A Novel Biosynthetic Pathway of Archaetidyl-myo-inositol via Archaetidyl-myo-inositol Phosphate from CDP-archaeol and d-Glucose 6-Phosphate in Methanoarchaeon Methanothermobacter thermautotrophicus Cells

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[¹⁴C]inositol 1-phosphate, which was enzymatically prepared from [¹⁴C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.     

Ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in Bacteria and Archaea,which contain inositol phospholipid

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles

The activity of phosphatidylserine (PS) synthase (CDP-1,2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8.8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Polysaccharides from the pulp of cupuassu (Theobroma grandiflorum): Structural characterization of a pectic fraction

Cupuassu (Theobroma grandiflorum, Schumann) is a Brazilian Amazonian fruit whose pulp contains volatile compounds that have been extensively studied. In this work, the pulp from fruits of cupuassu was ground, treated with MeOH–H₂O, and defatted with p-Tol–EtOH. The residue (4% in relation to the fresh pulp) was submitted to sequential extractions with water, aqueous citric acid and aqueous NaOH, resulting in polysaccharide fractions with 0.3–15% yield. The main pectic fraction (7% yield) was obtained with water at 25 °C (W-1 fraction) and was chosen to be better characterized. Chemical and spectroscopic analyses showed that W-1 is composed mainly of a homogalacturonan highly esterified (DE 53%; DA 1.7%) with some rhamnogalacturonan insertions, carrying side chains containing galactose and arabinose.     

Isolation and characterisation of lignin-carbohydrate complexes from the milled-wood lignin fraction of Pinus densiflora sieb. et zucc.

The lignin-carbohydrate complex (LCC-W), isolated from the milled-wood, lignin fraction of Pinus densiflora Sieb. et Zucc., comprised three fractions (W-1,2,3) by gel filtration on Sepharose 4B. W-1 was eluted at the void volume, whereas W-2 and W-3 were included in the gel and had apparent weight-average molecular weights of 5.0 × 10⁵ and 5.0 × 10³, respectively. W-2 and W-3 were homogeneous in ultracentrifugal and electrophoretic analyses. The sedimentation coefficients of W-2 and W-3 were 25.7 and 0.4S, respectively. The chemical composition of W-2 was 38.0% of neutral sugar, 6.2% of uronic acid, 51.5% of lignin, and the corresponding values for W-3 were 73.1, 11.0, and 22.2%. The neutral carbohydrate residues of W-2 and W-3 were l-arabinose, d-xylose, d-mannose, d-galactose, and d-glucose in the ratios 15.8:16.2:37.3:16.7:14.0 and 27.6:16.5:26.1:19.3:10.5, respectively. Based on the results of methylation and Smith-degradation analyses, the carbohydrate moiety of the LCC-W fractions was found to be multiply branched. The major backbone structure was composed of (1→4)-linked d-mannopyranosyl residues. By hydrophobicinteraction chromatography on Phenyl- and Octyl-Sepharsoe CL-4B gels, it is concluded that the LCC-W fractions have a hydrophobic property that is exclusively ascribed to the lignin moiety.     

Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase

Myo-inositol may be incorporated in the formation of phosphatidylinositol by two mechanisms. One reaction utilizes CDP-diacylglycerol and is catalyzed by phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). The second reaction is the phosphatidylinositol: myo-inositol exchange reaction, in which a free inositol is exchanged for an existing inositol headgroup. This characterization of inositol incorporation into phosphatidylinositol in the green alga Chlamydomonas reinhardtii provides evidence for the presence of both reactions. The transferase reaction required a divalent cation and exhibited its maximum activity at 2.0 mM Mn²⁺. The optimal pH for this reaction was 8.5–9.0. The best substrate concentrations were 0.5 mM CDP-diacylglycerol and 1.2 mM myo-inositol, with an estimated K_m for myo-inositol of 0.2 mM. The exchange reaction also required Mn²⁺ for activity, but became saturated at 0.5 mM Mn²⁺. The optimal pH of the exchange reaction was 8.0, the optimal myo-inositol concentration was 0.3 mM, and the estimated K_m for myo-inositol in this reaction was 0.015 mM. Measurement of the transferase reaction in cell fractions of C. reinhardtii indicated that the activity occurred primarily in the microsomal fraction, with little or no activity in the plastids.     

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae

Membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC2.7.8.8.) and CDP-diacylglycerol:myo-inositol phosphatidyltransferase (phosphatidylinositol synthase, EC 2.7.8.11) were solubilized from the microsomal fraction of Saccharomyces cerevisiae. A variety of detergents were examined for their ability to release phosphatidylserine synthase and phosphatidylinositol synthase activities from the microsome fraction. Both enzymes were solubilized from the microsome fraction with Renex 690 in yield over 80% with increase to specific activity of 1.6-fold. Both solubilized enzymatic activities were dependent on manganese ions and Triton X-100 for maximum activity. The pH optimum for each reaction was 8.0. The apparent Km values for CDP-diacylglycerol and serine for the phosphatidylserine synthase reaction were 0.1 and 0.25 mM, respectively. The apparent Km values for CDP-diacylglycerol and inositol for the phosphatidylinositol synthase reaction were 70 microM and 0.1 mM, respectively. Thioreactive agents inhibited both enzymatic activities. Both solubilized enzymatic activities were thermally inactivated at temperatures above 30 degrees C.     

Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles

The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [³H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [³H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed.     

Inhibition by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide,a calmodulin inhibitor,of dopamine release from rat brain synaptosomes

The release of preloaded [³H]dopamine by the synaptosomal fraction prepared from rat forebrain was examined in the presence and absence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor. The release induced by high K⁺ was blocked by W-7 in a concentration-dependent manner after the pretreatment with and in the presence of the inhibitor. The inhibition by W-7 may specifically involve calmodulin, because little effects were seen with N-(6-aminohexyl)-naphthalenesulfonamide, an analog of W-7 with only a low affinity for calmodulin. W-7 may not affect the voltage-dependent Ca²⁺ channel of synaptosomal plasmalemma, since the inhibitor produced no change in the synaptosomal ⁴⁵Ca²⁺ uptake induced by high K⁺ depolarization. Thus, calmodulin may play a role in transmitter release and may function at the step(s) after the increase of free Ca²⁺ concentration in the cytosol of the nerve terminal. W-7 affected only to a small extent [³H]dopamine release in the presence of A23187 plus Ca²⁺.     

Regulation of Plasma Membrane beta-Glucan Synthase from Red Beet Root by Phospholipids : Reactivation of Triton X-100 Extracted Glucan Synthase by Phospholipids

Extraction of red beet root plasma membranes with the detergent Triton X-100 at a level of 2.0% (weight/volume) resulted in the depletion of over 90% of total membrane phospholipid and the reduction of glucan synthase activity by 80 to 90%. Reconstitution of the delipidated Triton X-100, 100,000g fraction in the presence of phospholipids restored glucan synthase activity. The most effective phospholipid was phosphatidyl-ethanolamine, which restored 110 to 144% of the original activity at 0.5% (weight/volume). Glucan synthase in the phospholipid-reactivated Triton X-100-treated fraction was enriched 9-fold in specific activity relative to microsomal membranes but was unstable in digitonin. These results support the hypothesis that glucan synthase activity is regulated by its phospholipid environment.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. II. Phospholipids of ciliary and other membranes

The phospholipids of cilia and deciliated bodies of Paramecium tetraurelia were isolated and characterized. 1-alkyl-2-acyl-sn-glycero-3-(2′-aminoethyl) phosphonate (GAEPL), phosphatidylethanolamine, and 1-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (GPC) were the major lipids of Paramecium, and the minor lipids included phosphatidylinositol, cardiolipin, ceramide-(2-aminoethyl) phosphonate (CAEP), ceramide phosphorylethanolamine (COPE) and four sphingolipids whose identity was not established. The deciliated bodies contained 4% cardiolipin, 15% GAEPL, 41% phosphatidylethanolamine, 30% GPC and 3% each of CAEP and phosphatidylinositol; the cilia contained no cardiolipin, 24% GAEPL, 37% phosphatidylethanolamine, 15% GPC, 15% CAEP, 3% phosphatidylinositol, 2% COPE and small amounts (approx. 1%) of the four uncharacterized sphingolipids. No alteration in phospholipid composition was found among cells harvested in the various stages of growth. The phospholipids of six Paramecium mutants of three distinct phenotypes (pawn, paranoiac and fast) were also examined. Only one significant difference was found on comparison of the whole cell, deciliated body and cilia fraction of the mutants with the analogous fractions from wild type cells: the fast mutant, fA 97, had two extra, minor phospholipids (approx. 2%) in the deciliated body fraction that were tentatively identified as 1,2-diacyl-sn-glycero-3-(2′-aminoethyl) phosphonate (AEPL) and 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine (GPE).     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Ram-induced oestrus and ovulation in lactating and weaned Corriedale ewes

Two experiments were conducted in consecutive years in which recently (Experiment 1) or temporarily (Experiment 2) weaned ewes and matched post-partum non-lactating flockmates (DRY) were exposed to a stimulus group of rams and oestrous ewes (10 and 20 in Experiment 1, 20 and 20 in Experiment 2) for 28 days in spring. Lactating ewes (n = 130) in Experiment 1 were isolated from their lambs 4 (W-4), 2 (W-2), 1 (W-1) or 0 (W-0) days in advance and exposed along with a group of 32 DRY flockmates. Lactating ewes in Experiment 2 (n = 230) were allocated to an unreplicated factorial of two levels of temporary weaning before stimulation (B0: control; B24: lambs removed 24 h before stimulation) by four levels of ewe-lamb contact imposed at the start of the stimulation (A0: control; A12, A24 and A36: lamb-ewe separation during the initial 12, 24 or 36 h of exposure); DRY ewes (n = 54) acted as an augmented factorial control. Oestrus (rump marks) and ovulation (laparoscopy on day 5 and on day 28 (Experiment 1) or day 32 (Experiment 2)) were recorded. Ovulation and oestrous responses in Experiment 1 were similar for DRY (90.6% and 55.2%, respectively) and recently weaned ewes (83.8% and 53.7%, respectively). Amongst recently weaned ewes, the immediate ovulation response to the rams and the proportion of ewes still cycling by day 28 tended to be lower (P = 0.065 and P = 0.011) in ewes weaned on the day of ram exposure (71.9% and 54.8% v. 87.8% and 80.0%, respectively). Ovulation rate was lower (P < 0.003) in W-2 ewes (1.3 ± 0.10) than in the other recently weaned groups. In Experiment 2, ovulation (83.3%) and oestrous (68.9%) responses in DRY ewes were higher (P = 0.022 and P = 0.053, respectively) than in lactating ewes (66.2% and 51.0%, respectively). More ewes ovulated (P = 0.036) in B24 (70.5%) than in B0 (61.8%). Ewes having their lambs returned 12 h after the onset of stimulation (A12) had poorer ovulation responses (54.9%) than control ewes (A0, 72.9%, P < 0.05); this was probably associated to lamb restitution after the sunset. Main conclusions were that (i) the presence of the lambs is a depressing factor of both ovulation and oestrous responses to the ram effect in lactating ewes, (ii) the ovulation response of lactating ewes will probably benefit from removing lambs for a period of 24 h before the onset of stimulation, (iii) until additional information becomes available, temporary weaning protocols should be designed avoiding lamb restitution during the night.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Cloning of Arabidopsis thaliana phosphatidylinositol synthase and functional expression in the yeast pis mutant

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg²⁺ ions for full activity.     

Design,synthesis and evaluation of 2-amino-imidazol-4-one derivatives as potent β-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors

Inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) to prevent brain β-amyloid (Aβ) peptide’s formation is a potential effective approach to treat Alzheimer’s disease. In this report we described a structure-based optimization of a series of BACE1 inhibitors derived from an iminopyrimidinone scaffold W-41 (IC₅₀ = 7.1 μM) by Wyeth, which had good selectivity and brain permeability but low activity. The results showed that occupying the S₃ cavity of BACE1 enzyme could be an effective strategy to increase the biological activity, and five compounds exhibited stronger inhibitory activity and higher liposolubility than W-41, with L-5 was the most potent inhibitor against BACE1 (IC₅₀ = 0.12 μM, logP = 2.49).     

Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [³H]phosphatidylinositol 4,5-bisphosphate (PIP₂). Based on release of [³H]inositol phosphates, the plasma membrane exhibited a PIP₂-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast `bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [³H]inositol phosphates released were recovered as [³H]inositol trisphosphate (IP₃). These results suggest a plausible mechanism for the rapid breakdown of PIP₂ and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [³H]inositol phospholipids during these assays revealed that some of the [³H]-PIP₂ was converted to [³H]phosphatidylinositol 4-monophosphate (PIP) and to [³H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP₂/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca²⁺]. We also report here that 100 micromolar GTPγS stimulates phosphospholipase C activity over a range of free [Ca²⁺]. Together, these results provide evidence that the plasma membrane PIP₂-phospholipase C of D. salina may be subject to Ca²⁺ and G-protein regulation.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.