Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Cationic dependency of high affinity prostaglandin F2α receptors in bovine corpus luteum cell membranes

The specific binding of [³H] Prostaglandin (PG) F₂α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca²⁺ (HCa²⁺) was examined. The membranes prepared in H-EDTA buffer bound less [³H] PGF₂α and had a single class of PGF₂α receptors with an apparent dissociation constant (Kd) of 2.7 × 10^?8M. The addition of Ca²⁺ to these membranes resulted in increased binding with the appearance of new PGF₂α receptors of Kd = 4.3 × 10^?9M. The membranes prepared in HCa²⁺ buffer contained two classes of receptors with Kds = 2.9 × 10^?9M and 2.9 × 10^?8M. The removal of Ca²⁺ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10^?9M. These results suggest the dependency of high affinity PGF₂α receptors, in bovine corpus luteum cell membranes, on cations.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.     

A naphthalene derivative as ‘turn‐on’ fluorescent chemosensor for the highly selective and sensitive detection of Al3+

A Schiff base compound derived from naphthalene has been synthesized and characterized as an Al³⁺‐selective fluorescent probe. The chemosensor ( L ) exhibits high selectively for Al³⁺ in aqueous solution, even in the presence of biologically relevant cations such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Pb²⁺ and several transition metal ions. There was no observed interference from anions like Br^?, Cl^?, HSO₃^?, SO₃^2?, S₂O₃^2?, NO₂^?, CO₃^2? and AC^?. The lowest detection limit for the chemosensor L was found to be 1.89 × 10^?8 M with a linear response towards Al³⁺ over a concentration range of 5 × 10^?6 to 4 × 10^?5 M. Furthermore, the proposed chemosensor has been used for imaging of Al³⁺ in two different types of cells with satisfying results, which further demonstrates its value for practical application in biological systems.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^?1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

Nucleus Basal Body Connector of Dunaliella: Threshold Concentration of Calcium Necessary for in vitro Contraction*

Detergent-isolated flagellar apparatuses of the flagellate green alga Dunaliella bioculata retain remnants of the nucleus (the karyoskeleton) which are linked to the basal bodies by the centrin-containing nucleus basal body connectors (NBBC). Such complexes were subjected to different calcium concentrations (1 × 10^?9 M ? 5 × 10^?4 M Ca²⁺) and the distance between the basal bodies and the karyoskeleton was measured by light microscopy. The threshold concentration of Ca²⁺ for NBBC contraction was determined to be around 5 × 10^?8 M Ca²⁺. At > 10^?7 M Ca²⁺ NBBC were maximally contracted and the distance between the basal bodies and the karyoskeleton was only about 50% of the initial distance. Using a polyclonal antibody generated against centrin (Salisbury et al., 1984), the NBBC were visualized by indirect immunofluorescence in both the extended and contracted state. Our results demonstrate that in vitro contraction of centrin-containing filaments in green algae is initiated at about the same free Ca²⁺ concentration in three different centrin-containing basal apparatus components (i.e. the distal connecting fibre, the flagellar transitional region and the NBBC).     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Effects of some steroid hormones on head-to-head association in bovine spermatozoa

In the presence of 2 × 10^?6 M Ca²⁺ in Tris-buffered medium 0.5 × 10^?6 M, oestradiol-17β or corticosterone significantly increased the head-to-head association of washed bull spermatozoa; in the same concentration, testosterone and 5α-dihydrotestosterone had no significant effects, whereas progesterone significantly dissociated the associated spermatozoa. At 8 × 10^?6 M Ca²⁺ in the same medium, all five hormones increased the association to about the same level. In Tyrode solution with a Ca²⁺ concentration of 1.4 × 10^?3 M, oestradiol-17β and corticosterone acted as above, whereas progesterone and the two testosterones effected dissociation. In Tyrode solution each of the dissociating hormones was combined with oestradiol-17β. In each case a sum of the effects of the two hormones was obtained without any stimulation or inhibition. All five hormones still produced significant effects at 5 × 10^?7 M in Tyrode solution. A corresponding value for ATP was found at 1 × 10^?5 M.     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

L'adeninephosphoribosyltransferase des pousses de topinambour Helianthus tuberosus

An extract from Jerusalem artichoke shoots exhibited important adeninephosphoribosyltransferase activity. Only partial purification was possible because of the great instability of the enzyme. Phosphate ions and thiol reducing substances were necessary to stabilize it. The optimal temperature and pH were 40–45° and 5.5 to 6.5. The enzyme showed an absolute requirement for divalent cation: Mn²⁺ > Mg²⁺ = CO²⁺ = Zn²⁺ ? Ca²⁺. Kinetic studies gave K_m values of 6.4 × 10^?1 M for phosphoribosylpyrophosphate (PRPP) and 5.5 × 10^?6 M for adenine. AMP exercised a strong product inhibition, competitive towards PRPP (K_i = 10^?4 M). Inhibition by phosphate and pyrophosphate ions was also observed. The results suggested that the adeninephosphoribosyltransferase of Helianthus tuberosus has a key role in the purine salvage pathway.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

Activating effect of destomycin A on adenylate cyclase from several animal tissues

Highly purified sarcolemmal membranes were prepared from pig heart homogenates by differential and density gradient centrifugations. The membrane fragments exhibit ATP-dependent Ca²⁺-transport and Na⁺/Ca²⁺-exchange activities. ATP-dependent Ca²⁺-transport (K^Ca2+_0.5 = 0.3 μM; V_max = 4.6 nmol Ca²⁺?mg protein^?1 ?min^?1)_is not stimulated by oxalate. Ca²⁺-uptake is also not supported by p-nitrophenylphosphate. Preincubation of sarcolemma with MgATP, calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase stimulates active Ca²⁺-transport 1.8-fold. The effects of calmodulin and catalytic subunit are potentiating rather than additive. A large portion of the Ca²⁺ additionally accumulated after prephosphorylation of membranes is exchangable for Na⁺ via the Na⁺/Ca²⁺-exchange system.     

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca²⁺. At a constant benzyladenine (BA) level (4.4×10^?5 M) 10^?4 to 10^?2 M Ca²⁺ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10^?6 M) concentration of external Ca²⁺, Chl synthesis was relatively more Ca²⁺-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca²⁺ from external sources to cytosol, and/or (b) interacted with Ca²⁺ directly in the pathway of Chl biosynthesis. The need of external Ca²⁺ was evidenced in experiments with modulators of Ca²⁺-transport systems. The reduction of the inward current of Ca²⁺ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N′N′-tetraacetic acid, EGTA) and Ca²⁺-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca²⁺. Inhibitory action of Ca²⁺-channel blockers depended on their kind and concentration: at the 10^?5 M concentration La³⁺>verapamil>nifedipine inhibited Chl formation. In the presence of Ca²⁺, the Ca²⁺-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.     

Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes

Prostaglandin (PG) F_2α increased [³H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner with ED₅₀ values of 2.0 × 10^?8 M, 4.6 × 10^?8 M, and 7.5 × 10^?8 M, respectively. The increase in [³H]thymidine incorporation with PGF_2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca²⁺]_i increase with PGF_2α was still obvious in the absence of extracellular Ca²⁺ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF_2α, which was sensitive to GTP_γS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF_2α-specific Cl^? current when examined by voltage clamp. This Cl^? current was also insensitive to IAP pretreatment and not affected by extracellular Ca²⁺ concentration ([Ca²⁺]_o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF_2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca²⁺ via an IAP-insensitive G-proteins(s), 2) that this PGF_2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF_2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.