Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Preparation and properties of 5′-nucleotidases from bovine milk fat globule membranes

The 5′-nucleotidase (5′-ribonucleoside phosphohydrolase, EC 3.1.3.5) from bovine milk fat globule membranes was partially purified. Two separate peaks of activity were obtained from a Sepharose column and the two fractions, designated V and VI in order of elution, were collected and characterized separately. Both V and VI exhibited pH optima between 7.0 and 7.5 for AMP, GMP and CMP in the absence of metal ions. In the presence of Mg²⁺, a second pH optimum at 10.0 was observed with both fractions. Low concentrations of MnCl₂ activated Fraction V but not Fraction VI. HgCl₂ was a potent inhibitor of both fractions. The relative rates of hydrolysis of various 5′-mononucleotides differed comparing the two fractions. Optimum temperature for P_i release was 69 °C for both fractions. Activation energies were 10 400 cal/mole and 9600 cal/mole for Fractions V and VI, respectively. For V, calculated K_m values for AMP, GMP and CMP were 0.94, 2.5 and 1.16 mM, respectively. Calculated K_m values for Fraction VI for AMP, GMP and CMP were 5.0, 3.95 and 1.73 mM, respectively. ATP was a competitive inhibitor of AMP hydrolysis by Fraction V and a noncompetitive inhibitor of AMP hydrolysis by Fraction VI. Both fractions contained chloroform-methanol-extractable phospholipid. The phospholipid distribution pattern of Fraction VI was similar to that of milk fat globule membranes. Fraction V contained only sphingomyelin and phosphatidylcholine. It is proposed that milk fat globule membranes contain two separate 5′-nucleotidases.     

Ca2+ transporting activity of membrane fractions isolated from the post-mitochondrial supernatant of rat liver

The post-mitochondrial supernatant of rat liver contains two vesicular fractions which transport Ca2+ actively. The heavier fraction, sedimenting at 17.500 xg, 20 min, is enriched in plasma membrane markers and apparently contains both a Ca2+ pumping ATPase and a Na+/Ca2+ exchanger. These activities have been attributed to the plasma membrane vesicles. The lighter fraction, sedimenting at 100.000 xg, 60 min, is enriched in endoplasmic reticulum markers, and contains only a Ca2+ pumping ATPase, which can be differentiated from that of the heavier fraction on the basis of the sensitivity to vanadate. The Ca2+ pumping activity of endoplasmic reticulum appears to be regulated by both a cAMP-dependent, and a calmodulin-dependent system. The former system involves a heat-stable protein fraction from the cytosol. The regulation by the cAMP and the calmodulin-dependent systems involves the phosphorylation of several proteins in the endoplasmic reticulum membrane.     

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Hydrodynamic studies on defined heterochromatin fragments support a 30-nm fiber having six nucleosomes per turn

We have compared the physical properties of a 15.51-kb constitutive heterochromatin segment and a 16.17-kb facultative heterochromatin segment that form part of the chicken β-globin locus. These segments were excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus, thus allowing measurement of the sedimentation coefficients by use of calibrated sucrose gradients. A determination of the buoyant density of the cross-linked particle in CsCl led to the total mass of the particles and their frictional coefficients, f. Despite the slight differences in nucleosome density, the measured value of f for both fragments was consistent with a rodlike particle having a diameter of 33-45 nm and a length corresponding to approximately six to seven nucleosomes per 11-nm turn. At higher ionic strengths we found no evidence of any abrupt conformational change, demonstrating that these chromatin fragments released from the nucleus did not assume the more compact conformations recently described for some reconstituted structures.     

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.     

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase-aldolase complex is located on alpha-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase-aldolase and FBPase-alpha-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.     

Ca2+-dependent effect of acetylphosphate on spin-labeled sarcoplasmic reticulum.

Acetylphosphate produces a definite change in the spectrum of an iodoacetamide spin probe covalently bound to sarcoplasmic reticulum ATPase. The observed change, which is Ca²⁺ dependent and reversible, is attributed to a protein conformational change occurring during the Ca²⁺ transport cycle.     

The effect of testosterone on ascorbic acid synthesis in rooster comb

The level of “total” ascorbic acid (ascorbate+dehydroascorbate) has been measured in the mucoid layer of combs from normal roosters, capons and capons treated with testosterone. The “total” ascorbate level in capon comb was lower than the value obtained from combs from normal roosters. This value returned towards normal in combs from capons treated with testosterone. The specific activity of L-gulonate-NADP⁺ oxidoreductase, an enzyme in the pathway of ascorbate biosynthesis, also was measured. The specific activity levels followed a pattern similar to the ascorbate levels in the three types of combs utilized. The results are consistent with the possible role of L-ascorbic acid as a cofactor in the synthesis of collagen, a process which also appears to be dependent on the level of testosterone in the comb mucoid layer.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Lysosome destabilization by cytosolic extracts, putative involvement of Ca(2+)/phospholipase C

Lysosomal disintegration is a crucial event for living cells, but mechanisms for the event are still unclear. In this study, we established that the cytosolic extracts could enhance lysosomal osmotic sensitivity and osmotically destabilize the lysosomes. The cytosol also caused the lysosomes to become more swollen in the hypotonic sucrose medium. The results indicate that the cytosol induced an osmotic shock to the lysosomes and an influx of water into the organelle. Since the effects of cytosol on the lysosomes could be abolished by O-tricyclo[5.2.1.0(2,6)]dec-9-yl dithiocarbonate potassium salt (D609), a specific inhibitor of cytosolic phospholipase C (PLC), the PLC might play an important role in the lysosomal osmotic destabilization. The activity of cytosolic PLC and the extent of enzyme latency loss of the cytosol-treated lysosomes exhibited a similar biphasic dependence on the cytosolic Ca2+ concentration. In addition, the cytosol did not osmotically destabilize the lysosomes until the cytosolic calcium ions rose above 100 nM. It suggests that the destabilization effect of cytosol on the lysosomes is Ca(2+)-dependent.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

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.