Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on l-leucyl-β-naphthylamidase, alkaline phosphodiesterase I and Ca²⁺- or Mg²⁺-ATPase, but substantial proportions of the alkaline phosphatase and 5′-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not excluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.     

Stimulus-response coupling in a cell-free platelet membrane system. GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3

The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP-ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.     

Dicyclohexylcarbodiimide interaction with sarcoplasmic reticulum. Inhibition of Ca2+ efflux

Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + Pi, or membrane modification with acetic anhydride. Under the conditions used, the same concentrations of DCCD were required for inhibition of Ca2+ release, Ca2+-ATPase activity, and Ca2+ uptake. On the other hand, free Ca2+ or alkaline pH prevented the inhibition by DCCD of Ca2+-ATPase and coupled Ca2+ transport but not that of Ca2+ release. Moreover, several hydrophilic carboxyl reagents inhibited Ca2+-ATPase but not Ca2+ release. We suggest that a carboxyl residue(s), located in a hydrophobic region of a protein(s), is involved in the control of Ca2+ release, where DCCD interaction with this group blocks Ca2+ release. This group is distinct from the one involved in the inhibition of Ca2+-ATPase. DCCD also inhibited [3H]ryanodine binding to junctional sarcoplasmic reticulum membranes. The presence of Ca2+ or an alkaline pH only slightly affects the degree of inhibition of ryanodine binding by DCCD. Incubation of the membranes with [14C]DCCD resulted in labeling of 350-, 170-, 140-, 53-, and 30-kDa proteins in addition to the Ca2+-ATPase. The involvement of one or all of the DCCD-labeled proteins in Ca2+ release and ryanodine binding is discussed.     

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplasmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (less than 100 micrograms/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28-50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (greater than 100 micrograms/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20-200 micrograms/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Cross-linking of IgG receptors inhibits membrane immunoglobulin- stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor- induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg- mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2- loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

Causes of excitation-induced muscle cell damage in isometric contractions: mechanical stress or calcium overload?

Prolonged or unaccustomed exercise leads to muscle cell membrane damage, detectable as release of the intracellular enzyme lactic acid dehydrogenase (LDH). This is correlated to excitation-induced influx of Ca2+, but it cannot be excluded that mechanical stress contributes to the damage. We here explore this question using N-benzyl-p-toluene sulfonamide (BTS), which specifically blocks muscle contraction. Extensor digitorum longus muscles were prepared from 4-wk-old rats and mounted on holders for isometric contractions. Muscles were stimulated intermittently at 40 Hz for 15-60 min or exposed to the Ca2+ ionophore A23187. Electrical stimulation increased 45Ca influx 3-5 fold. This was followed by a progressive release of LDH, which was correlated to the influx of Ca2+. BTS (50 microM) caused a 90% inhibition of contractile force but had no effect on the excitation-induced 45Ca influx. After stimulation, ATP and creatine phosphate levels were higher in BTS-treated muscles, most likely due to the cessation of ATP-utilization for cross-bridge cycling, indicating a better energy status of these muscles. No release of LDH was observed in BTS-treated muscles. However, when exposed to anoxia, electrical stimulation caused a marked increase in LDH release that was not suppressed by BTS but associated with a decrease in the content of ATP. Dynamic passive stretching caused no increase in muscle Ca2+ content and only a minor release of LDH, whereas treatment with A23187 markedly increased LDH release both in control and BTS-treated muscles. In conclusion, after isometric contractions, muscle cell membrane damage depends on Ca2+ influx and energy status and not on mechanical stress.     

Increased permeability of mitochondria during Ca2+ release induced by t-butyl hydroperoxide or oxalacetate. the effect of ruthenium red

Ca2+ release from mitochondria induced by oxalacetate or t-butyl hydroperoxide is accompanied by loss of endogenous Mg2+ and K+, swelling, loss of membrane potential, and other alterations which indicate that Ca2+ release is a result of increased inner membrane permeability. When ruthenium red is added after Ca2+ uptake, but before the releasing agent, the extent of Ca2+ release is diminished as is the extent of Mg2+ and K+ depletion and the extent of swelling. Under these conditions, the membrane potential appears to remain at a high value. When Ca2+ release is induced by oxalacetate or t-butyl hydroperoxide and ruthenium red is added subsequently, an apparent regeneration of membrane potential is observed providing that the associated swelling and Mg2+ loss had not been completed at the time ruthenium red was added. Under these conditions subsequent swelling and Mg2+ loss are inhibited.l Ultrastructural observations show the mitochondria become permeable in response to Ca2+ plus oxalacetate or Ca2+ plus t-butyl hydroperoxide in a heterogeneous manner. Conditions which appear to separate Ca2+ release from a decline in membrane potential or to produce an apparent recovery of membrane potential following partial collapse are shown to prevent a subpopulation of the mitochondria from becoming permeable. It is shown that membrane potential probes will not indicate a decline in potential or the presence of a permeable fraction under these conditions. It is concluded that the presence of Ca2+ accumulation inhibitors does not separate Ca2+ release from the development of increased inner membrane permeability.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell.

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris--HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-napthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.     

Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GPI)-anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysoPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca²⁺-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein. J. Cell. Biochem. 65:550–564. © 1997 Wiley-Liss Inc.     

Mn、Ca阳离子在菠菜PSII颗粒氧化水中的作用

用Triton X-100处理菠菜叶绿体,获得一个基本不含PSⅠ成分、而具放氧活性的PSⅡ颗粒。最适pH移至6.9,超过pH7.2就发生凝集,在照光下只形成很小或不形成H~+梯度,只有微弱的毫秒延迟荧光发射,老化和解联剂都不加速电子传递。 Mn、Ca阳离子促进PSⅡ颗粒的放氧和H~+释放,两者作用不能叠加。Mn离子只作用于活化的PSⅡ颗粒,对叶绿体和部分失活的PSⅡ颗粒无效。Ca离子对叶绿体、PSⅡ颗粒或部分失活的PSⅡ颗粒,都有相同程度的促进效应。     

Calcium ionophoretic activity of chemically synthesized oligomeric derivatives of prostaglandin B1

Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.