Influx and efflux of p in roots of intact maize plants : double-labeling with p and p

Rates of P influx and efflux were determined in whole plants at ambient P concentrations comparable to those found in soil solutions. Maize (Zea mays L. var NC+59) seedlings were trimmed (endosperm and adventitious roots removed) and grown in a greenhouse in solution cultures at P concentrations of approximately 0.4 and 1.8 micromolar. Roots of intact plants previously exposed to ³²P-labeled solutions at 0.2 and 2.0 micromolar P for 48 hours were rinsed 10 minutes in P-free solution and exposed to ³³P solutions at 0.2 and 2.0 micromolar for 10 minutes. Net depletion of ³³P from and appearance of ³²P in the ambient solution were used to measure influx and efflux. The ration of ³²P efflux to ³³P influx was about 0.68 at 0.2 micromolar and 0.08 at 2.0 micromolar. When plants were allowed to deplete P from solutions, the P concentration in the medium dropped to about 0.15 micromolar within 24 hours and 0.05 micromolar within 60 hours. Results indicate that P efflux is a substantial component of net P accumulation at P concentrations normally found in soil solutions.     

Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants

Phosphate in the xylem exudate of tomato (Lycopersicon esculentum) plants was 70 to 98% inorganic phosphate (Pi), 2 to 30% P-choline, and less than 1% P-ethanolamine. Upon adding ³²Pi to the nutrient, Pi in xylem exudate had the same specific activity within 4 hours. P-choline and P-ethanolamine reached the same specific activity only after 96 hours. The amount of Pi in xylem exudate was dependent on Pi concentration in the nutrient and decreased from 1700 to 170 micromolar when Pi in the nutrient decreased from 50 to 2 micromolar. The flux of 0.4 nmoles organic phosphate per minute per gram fresh weight root into the xylem exudate was not affected by the Pi concentration in the nutrient solution unless it was below 1 micromolar. During 7 days of Pi starvation, Pi in the xylem exudate decreased from 1400 to 130 micromolar while concentrations of the two phosphate esters remained unchanged.

The concentration of phosphate esters in the xylem exudate was increased by addition of choline or ethanolamine to the nutrient solution, but Pi remained unchanged. Upon adding [¹⁴C]choline to the nutrient, 10 times more [¹⁴C]P-choline than [¹⁴C]choline was in the xylem exudate and 85 to 90% of the ester phosphate was P-choline. When [¹⁴C]ethanolamine was added, [¹⁴C]P-ethanolamine and [¹⁴C]ethanolamine in the xylem sap were equal in amount. P-choline and P-ethanolamine accumulated in leaves of whole plants at the same time and the same proportion as observed for their flux into the xylem exudate. No relationship between the transport of P-choline and Pi in the xylem was established. Rather, the amount of choline in xylem exudate and its incorporation into phosphatidylcholine in the leaf suggest that the root is a site of synthesis of P-choline and P-ethanolamine for phospholipid synthesis in tomato leaves.

     

Root absorption and transport behavior of technetium in soybean

The absorption characteristics and mechanisms of pertechnetate (TcO₄⁻) uptake by hydroponically grown soybean seedlings (Glycine max cv Williams) were determined. Absorption from 10 micromolar solutions was linear for at least 6 hours, with 30% of the absorbed TcO₄⁻ being transferred to the shoot. Evaluation of concentration-dependent absorption rates from solutions containing 0.02 to 10 micromolar TcO₄⁻ shows the presence of multiphasic absorption isotherms with calculated K_s values of 0.09, 8.9, and 54 micromolar for intact seedlings. The uptake of TcO₄⁻ was inhibited by a 4-fold concentration excess of sulfate, phosphate, selenate, molybdate, and permanganate; no reduction was noted with borate, nitrate, tungstate, perrhenate, iodate, or vanadate. Analyses of the kinetics of interaction between TcO₄⁻ and inhibiting anions show permanganate to be a noncompetitive inhibitor, while sulfate, phosphate, and selenate, and molybdate exhibit characteristics of competitive inhibitors of TcO₄⁻ transport suggesting involvement of a common transport process.     

Characterization of the inhibition of k absorption in oat roots by salicylic Acid

The phenolic compounds salicylic acid (o-hydroxybenzoic acid) and ferulic acid (4-hydroxy-3-methoxycinnamic acid) inhibited K⁺ (⁸⁶Rb⁺) absorption in excised oat (Avena sativa L. cv. Goodfield) root tissue. Salicylic acid was the most inhibitory. The degree of inhibition was both concentration- and pH-dependent. With decreasing pH, the inhibitory effect of the phenolic increased. During the early stages of incubation, the time required to inhibit K⁺ absorption was also pH- and concentration-dependent. At pH 4.0, 5×10⁻⁴ molar salicylic acid inhibited K⁺ absorption about 60% within 1 minute; whereas, at pH 6.5, this concentration affected absorption only after 10 to 15 minutes. However, at 5 × 10⁻³ molar and pH 6.5, salicylic acid was inhibitory within 1 minute. The capacity of the tissue to recover following a 1-hour treatment in 5 × 10⁻⁴ molar salicylic acid ranged from no recovery at pH 4.5 to complete recovery at pH 7.5. The absorption of salicylic acid was pH-dependent, also. As pH decreased, more of the phenolic compound was absorbed by the tissue. The increased absorption of the compound at low pH most likely contributed to apparent tissue damage at pH 4.5 and might have accounted for the lack of recovery of K⁺ absorption as pH decreased.     

Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots

Active K⁺ influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) aminocarbonyl]benzenesulfonamide).

Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K⁺ uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K⁺ influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K⁺ influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the `washing' effect.

     

Association of Potassium and Some Other Monovalent Cations with Occurrence of Polyphosphate Bodies in Chlorella pyrenoidosa

Phosphate-starved Chlorella pyrenoidosa cells formed polyphosphate bodies (PB) upon transfer into nutrient solutions containing phosphate and potassium, or another monovalent cation, such as Na⁺, NH₄⁺, Li⁺, or Rb⁺. The phenomenon was studied by chemical analyses, light microscopy, and electron microscopy.

When the P-starved cells were transferred into a complete nutrient solution containing 100 micromolar P, they accumulated large quantities of P and K within several hours. The accumulation was accompanied by a corresponding appearance of PB in the cells. The absence of K from the medium prevented appreciable P accumulation and PB formation, but omitting Ca or Mg did not.

The P-starved cells exposed to a simple solution of at least 20 micromolar H₃PO₄ and 100 micromolar KHCO₃ responded in a similar manner as the cells exposed to the complete nutrient solution. However, the PB appeared structurally different.

It is proposed that monovalent cations are essential for PB formation in C. pyrenoidosa. K is suggested to be a major component of PB formed in K-sufficient media.

     

Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings

The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca²⁺ (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg²⁺ (1.8 millimolar) and La³⁺ (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V_max reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma.     

Dependency of Iron Reduction on Development of a Unique Root Morphology in Ficus benjamina L

The activity of the Fe³⁺ reductase of excised adventitious roots of Ficus benjamina L., grown in hydroponic culture without iron, was determined by a colorometric assay simplified by the use of a microplate reader. Reductase activity remained the same from pH 4.5 to 6.5 and decreased sharply above pH 6.5. Acetate buffer inhibited reduction. During early stages of root growth, excised roots did not exhibit Fe³⁺ reductase activity. After several weeks and extensive root system development, Fe³⁺ reduction still was not detectable in primary roots, but intermediate and high rates of reduction occurred in lateral and newly formed root clusters, respectively. Clustered roots only developed on plants grown at 0 or very low (<1 micromolar) iron. Microscopic examination revealed the root cluster to be composed of up to 30 lateral roots, usually less than 1 millimeter in diameter and 1 centimeter in length, that were completely covered with root hairs.     

Assessing the Rhizotoxicity of the Aluminate Ion, Al(OH)(4)

Dissolved aluminum (III) in acidic soils or culture media is often rhizotoxic (inhibitory to root elongation). Alkaline solutions of Al are also sometimes rhizotoxic, and for that reason toxicity has been attributed to the aluminate ion, Al(OH)₄⁻. In the present study, seedlings of wheat (Triticum aestivum L. cv Tyler) and red clover (Trifolium pratense L. cv Kenland) were cultured in aerated aluminate solutions at pH 8.0 to 8.9. The bulk phases of these solutions were free of reactive polynuclear hydroxy-Al (including the extremely toxic species AlO₄Al₁₂[OH]₂₄[H₂O]⁷⁺₁₂ [Al₁₃]) according to the ferron (8-hydroxy-7-iodo-5-quinolinesulfonic acid) assay. At an aluminate concentration of 25 micromolar (23 micromolar activity) and a pH of 8, root elongation was less than 40% of Al-free controls, but at pH 8.9 elongation was 100% of controls. The hypothesis is offered that aluminate is nontoxic and that the inhibition at lower pH values is attributable to Al₁₃ postulated to have formed in the acidic free space of the roots where the ratio /{Al³⁺/}//{H⁺/}³ may rise above 10¹⁰. At this value hydroxy-Al in over-saturated, alkaline solutions begins to undergo rapid conversion to polynuclear species.     

Effect of pH on Orthophosphate Uptake by Corn Roots

Orthophosphate (Pi) influx in washed corn roots was studied with experimental conditions allowing a distinction of pH effects on Pi ionization in the medium and on the transport system itself. There appeared to be no relationship between the pH dependencies of membrane potential, H⁺ secretion, and ³²Pi influx. The Pi uptake versus pH curves were compared to the calculated ones describing the concentrations of the different ionized Pi forms in the medium and in the cell walls; the latter were obtained using the theoretical model described by Sentenac and Grignon (1981) Plant Physiol 68: 415-419). The conclusion was that the transported form is H₂PO₄⁻ and the concentration sensed by the transport system is the local one. The ionic compositions of experimental media were manipulated to ensure constant pH and various H₂PO₄⁻ concentrations, or constant H₂PO₄⁻ concentration and various pH values in the walls. The kinetic analysis of the results in the micromolar range showed that the transport system has an intrinsic sensitivity to pH, and is switched from a low activity state at pH > 6 to a high activity one at pH < 4 (pH in the walls). This change could be triggered by the protonation of a group with pK 5.5.     

Phosphate uptake in Chara: membrane transport via Na/Pi cotransport

Phosphate uptake in the freshwater charophyte plant Chara corallina was found to be strongly dependent on the presence of Na in the external medium. Based on the reciprocal stimulations of ³²Pi uptake by Na and ²²Na uptake by Pi, the logical mechanism for Pi uptake appears to be a nNa/Pi symport with a half‐maximal stimulation (K_m) for Na of approximately 300 μM and a K_m for Pi of approximately 10 μM . Comparison of the stimulations of ³²Pi and ²²Na influxes at pH 6 gives a stoichiometry of Na : Pi of 5·68. The reduction in Pi influx with increasing pH is consistent with the transported species being the monovalent H₂PO₄^?. In voltage‐clamp experiments, currents elicited by Pi in the presence of Na were equivalent to an influx of positive charge which exceeded the measured influxes of ³²P by a factor of 6·26. Intracellular perfusion was used to examine the dependence of Pi influx on ATP and Na. In perfused cells, Pi influx was low when ATP was absent from the internal medium or Na was absent from the external medium. Addition of ATP alone had little effect whereas addition of Na alone increased the ³²Pi influx slightly. Addition of both ATP and Na together restored Pi influx to rates comparable to those of intact cells. It is suggested that the ATP is required for membrane hyperpolarization which in turn drives the highly electrogenic flux of Pi with up to 6 Na. However, consideration of the electrochemical potential differences for Na and Pi at pH less than 6 shows that nNa/Pi would not be feasible. It is suggested that at low pH, H⁺ may substitute for Na.     

Conformational Changes of Myoglobin Upon Interaction with Negatively-Charged Phospholipid Vesicles

Abstract

The interaction between myoglobin and negatively-charged liposomes composed of phosphatidylcholine/phosphatidylglycerol (1:1) was studied at low ionic strength under acidic conditions. Changes in the absorbance and the fluorescence spectra of myoglobin were recorded upon addition of liposomes to partially unfolded (pH 3.5) and native (pH 4.5 and pH 6.5) myoglobin. Association of myoglobin with liposomes was a relatively fast process at pH 3.5 and pH 4.5. Although at pH 3.5 myoglobin was unfolded partially before the addition of the liposomes while at pH 4.5 before the addition of liposomes myoglobin retained its native form, similar interaction patterns of myoglobin with liposomes were observed. The fluorescence and absorption spectra in the Soret region of myoglobin clearly indicated that at these pH values myoglobin was associated with the liposomes in a (partially) unfolded state. At pH 6.5 the kinetics of myoglobin association with liposomes was much slower than at pH 3.5 and 4.5. The spectroscopic measurements also indicated that the interaction of myoglobin with liposomes at pH 6.5 followed a different pattern and resulted in different protein structures in comparison with pH 3.5/4.5.     

Kinetics of phosphorus absorption by mycorrhizal and nonmycorrhizal tomato roots

Kinetics of P absorption were investigated in mycorrhizal (Glomus fasciculatus) and nonmycorrhizal tomato (Lycopersicon esculentum) roots to determine why increased ion absorption by mycorrhizae occurs. Initial rates of absorption of ³²P were measured at 1 to 100 micromolar KH₂PO₄ (pH 4.6). Absorption rates of mycorrhizae were about twice those of control roots. Augustinsson-Hofstee analysis yielded two linear phases; V_max and K_m were calculated for each phase. In the low phase (1 to 20 micromolar), V_max values for the mycorrhizal and nonmycorrhizal roots were each 0.10 micromoles P per gram fresh weight per hour while K_m values were 1.6 and 3.9 micromolar KH₂PO₄, respectively. For the high phase (30 to 100 micromolar), V_max values for mycorrhizal and nonmycorrhizal roots were 0.32 and 0.25 micromoles P per gram fresh weight per hour and K_m values were 35 and 42 micromolar, respectively. These results indicate that at the lower phase concentrations, similar to those expected in most soil solutions, a major factor contributing to the increased uptake was an apparent greater affinity of the absorbing sites for H₂PO₄⁻ (lower K_m).     

Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells: Measurement with Fluorescent pH Indicator and P-Nuclear Magnetic Resonance

Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2′,7′-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and ³¹P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by ³¹P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested.     

模拟酸雨对柚木幼苗生长、光合与水分利用的影响

模拟pH6.5(对照)、4.5和2.5三个酸雨梯度,研究其对1a生组培柚木(TectonagrandisL.f.)幼苗生长、光合与水分利用的影响。结果表明,尽管不同处理间的各项生理指标差异不明显,但模拟酸雨对柚木形态构件参数造成较严重的影响。pH4.5和pH2.5处理组柚木基径(D)和树高(H)增长明显下降,使得D2H下降更加显著;不同处理下柚木叶片净光合速率(Pn)和蒸腾速率(E)日变化趋于一致,气孔导度(gs)日变化与对应的叶片净光合速率日变化十分相似,同时,对照与两个处理的Pn与gs之间都表现正相关(p<0.01),且在pH4.5处理表现更为显著,但是对照和两个处理的E与gs的线性关系不显著;pH4.5和2.5处理的水分利用效率(WUE)日变化趋于一致;对照胞间CO2浓度与大气CO2浓度比(Ci/Ca)均值最低,表明对照柚木对CO2利用最有效。     

Gravitropic Responses of Partially Decapitated Corn Coleoptiles with and without Applied [C]Indoleacetic Acid

The curvature of corn seedling (Zea mays L. Mo17 × B73) coleoptiles which had been half-decapitated and supplied with [¹⁴C]indoleacetic acid (IAA) (3.2 micromolar, 51 milliCuries per millimole) was determined during a 3-hour period of gravitational stimulation. Curvature of such half-decapitated coleoptiles was found to be similar in rate and extent to that of intact coleoptiles responding to gravity. Gravitational stimulation was accomplished by reorienting seedlings to a horizontal position, either up or down with respect to the removed half of the coleoptile tips.

The first set of experiments involved placing aluminum foil barriers along one of the two cut surfaces to restrict the movement of IAA into tissues. The initiation and extent of curvature of these half-decapitated coleoptiles was dependent upon the orientation of the removed half-tip and the accompanying barrier. The distribution of radioactivity from [¹⁴C] IAA after 3 hours indicated that the specific lateral movement of label was also dependent upon orientation of the removed half-tip of the coleoptile. A specific movement to the lower side of approximately 14% of the total recovered radioactivity was found in coleoptiles in which the [¹⁴C]IAA was supplied across a transverse cut surface. In contrast, specific movement of only 4% was found for application across a longitudinal cut surface.

A second series of experiments was conducted using 1.0 and 3.2 micromolar [¹⁴C]IAA (51 milliCuries per millimole) supplied to half-decapitated coleoptiles without inserted barriers. The 3.2 micromolar concentration adequately replaced the removed coleoptile half-tips in terms of straight growth, but it did not result in as much curvature as shown by coleoptiles of intact seedlings. The 1 micromolar concentration was not adequate to replace the removed half-tip in straight growth, but resulted in gravitropic curvature nearly as great as that produced by the higher concentration.

The data presented here suggest that strong auxin gradients are not produced in response to gravity stimulation based on the recovered radioactivity from [¹⁴C]IAA. However, it is evident that auxin is required for the development of normal gravitropic responses. It is possible, therefore, that an important early role of this movement is not to cause a large stimulation of growth on the lower side but to decrease growth on the upper side of a gravitropically responding coleoptile.

     

Plasma membrane ATPase of red beet forms a phosphorylated intermediate

When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [γ-³²P] ATP, 40 micromolar MgSO₄ at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.     

Purification and properties of adenosinetriphosphatase from Zea mays seedling microsomes

A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg²⁺°. Mg²⁺ and Ca²⁺ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na⁺ and K⁺ brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na⁺ and K⁺, and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na⁺ and K⁺ ions.     

Is the Cytosolic Pi Concentration a Limiting Factor for Plant Cell Respiration?

The substrate-dependent O₂ uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O₂ uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. ³¹P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O₂ uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria.     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.