Abstract: Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.

Abstract: The effect of growth hormone (GH) and cadmium (Cd) on metallothionein (MT) expression was investigated in hepatoma cells. In fish the constitutive isoform MT-B and the metal-responsive MT-A are expressed. Real-time RT-PCR revealed that: Cd up-regulates mostly MT-A, GH slightly induces MT-B and the GH/Cd combination induces synergistically both MTs. Perturbations in Ca2+ levels suppressed or reduced the Cd-induction of MTs and abolished the GH/Cd synergy. Similar results were obtained by inhibition of tyrosine kinases. Also the signaling molecules recruited by the GH receptor responded differently to GH and Cd, with ERKs showing a synergistic activation upon GH/Cd. The following conclusions can be drawn: (1) cytosolic Ca2+ is mainly involved in MT-A regulation; (2) both Ca2+ and tyrosine phosphorylation are essential for Cd-induction and GH/Cd synergy on MTs. The synergy could depend on interactions in different signaling pathways, leading to a differential recruitment of MTF-1 and AP-1 transcription factors.

Abstract: PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.

Abstract: To achieve our aim of understanding the interactions between direct current and enzymes in solution, we exposed reconstituted Crotalus atrox venom to direct electric current by immersing two platinum thread electrodes connected to a voltage generator (between 0 and 8 V) into a reaction mixture for a few seconds. Then, we assayed the residual activity of phospholipases A(2) (PLA(2)),metalloproteinases, and phosphodiesterases, abundant in crotaline snake venoms and relevant in the pathophysiology of envenomation, characterized by hemorrhage, pain, and tissue damage. C. atrox venom phospholipase A(2) and metalloproteinases were consistently and irreversibly inactivated by direct current (between 0 and 0.7 mA) exposure. In contrast, C. atrox venom phosphodiesterases were not affected. Total protein content and temperature of the sample remained the same. Secretory pancreatic phospholipase A(2), homologue to snake venom phospholipases A(2), was also inactivated by direct current treatment. In order to understand the structural reasoning behind PLA(2) inactivation, circular dichroism measurements were conducted on homogeneous commercial pancreatic phospholipase A(2), and it was found that the enzyme undergoes structural alterations upon direct current exposure.

Abstract: Adenylate kinases (AKs) are ubiquitous phosphotransferases that contribute to homeostasis of adenine nucleotide composition in cells. Six AK isoforms were found in vertebrates. We report that soluble AK isoform 1 is expressed in the cytosol of bovine retina consistently devoid of rod outer segments. Immunoblotting analysis with a polyclonal antibody raised against soluble adenylate kinase and subsequent sequencing of eluted peptide by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry allowed enzyme isolation by joining purification methods to two-dimensional electrophoresis. In this study, we found that cytosolic adenylate kinase isoform 1 is expressed in bovine retina. Cytoplasmic AK1 would physiologically contribute to retinal energy metabolism.

Abstract: Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis.

Abstract: Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 +/- 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.

Abstract: The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54-61% with field amplitudes above a threshold of 73-151 microT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.

Abstract: We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+ or Cd2+ affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+ or Cu2+ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+ prior to GH produced a considerable increase of the [Ca2+]i variation produced by either element, while using Cu2+ or Cd2+ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+ is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+ signaling; Cu2+ is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+ is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction.

Abstract: Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg(2+) and Cu(2+)in the microM range, and H(2)O(2) in the mM range, induced tyrosine phosphorylation. The effect of Cu(2+)was prevented by pre-incubation with genistein, while those of Hg(2+)and H(2)O(2) were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H(2)O(2), Hg(2+), and Cu(2+) plus low H(2)O(2). Cell pre-incubation with p38 inhibitors indicated that ERK activation by H(2)O(2) is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H(2)O(2) only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects.

Abstract: ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca(2+) ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca(2+)-ATPase previously found on the disk membranes.

Abstract: The mechanism of lysosome activation by 17beta-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+ -dependent PLA2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+ -independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+](i) and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+ -dependent mechanism involving activation of Ca2+ -dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.

Abstract: Previously we have shown that Fe3+/ascorbate-induced Ca2+ release from scallop sarcoplasmic reticulum (SR) is due to Ca2+-channel gating by free radicals. This study is aimed at demonstrating that Ca2+-induced Ca2+ release (CICR) plays a role in this kind of Ca2+ release. Scallop SR vesicles were incubated with fluo-3 and exposed to Fe3+/ascorbate. Fluorimetric recordings showed massive Ca2+ release, with maximum rate and 50% release occurring at 30 min after exposure. Conversely, the use of the probe for reactive oxygen species dihydrorhodamine or the assay of malondialdehyde allowed oxyradical production to be traced for approximately 5 min only. Hence, although Ca2+ release started just after exposure to Fe3+/ascorbate, most release occurred after free radical exhaustion. Ruthenium red addition after Fe3+/ascorbate slowed down the Ca2+ release, whereas cyclic adenosine 5'-diphosphoribose addition accelerated it, indicating that the free radical-induced Ca2+ release from SR vesicles triggers a mechanism of CICR that dramatically increases the initial effect.

Abstract: Metallothioneins (MTs) are sulfhydryl-rich proteins binding essential and non-essential heavy metals. MTs display in vitro oxyradical scavenging capacity, suggesting that they may specifically neutralize hydroxyl radicals. Yet, this is probably an oversimplified view, as MTs represent a superfamily of widely differentiated metalloproteins. MT antioxidant properties mainly derive from sulfhydryl nucleophilicity, but also from metal complexation. Binding of transition metals displaying Fenton reactivity (Fe,Cu) can reduce oxidative stress, whereas their release exacerbates it. In vertebrates, MT gene promoters contain metal (MRE) and glucocorticoid response elements (GRE), Sp and AP sequences, but also antioxidant response elements (ARE). MT neosynthesis is induced by heavy metals, cytokines, hormones, but also by different oxidants and prooxidants. Accordingly, MT overexpression increases the resistance of tissues and cells to oxidative stress. As for invertebrates, data from the mussel show that MT can actually protect against oxidative stress, but is poorly inducible by oxidants. In yeast, there is a Cu(I)-MT that in contrast to mammalCu-MT exhibits antioxidant activity, possibly due to differences in metal binding domains. Finally, as the relevance of redox processes in cell signaling is becoming more and more evident, a search for MT effects on redox signaling could represent a turning point in the understanding of the functional role of these protein.

Abstract: Purified disk membranes from rod outer segments of the bovine retina were able to synthesize ATP with a maximal activity (about 52 nmoles ATP/min/mg of protein) at physiological calcium concentrations. This activity was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting the reversal functioning of the disk Ca(2+)-ATPase, which would act as a ATP synthesizer at the expense of the calcium gradient between the disks and the cytoplasm of the rod outer segment. The results are discussed in terms of the need of an immediate source of ATP on the disk membranes where the energy is required to supply the rapid reactions of the photoreception processes.

Abstract: In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.

Abstract: We studied the in vivo and in vitro effects of Hg2+ and Cu2+ on the activity of phospholipase C (PLC), specific for phosphatidylinositol 4,5-bisphosphate, in the mussel (Mytilus galloprovincialis Lam). The enzyme activity was assayed in tissue homogenates from gills and digestive gland. The toxic effect of Hg2+ appeared to be stronger than that of Cu2+ both in vitro and in vivo, especially for the digestive gland. In in vitro tests, Hg2+ was able to inhibit PLC activity when added directly to the reaction mixture. Conversely, Cu2+ was effective only after preincubation, suggesting that the effect of the metal may be derived from lipid peroxidation due to Cu2+-induced oxyradical production. Treatment of mussels with sublethal concentrations of Hg2+ or Cu2+ in vivo produced significant PLC inhibition after 1 or 4 days, respectively. A recovery was reached after 7 days of in vivo metal incubation. Data indicate that in mussel gills and digestive gland heavy metals impair PLC activity, thereby affecting IP3-dependent Ca2+ signaling.

Abstract: Cyclic ADP-ribose (cADPR) elicits calcium-induced calcium release (CICR) in a variety of cell types. We studied the effect of cADPR on Ca2+ release in muscle cells by incubating SR vesicles from scallop (Pecten jacobaeus) adductor muscle in the presence of the Ca2+ tracer fluo-3. Exposure of SR to cADPR (20 microM) produced Ca2+ release, which was a function of free [Ca2+] in a range between about 150 and 1000 nM, indicating an involvement of ryanodine-sensitive Ca2+ channels. This Ca2+ release was not significantly enhanced by calmodulin (7 micrograms/ml), but it was enhanced by equimolar addition of noncyclic ADPR. Also, the Ca2+ release elicited by cADPR/ADPR was a function of free [Ca2+] in a range between about 150 and 3000 nM, over which Ca2+ was inhibitory. cADPR self-inactivation was observed at low free [Ca2+] (about 150 nM), but it tended to disappear upon [Ca2+] elevation (about 250 nM). Caffeine or ryanodine induced a Ca2+ release which was ruthenium red (2.5 microM) sensitive at low [Ca2+]. However, the Ca2+ release induced by either ryanodine or cADPR was no longer ruthenium red sensitive when free [Ca2+] was increased. Based on these data, a model is proposed for Ca2+ signaling in muscle cells, where a steady-state cADPR level would trigger Ca2+ release when free [Ca2+] does reach a threshold slightly above its resting level, hence producing cascade RyR recruitment along SR cisternae from initial Ca2+ signaling sites.

Abstract: (Na+,K+ )ATPase activity was evaluated in membranes from rat hippocampal slices after in vitro hypoxia and reoxygenation. Membranes were prepared with two different methods, one using an isotonic medium and another using a hypotonic one. The changes that were found after hypoxia went into opposite directions in the two cases. Membranes prepared in a hypotonic medium are probably more suitable for these measurements. Using these membranes, hypoxia results in a slight decrease of (Na+,K+)ATPase activity and in a further decrease after reoxygenation. We also found that expressing (Na+,K+)ATPase activity as a percent of total ATPase activity is appropriate for membranes prepared under hypotonic conditions and can unveil (by reducing variability between experiments) significant changes that may be masked in small samples like ours.

Abstract: Two forms of Ca(2+)-pump were identified in bovine brain synaptic membranes as aspartylphosphate intermediates and were characterized. The 140 kDa and 97 kDa phosphoproteins were digested by calpain, producing two phosphorylated fragments, of M.W. 124 and 80 kDa respectively, not inhibited by thapsigargin, and displayed a trypsin digestion pattern with the formation of one phosphorylatable fragment of about 80 kDa. These results suggest that both pumps belong to the Plasma Membrane-type of Ca2+ ATPases, differing from the Sarco- or Endoplasmic Reticulum kind. A plasma membrane Ca(2+)-ATPase proteinaceous inhibitor with molecular weight between 6,000 and 10,000 Da was resolved from synaptic terminal cytosol, where it is enriched by fourfold with respect to frontal cortex brain cytosol. Such enrichment is already evident in the correspondent crude fractions. The presence of calcium pump and its proteinaceous inhibitor inside the synaptic terminals from bovine brain is discussed in terms of free calcium level regulation in neuron synaptoplasm.

Abstract: Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.

Abstract: The existence of a Ca2+ pump in rod outer segment disks of bovine retina is strongly suggested by the isolation on sodium dodecyl sulfate polyacrylamide gel electrophoresis of a hydroxylamine-sensitive phosphorylated intermediate (E-P) of molecular mass of about 100 kDa as well as by measurements of active calcium transport and adenosine 5'-triphosphate (ATP) hydrolysis. Active Ca2+ uptake by disks was dependent on the presence of Mg(2+)-ATP, was inhibited by vanadate or lanthanum and appeared poorly sensitive to calmodulin. ATP hydrolysis by disk membranes was a function of free Ca2+ concentration in the absence of exogenous Mg2+. The presence of a Ca2+ pump on disk membranes is discussed in terms of its possible role in Ca2+ ion buffering during photoreceptor cell functioning.

Abstract: The Ca(2+)-pump isolated in rod outer segment disk membranes as aspartylphosphate intermediate E-P has been characterized: the 100 kDa phosphoprotein was completely inhibited by thapsigargin, was not sensitive to digestion by calpain, and displayed a tryptic digestion pattern with the formation of two autophosphorylatable fragments of about 55 and 35 kDa. These results are typical of the calcium pumps of sarcoplasmic or endoplasmic reticulum calcium and differ from those of plasma membrane, such as the Ca(2+)-ATPase of the red blood cells, here shown as controls. The physiological role of calcium pump in disk membranes of vertebrate photoreceptors is discussed in terms of intracellular calcium buffering.

Abstract: 1. Polyphosphoinositide-specific phosphodiesterase (phospholipase C, PLC) activity against phosphatidylinositol 4,5-bisphosphate, present in gill and digestive gland homogenates of mussel (Mytilus galloprovincialis Lam.), has been biochemically characterized. 2. The enzyme was strictly modulated by free calcium ion concentration in both tissues and maximally activated at 10(-5) M Ca2+ (19 +/- 4 and 11 +/- 2 nmol phosphatidylinositol 4,5-bisphosphate hydrolysed/min/mg of protein for gill and digestive gland PLC, respectively, at 19 degrees C). Optimum pH at 10(-5) M Ca2+ was around 7.0 in both cases. The Ca(2+)-stimulated PLC activity showed high specificity for PIP2; the KMa for PIP2 were 150 and 170 microM for the gills and digestive gland, respectively. 3. Good substrate dispersion was obtained in the presence of sodium deoxycholate; the concentration routinely used in the assay (0.08%) produced a 9-fold activation of both gill and digestive gland PLC, consistent with previous reports. 4. The possible biochemical and physiological role of the enzyme in mussel tissues is discussed.

Abstract: Polyphosphoinositide-specific phosphodiesterase (phospholipase C) activity against phosphatidylinositol 4,5-bisphosphate has been examined in disrupted bovine retinal rod outer segments. The enzyme was strictly modulated by free calcium ion concentration and maximally activated at 10(-5) M Ca2+ (91 +/- 4 nmoles phosphatidylinositol 4,5-bisphosphate hydrolyzed/min/mg of protein). Guanine nucleotides did not affect in vitro phospholipase C activity either in the presence or absence of light, carbachol or epinephrine. The pH optimum at 10(-5) M Ca2+ in the presence of sodium deoxycholate was 6.5. The enzyme of bovine rod outer segments was concluded to be indirectly regulated by the phototransduction events.

Abstract: A Ca(2+)-pumping ATPase activity is present in bovine retinal rod outer segment purified disks. The ATPase has a high Ca2+ affinity (KM = 25 microM). Low Ca2+ (n-microM) concentrations stimulate an ATP-dependent Ca2+ uptake and the ATP hydrolysis in the absence of exogenous Mg2+. Electrophoretic analysis of disk proteins after treatment with (gamma-32P)ATP shows the existence of the enzyme-phosphate acid-stable, hydroxylamine-sensitive intermediate complex of molecular mass of about 135 kDa. The results would indicate the presence of an inwardly directed Ca(2+)-ATPase pump acting on the disk membrane, that could be involved in the regulation of cytosolic free Ca2+ levels inside ROS.

Abstract: Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes.

Abstract: In the rod outer segment membranes of the bovine retina at least two members of the small molecular weight guanine nucleotide binding proteins were identified by means of the technique of binding radiolabeled GTP to nitrocellulose Western blots of proteins separated by sodium dodecyl sulphate gel electrophoresis. Such proteins, of 23 and 25 kDa, are able to specifically bind guanine nucleotides after denaturing treatments, and are not labeled by pertussis or cholera toxin-catalyzed ADP-ribosylation. The binding site is specific for GTP.

Abstract: A possible secondary structure of the globular part of the histone H1 was obtained with a statistical approach based on the GOR method. The results of circular dichroism measurements on the protein were taken into account in order to choose between theoretically equivalent structures.

Abstract: Bovine retinal rod outer segment membranes are enriched in a phosphoinositide-specific phosphodiesterase (phospholipase C) activity strictly modulated by free calcium ion concentration. The enzyme(s) was highly active on phosphatidylinositol 4,5-bisphosphate: maximal hydrolysis rate was attained at 10(-5)M Ca2+ and accounted for 91 +/- 4 nmoles hydrolyzed/min/mg of protein. The results support the notion that in vivo the enzyme(s) is regulated so as to conform to the phototransduction events.

Abstract: Light activation of guanylate cyclase at different calcium concentrations was studied in the rod outer segments of the toad retina. The enzyme becomes sensitive to calcium ions after a flash of light, showing an enhancement of its activity when Ca2+ concentration is lowered from 10(-4) M to 10(-8) M. A possible pathway of guanylate cyclase activation by light was also investigated by means of the antibody 4A to transducin. When added in excess to transducin, the antibody inhibits light activation of phosphodiesterase as well as of cyclase, suggesting a possible coupling of the two enzymes.

Abstract: Proteins of purified rod outer segments from toad retina were analysed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The binding of proteins with calmodulin and with guanosine triphosphate was studied by electroblotting the proteins resolved by electrophoresis onto nitrocellulose sheets and by incubating the blots with labelled ligands. The results indicate that rod outer segments from toad retina contain nine proteins which bind to calmodulin and one protein, different from transducin, that binds to guanosine triphosphate.

Abstract: Guanylate cyclase activity was measured in disrupted rod outer segments of the toad retina. The experiments showed that cGMP is synthesized from GTP at a rate of 3 +/- 1 nmol/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, while it is enhanced by flashes of light of increasing intensity upon lowering Ca from 10-5 to 10-8 M. In view of recent observations that shortly after a flash of light calcium activity inside the photoreceptor cell decreases, it seems likely that calcium plays a regulatory role in cGMP metabolism in visual excitation.

Abstract: The rod outer segments of toad retina contain a guanylate cyclase activity of about 3 +/- 1 nmol of cGMP formed/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, although it is enhanced by light upon lowering the Ca2+ concentration from 10(-5) to 10(-8) M. The activating effect of light on cyclase at low Ca2+ concentrations is enlarged upon increasing the light intensity. With a flash of light bleaching 7 X 10(-2) percent of rhodopsin, cyclase activity increased by a factor of 30 when Ca2+ levels dropped from 10(-5) to 10(-8) M. In view of recent observations that shortly after a flash of light the calcium activity inside the photoreceptor cell decreases, it seems likely that Ca2+ plays a regulatory role on cGMP metabolism in visual excitation.