Factors required for calcium dependent acetylcholine release from isolated torpedo synaptic vesicles.

The release of acetylcholine (Ach) from Torpedo synaptic vesicles has been investigated. Factors have been found which induce Ca⁺² dependent Ach release from the synaptic vesicles. In the absence of these factors, the vesicles are not affected by Ca⁺². Addition of a soluble factor to the vesicles induces a Ca⁺²-dependent release of their Ach. This secretion is enhanced by a non-vesicular membranous component which, by itself, does not induce the Ca⁺²-dependent release. These results demonstrate that vesicular Ach release may be studied $\text{in vitro}$ and thus will enable the study, at the molecular level, of the biochemical events underlying neurotransmission.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Phosphate induced swelling, inhibition and partial uncoupling of oxidative phosphorylation in heart mitochondria in the absence of external calcium and the presence of EGTA

Short-term (0–1 h) incubations of rat hepatocytes with oleate (2 mM) resulted in a decrease in the rate of cholesterol synthesis compared to controls incubated in the absence of fatty acid. However, during this period the activity of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase was higher in the oleate-incubated cells. After longer incubation periods in the presence of oleate there was a higher rate of cholesterol synthesis than in the corresponding non-oleate controls and HMG-CoA reductase activity remained elevated. This biphasic effect provides an explanation for previous contradictory reports concerning the effect of exogenous fatty acids on the rate of cholesterol synthesis in liver. The present studies also suggest that in some physiological situations, the rate of cholesterol synthesis is determined by substrate supply rather than by HMG-CoA reductase activity.     

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.     

Conformational changes of F-actin-ε-ADP in thin filaments in myosin-free muscle fibers induced by Ca2+

Polarized fluorescence from F-actin-ε-ADP in thin filaments reconstituted in a myosin-free single muscle fiber was measured at various concentrations of Ca²⁺. Four components of polarized fluorescence changed with increasing Ca²⁺ concentration at pCa values of around 7 to 6, concomitant with a change of the tension generated by the fiber irrigated with myosin in the presence of Mg-ATP. From analysis of observed values of the four components, it was found that the flexibility of the thin filament increased, or the elastic modulus for bending decreased from 5.7 × 10^?17 dyn cm² to 4.7 × 10^?17 dyn cm², when the pCa value decreased from 7 to 6. In the same range of pCa values, the angles of absorption and emission dipoles of ε-ADP changed, suggesting a small rotation of the base-plane of ε-ADP around an axis perpendicular to the F-actin axis.     

Protective effect of EGTA on rat gastric membranes enriched in (H+-K+)-ATPase

Rat gastric membranes enriched in (H⁺-K⁺)-ATPase, when prepared in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid, showed the ability to accumulate H⁺ ions upon addition of ATP, KCl, and valinomycin. The membranes were largely impermeable to K⁺ and Cl^?. In contrast, the rat membranes prepared without the Ca²⁺ chelator lost the ability to develop a pH gradient because of the membrane leakiness to H⁺. A majority of these membrane vesicles became also permeable to K⁺. We suggest that the calcium chelator preserved the gastric membrane permeability barrier during isolation by inhibiting various Ca²⁺-dependent phospholipases in rat gastric mucosa.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Opposing actions of Ca++ and ATP plus Mg++ in controlling the kynurenine aminotransferase activity of isolated rat kidney mitochondria.

When human spermatozoa are extracted in the presence of 0.05 M benzamidine, the resulting solutions show a time dependent, sigmoidal increase of trypsin-like activity upon incubation at pH 8. Gel permeation chromatography of these extracts separates two species, P₁ and P₂, with apparent molecular weights of 75,000 and 42,000 respectively. P₁ and P₂ are both autoactivatable at pH 7–8 and the kinetic parameters of activated P₁ and P₂ are indistinguishable from those of human acrosin. That P₁ and P₂ are inactive precursors of human acrosin is shown by the fact that, in the presence of benzamidine, they are obtained instead of and in greater yield than acrosin. That P₁ and P₂ are zymogens is shown by the features of the activation process.     

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.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Effects of chelating agents on the initial interaction of phytohemagglutinin with lymphocytes and the subsequent stimulation of amino acid uptake

The chelating agents, ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) and EDTA, had no effect on the initial interaction of phytohemagglutinin with lymphocytes at concentrations which have been shown previously to inhibit the development of the phytohemagglutinin response completely. However, they had a marked inhibitory effect on uptake of the amino acid analog, α-aminoisobutyric acid in both unstimulated and phytohemagglutinin-stimulated cells. The inhibition of amino acid uptake by EGTA could be reversed by adding Ca²⁺ but not Mg²⁺. These results demonstrated that Ca²⁺ is not essential to the initial interaction of phytohemagglutinin with the cell, but does influence amino acid transport which may be a critical preparatory event for later increased protein synthesis.     

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.     

Some kinetic properties of phosphorylated ATPase of sarcoplasmic reticulum formed in the absence of added alkali metal salts.

In the rat both hypothyroidism and diabetes decrease heparin-releasable liver lipase activity. This defect may be reversed by feeding a diet rich in polyunsaturated fatty acids. It is suggested that a diet-induced increase of membrane fluidity restores liver lipase activity, which contributes to the hypolipidemic effect of polyunsaturated fatty acids.     

Specificity of association of a Ca2+/Mg2+ ATPase with cholinergic synaptic vesicles from Torpedo electric organ.

Cholinergic synaptic vesicles from the electric organ of $\text{Torpedo}$$\text{californica}$ have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase in the cholinergic synaptic vesicle.     

Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system.     

Polyamine-stimulated phosphorylation of proteins from corn (Zea mays L.) coleoptiles

The effect of polyamines (spermine, spermidine and putrescine) on in vitro phosphorylation of proteins from corn coleoptiles was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine promoted the phosphorylation of several membrane and soluble proteins and most of the proteins phosphorylated were different from those phosphorylated in the presence of calcium. Spermidine promoted the phosphorylation to a lesser extent and putrescine had very little stimulatory effect. Spermine-promoted phosphorylation of soluble proteins was dependent upon the presence of Mg2+ and was discernible at 100 microM spermine concentration.     

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 $\text{in}\text{vivo}$.     

Improved Medium for Extraction of Plasminogen Activator from Tissue

Quantitation of plasminogen activators present in tissue may depend to a large extent on the extraction procedure used to solubilize the enzymes. Potassium thiocyanate solution is known to be an efficient solubilizer, but it can inhibit assay systems other than fibrin plates. An equally effective acetate-detergent extractant is reported here which can be used with the highly sensitive azocase inolytic assay procedure. The results indicate that a threefold increase in activator activity can be extracted from selected tissues relative to that previously reported for a phosphate-detergent extractant. The extraction medium contains 75 mM K acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM EDTA, 0.25% Triton X-100, final pH 4.2.     

Detection of an initial burst of Ca2+ translocation in sarcoplasmic reticulum.

Rapid quench methods were used to determine Ca²⁺ uptake, ATPase phosphorylation and Pi production in the transient state of Sarcoplasmic Reticulum. It was found that within 20 milliseconds of the addition of ATP maximal levels of phosphorylated enzyme intermediate are reached and an initial burst of Ca²⁺ uptake is completed. This burst, kinetically distinct from the following transport activity, is related to the phosphorylated intermediate with a molar ratio of two.