Effect of Japanese Paramecium bursaria extract on photosynthetic carbon fixation of symbiotic algae

To investigate the relationship between the Japanese Paramecium bursaria host and its symbiont, we studied the effect of a host cell-free extract on carbon fixation and photosynthate release of the symbiont. The host extract enhanced symbiotic algal carbon fixation about 3-fold at an increased concentration; however, release of photosynthate hardly changed. Since the enhancing effect was not affected by elimination of carbon dioxide from the host extract, the existence of a host factor that stimulates algal carbon fixation was made clear. The host factor is a heat-stable, low molecular weight substance. In relation to the pH dependence, the extract improved carbon fixation at acidic and neutral pH and showed almost no effect at pH 9.0. Therefore, the stimulation of carbon fixation by the host factor is unlikely to be caused by intracellular pH change. The extract also improved carbon fixation of several Chlorella species, symbiotic and free-living, and apparently exhibited no species specificity. Therefore, the host seems to regulate the photosynthesis of the symbiont via a specific compound.     

Comparative studies on photosynthetic enzymes of the symbiotic Chlorella from Paramecium bursaria and other symbiotic and non-symbiotic Chlorella strains

The activities of ribulose 1,5-bisphosphate carboxylase and of carbonic anhydrase were studied in cell-free extracts of two symbiotic Chlorella strains isolated from Paramecium bursaria and from Spongilla sp., and of two nonsymbiotic strains of Chlorella (Chlorella fusca and Chlorella vulgaris) cultivated at varied CO₂-concentrations. The symbiotic Chlorella of Paramecium bursaria differs distinctly from the other Chlorella strains by a higher activity of ribulose 1,5-bisphosphate carboxylase, which is independent of the actual CO₂-concentration, and by a lack of carbonic anhydrase activity. These properties are discussed with respect to their ecological significance.Abbreviations CA carbonic anhydrase - Pbi Paramecium bursaria isolate - RuBP ribulose 1,5-bisphosphate Dedicated to Prof. Dr. André Pirson on the occasion of his 70th birthday     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

鼎湖山主要森林类型土壤交换性阳离子含量及其季节动态特征

研究鼎湖山自然保护区马尾松林、马尾松荷木混交林和季风常绿阔叶林三种代表性森林类型表层土壤(0~20cm)交换性阳离子含量及其季节动态。结果表明:土壤交换性阳离子含量因元素种类、森林类型和季节不同而异。三种森林土壤交换性阳离子含量都表现为:Al3+>H+>K+>Ca2+、Mg2+、Na+。几乎所有调查的阳离子含量在阔叶林显著高于马尾松林和混交林,但后两者之间大多数阳离子含量差异不显著。鼎湖山森林土壤可交换性阳离子含量虽然较高,但盐基饱和度却很低。马尾松林、混交林和阔叶林土壤可交换性阳离子含量在1997年6月份分别为:58.3、84.5和118.7mmolc/kg,盐基饱和度分别为:5.5%、3.2%和4.5%。三种森林土壤交换性Ca2+、Mg2+、K+和H+含量季节差异极显著(P<0.001),但交换性Al3+含量只在马尾松林土壤存在极显著的季节性差异(P<0.001)。同一元素季节变化大小程度趋向马尾松林>混交林>阔叶林。森林土壤交换性Ca2+、Na+和H+含量与土壤pH值相关关系不明显,但交换性Mg2+、K+和Al3+与土壤pH值间呈极显著负相关。     

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

Photosynthetic Shutdown in Chlorella NC64A Associated with the Infection Cycle of Paramecium bursaria Chlorella Virus-1

The effects of the algal virus Paramecium bursaria Chlorella virus-1 on the photosynthetic physiology of its host, Chlorella NC64A, was studied by observing changes in Chl fluorescence quenching and O2 exchange. Metabolic changes were calibrated against electron microscopic analysis of the morphological changes that occur during the infection cycle. It takes approximately 10 h from attachment of the virus to final lysis of the host cell, so a complete infection cycle can be observed continuously in one experiment. During the early stages of the infection cycle many rapid changes occurred in the host cell's metabolism and these were reflected in changes of photosynthetic and respiratory rates. The dramatic inhibition of photosynthesis in Chlorella NC64A cells by P. bursaria Chlorella virus-1 has facilitated the use of fluorescence quenching as an accurate measure of the first phase of viral infection (attachment and penetration of the host cell) and the extent to which a population of host cells is infected. Effects of temperature and cation requirement of the infection cycle are described. The relevance of our observations to the events observed during viral infection of higher plants is discussed.     

Infection of Alga-Free Paramecium bursaria with Strains of Chlorella, Scenedesmus, and a Yeast

SYNOPSIS. Twenty-one different stocks of Paramecittm bursaria , belonging t o 4 separate varieties (syngens), whose endosymbiotic chorellae had been removed, were tested for reinfection by several strains of Chorella , some previously isolated from P. bursaria , and others free-living. In addition, infection of P. bursaria by a single strain of the green alga Scenedesmus sp., and an unidentified strain of yeast was attempted. Most combinations involving Chlorella yielded infected paramecia, and all those with Scenedesmus or the yeast did so. The failures with Chlorella were attributed to low infectibility of the stocks of Paramecium concerned, rather than to inability of the Chlorella to survive inside the paramecia. Little evidence was found that the strains of P. bursaria differed genetically in ability to maintain the symbiotic organisms.     

Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

绿草履虫-小球藻共生系统中共生藻对宿主细胞超微结构的影响

应用透射电镜术显示了含小球藻绿草履虫和人工诱导获得的无小球藻绿草履虫细胞的超微结构特征,无小球藻绿草履虫细胞内有大量处于不同消化阶段的食物泡及膜性小泡,在细胞质内常见有线粒体聚集分布以及内质网分布其中,细胞大核内核仁数目增多,并聚集形成多个核仁区。含小球藻绿草履虫中细胞膜性结构较少见,细胞大核中核仁数目较少。结果表明,小球藻共生体可能影响了宿主草履虫细胞中所述细胞器的功能,数量和分布,并影响了核仁的功能,数量和分布。     

Two Ca2+ functions are demonstrated by the substitution of specific divalent and lanthanide cations for the Ca2+ required by the aggregation factor complex from the marine sponge, Microciona prolifera

Multivalent cations were tested for their ability to replace the Ca2+ requirements of aggregation factor (AF) complex in activity, stability, and integrity assays. The ability of each cation to replace the Ca2+ required for the cell aggregation-enhancing activity of AF was examined by replacing the usual 10 mM Ca2+ with the test cation at various concentrations in the serial dilution assay of the AF. The other alkaline earth cations, Mg2+, Sr2+, and Ba2+, could not replace Ca2+; two transition elements, Mn2+ and Cd2+, partially replaced calcium. All 15 of the available lanthanides (including La3+ and Y3+) produced normal activity but only at 10-400-fold lower cation concentrations than Ca2+. An AF preparation is stable and remains active for months in 1 mM Ca2+ but decays rapidly when Ca2+ is lowered. Sr2+ and Ba2+ at 20 mM but not at 1 mM could replace 1 mM Ca2+ and give long term stability. AF was not stable in the presence of Mg2+, even at 100 mM. High Mn2+ concentrations did not stabilize AF even though AF was partially active in Mn2+. Cd2+ gave full stability at 75 mM and La3+ at about 0.1 mM. When Ca2+ is chelated, the macromolecular subunits of the AF slowly dissociate. Permeation chromatography and analytical ultracentrifugation showed that the cations that stabilized activity maintained the integrity of AF complex while those that failed to stabilize activity allowed the complex to dissociate into subunits, indicating that these two Ca2+ requirements are related. The cation specificities for activity and for stability-integrity are different indicating that these are separate Ca2+-dependent functions.     

Purification and properties of phosphatidylglycerophosphate synthetase from mammalian liver mitochondria

The enzyme which catalyzes the synthesis of phosphatidylgly cerophosphate from an-glycerol-3-phosphated and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids. The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids. The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+,Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM. The phospholipik stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.