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.     

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.     

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.     

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 $\text{in}\text{vivo}$.     

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.     

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.     

The effect of oncomodulin on cAMP phosphodiesterase activity

Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43, 5390-5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.     

Na+, K+-ATPase: evidence for the binding of ATP to the phosphoenzyme

Highly purified Na⁺, K⁺-ATPase of the dog kidney was reacted with Mg²⁺+³²Pi or Mg²⁺+³²Pi + ouabain. ³²P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K_0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na⁺. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested.     

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.     

Detection of caldesmon in muscle and non-muscle tissues of the chicken using polyclonal antibodies

Polyclonal antibodies raised in rabbits against chicken gizzard caldesmon have been purified and used in immunoblotting experiments to study the distribution of this actin- and calmodulin-binding protein in diverse tissues of the chicken. Total homogenates and heat-treated homogenate supernatants derived from each tissue were subjected to sodium dodecyl sulfate-polyacrylamide gradient slab gel electrophoresis and immunoblotting using the horseradish peroxidase method. All chicken tissues examined contained caldesmon of Mr = 141,000. The amount of caldesmon found in the different tissues varied considerably and semi-quantitative comparison of stained immunoblots indicated the following relative caldesmon contents: gizzard greater than oesophagus greater than duodenum = small intestine greater than lung greater than aorta greater than heart = skeletal muscle greater than kidney = trachea greater than brain greater than liver. Each tissue revealed small amounts of lower Mr immunoreactive proteins, predominantly bands of Mr 94,000 and 70,000, which appear to be proteolytic fragments of caldesmon. Isolated caldesmon was found to be highly sensitive to proteolysis. The widespread distribution and similarity of caldesmon in different tissues of the chicken suggest its functional importance and structural conservation.     

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.     

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.     

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.     

Molecular diversity of calpastatin in mammalian organs

The crude homogenates of various human and porcine organs were subjected to immunoelectrophoretic blot analysis using affinity-purified anti-calpastatin antibody which specifically reacts with human erythrocyte 70 kDa calpastatin. Multiple immuno-reactive bands were revealed which ranged from 100 to 50 kDa. The results indicated the diversity of monomeric calpastatin molecules. The band patterns were different from one organ to the other. Among them, lung, heart and skeletal muscle were characterized by the predominance of 90-100 kDa calpastatin, having a common antigenicity to erythrocyte 70 kDa calpastatin. Such molecular diversity of calpastatins was also substantiated by enzymatic and chromatographic analyses.     

Vitamin A acid-induced activation of Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina

Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina was partially purified. Vitamin A acid (retinoic acid) stimulated this protein kinase in the presence of Ca2+, while other metabolites of vitamin A such as retinol or retinal were less effective. The order of the extent of phosphorylation of the various substrate proteins by this protein kinase was identical in the presence of vitamin A acid or phosphatidylserine. The major spots of the 32P labeled peptide from histone H1 phosphorylated in the presence of vitamin A acid by this protein kinase did not differ from those obtained from histone H1 phosphorylated in the presence of phosphatidylserine. Retinol caused a further enhancement of the enzymatic activity, whereas the addition of retinal inhibited the activation by vitamin A acid. Thus, vitamin A and its metabolites may play an important role in the regulation of Ca2+-activated, phospholipid-dependent protein kinase activity in the retina.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

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⁺.     

Ca2+-activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.     

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.