺ in the assay medium on the processes of electron transfer and H⁺ translocation associated with Site III are investigated. The replacement of K⁺ with Rb⁺ has little effect, if any, on the measured initial rates of H⁺ extrusion and electron transfer. The substitution of K⁺ by Li⁺ increases the initial rate of both processes simultaneously while the replacement of K⁺ by Na⁺ causes an enhancement on the rate of electron transfer with concomitant inhibition of the observed acidification. The presence of either Na⁺ or Li⁺ decreases the proton-leak rate of the inner membrane. These results suggest that the link between electron transfer and H⁺ translocation in Site III is weakened by the presence of Na⁺.     

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.     

Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100°C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitors(s) directed against factor X_a but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor X_a-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(β-maminoethyl ether)-N,N′-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfuorophosphate. The factor X_a inhibitors(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor X_a-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.     

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine>tetracaine>lidocaine>procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca²⁺ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.