Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.     

Immunochemical characterization of rat brain protein kinase C

Polyclonal antibodies against rat brain protein kinase C (the Ca2+/phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca2+, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca2+/phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of [3H]phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. This degraded form of the phorbol ester-binding protein still requires phospholipid for activity but, unlike the native enzyme, becomes less dependent on Ca2+. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca2+-independent/phospholipid-dependent phorbol ester-binding protein.     

Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions

Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLA2 activities were demonstrated: PLA2-H (200-500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1-100 microM Ca2+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2(+)-dependent manner. The association was completed within 5-10 min at 25 degrees C both at 10 microM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLA2, are present.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Effects of endothelin on sodium transport mechanisms: potential role in cellular Ca2+ mobilization

The effects of endothelin on cellular Ca2+ mobilization were examined in cultured rat vascular smooth muscle cells (VSMC). Endothelin (10(-8)M) induced a rapid transient increase of [Ca2+]i from 77 +/- 3 to 104 +/- 5 nM (p less than .05) in VSMC. Preincubation (60 min) with endothelin (2 x 10(-6)M) increased basal [Ca2+]i from 77 +/- 3 to 105 +/- 8 nM (p less than .05). Preincubation with endothelin also enhanced vasopressin (10(-7)M)-stimulated peak levels of [Ca2+]i (528 +/- 20 nM vs 969 +/- 21 nM, p less than .01). Endothelin (10(-7)M) induced an intracellular alkalinization (7.18 +/- 0.03 vs 7.37 +/- 0.04, p less than .01) which was blocked by pretreatment with amiloride. The biphasic effects of endothelin on [Ca2+]i were similar to those of an endogenous inhibitor of Na-K-ATPase that we examined in a previous study. Therefore, we examined the effects of endothelin on Na-K-ATPase in an enzyme preparation from hog cerebral cortex. At high concentrations, endothelin (10(-5)M) inhibited Na-K-ATPase in vitro. Thus, endothelin may exert its vasoconstrictor effects at least in part via alterations of cellular Ca2+ mobilization in VSMC. While the rapid transient increase of [Ca2+]i appears to reflect intracellular Ca2+ mobilization, the sustained effect on [Ca2+]i may be related to an increase of intracellular sodium mediated by inhibition of Na-K-ATPase and/or more likely by stimulation of the Na+/H+-antiport.     

A guanine nucleotide-binding protein participates in IgE receptor-mediated activation of endogenous and reconstituted phospholipase A2 in a permeabilized cell system

Activation of phospholipase A2 (PLA2) by the aggregation of receptors for immunoglobulin E (IgE) can be studied in streptolysin O-permeabilized rat basophilic leukemia cells. Under these conditions, 40 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) stimulates PLA2 activity 5-6-fold when free Ca2+ concentrations are buffered at 10(-7)-10(-5) M. Antigen-mediated cross-linking of receptors for IgE synergizes with low concentrations of GTP gamma S (0.1 microM) to cause similar stimulation. When the endogenous PLA2 activity is inactivated by chemical modification, we find that exogenously supplied PLA2 from porcine pancreas and Naja naja venom is also activated by the aggregation of cell-surface IgE receptors in these permeabilized cells. As with endogenous PLA2, GTP gamma S synergizes with IgE receptor-aggregation to activate exogenous PLA2 approximately 10-fold at 10(-7)-10(-6) M free Ca2+. These data indicate that receptor-mediated activation of a guanine nucleotide-binding protein can shift the Ca2+ dependence of PLA2 activity resulting in greatly enhanced activity at physiological concentrations of intracellular free Ca2+. The partial reconstitution of various PLA2 forms into such a broken-cell system offers a new approach for studying the mechanisms of G-protein-mediated activation of PLA2.     

Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.     

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.     

Bradykinin stimulated PGE2 production is independent of changes in intracellular calcium in MDCK cells

In order to assess whether changes in intracellular Ca2+ are necessary for bradykinin stimulated activation of phospholipase A2 and PGE2 production, MDCK cells were treated with phorbol myristate acetate (PMA) and Lanthanum (La3+). 100 nM PMA reduced peak BK (1 uM) stimulated Ca2+ to 44.0 +/- 11.4% of control, while 1 mM LaC13 reduced peak Ca2+ to 43.5 +/- 12.2% of control. Addition of both PMA and LaC13 reduced the BK stimulated change in intracellular Ca2+ to 8.3 +/- 1.0% of control. In contrast, La3+ did not reduce PGE2 production in response to 10(-7) M to 10(-5) M BK. PMA stimulated PGE2 production, as shown previously. Addition of both PMA and La3+ at doses capable of reducing Ca2+ changes to less than 10% of normal, failed to block BK induced PGE2 production. Therefore, BK stimulated PLA2 activation and PGE2 production can be dissociated from changes in intracellular Ca2+ and suggest that BK activates PLA2 through a mechanism other than by increases in intracellular Ca2+.     

Identification and purification of a calcium-binding protein from Bacillus subtilis

A Ca(2+)-binding protein was identified in Bacillus subtilis in the log phase of growth. The molecular mass of this protein is about 38 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS and by gel filtration. The protein was found to be resistant 10 min at 65 degrees C and was purified about 400 times, starting from heated crude extract, by conventional procedures. This novel protein is able to bind Ca2+ in the presence of an excess of MgCl2 and KCl both in solution and after SDS gel electrophoresis and electrotransfer. Since an impairment of the Ca2+ intake, in Bacillus subtilis, results in an impairment of chemotactic behavior (Matsushita, T. et al (1988) FEBS lett. 236, 437-440), 38 kDa protein may be involved in the regulation of chemotaxis.     

Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Distribution of the vasoconstrictor and vasorelaxant effects of norbormide along the vascular tree of the rat

Norbormide is a vasoconstrictor of rat peripheral arteries and a relaxant in rat aorta. To characterise norbormide actions within the rat vascular tree we have investigated its effects on the contractile function of rings from several arteries and veins. A maximal norbormide concentration (50 microM) failed to contract thoracic aorta and carotid artery, whereas in pulmonary artery, abdominal aorta, iliac, caudal, and femoral arteries it induced a contractile effect that was respectively 4.8 +/- 0.6, 18.4 +/- 1.5, 39 +/- 5, 144 +/- 7, and 260 +/- 22% of that induced by 90 mM KCl. In pulmonary, carotid, and iliac arteries, and in thoracic and abdominal aorta, 50 microM norbormide inhibited KCl-induced responses. Norbormide (50 microM) contracted all veins investigated. The effect, expressed as % of KCl-induced contraction, was 121 +/- 25, 154 +/- 14.5, 154 +/- 18.2, 203 +/- 19, and 267 +/- 33 for pulmonary vein, thoracic and abdominal vena cava, iliac and jugular veins, respectively. In jugular vein, as previously shown in rat caudal artery, norbormide contraction was abolished in Ca2+-free medium, was unaffected by the Ca2+ channel blocker nifedipine, and was relaxed by SK&F 96365, a blocker of store-operated Ca2+ channels. In conclusion: i) rat veins represent the main target for contractile norbormide action; ii) in both artery and veins norbormide contractions are generally inversely related to the calibre of the vessel; iii) norbormide-induced contraction is mediated by the same mechanism/s in arteries and veins; iiii) in norbormide-contracted arteries the drug activates both contractile and relaxing mechanisms.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.     

Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.     

Pharmacological action of the phospholipase A2 from the venom of Trimeresurus flavoviridis on the smooth muscle of the rat stomach

Phospholipase A2 (PLA2) from the venom of the snake Trimeresurus flavoviridis produced an increase in resting tension of isolated strips of rat stomach fundus. The contractions of the fundus strips induced by the PLA2 were significantly inhibited by treatment with 10(-6) M indomethacin and in Ca2+-free medium, while treatment of the fundus strips with nordihydroguaiaretic acid caused a marked potentiation of the PLA2-induced contraction. Atropine (10(-6) M), chlorpheniramine (10(-6) M) and methysergide (10(-6) M) had no effects on the contractions induced by PLA2, while tetrodotoxin (10(-6) M) significantly potentiated the contraction. From these results, it appears that exogenously applied PLA2 may cause contraction of the rat stomach fundus through the liberation of endogenous arachidonic acid which may then be transformed into prostaglandins.     

Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties

It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins.     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.