Evidence of phosphatidylinositol and diacylglycerol kinases in suspension cultured plant cells

Suspension cultured cells of Catharanthus roseus and Nicotiana tabacum, after two cycles of freezing and thawing, incorporated labeled phosphate from exogenous [γ-³²P]ATP into their phospholipid fraction. Quantitative thin layer chromatography (TLC) revealed strongly labeled phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and phosphatidic acid (PA), and less incorporation into phosphatidylinositol diphosphate (PIP₂). Neomycin and spermine affected the amount of phosphorylation into the different components in a similar way to that described for animal cells.     

Der Einfluß von CO2 und pH auf die 32P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht

Summary The effect of CO₂ on the ³²P-labelling of polyphosphates and acid-soluble organic phosphates is studied in synchronously grown cultures of the green alga Ankistrodesmus braunii, using trichloroacetic acid treatment and acid hydrolysis for the fractionation of the phosphorus compounds.Three per cent CO₂ in nitrogen causes an inhibition of the labelling of polyphosphates but a marked increase of ³²P in organic phosphates, whereas oxygen (CO₂-free air) produces the reverse effect. Polyphosphates and ATP are the fractions most stimulated by O₂, while stable organic phosphates show the strongest inhibition. Labelling of nucleic acids is relatively indifferent to both oxygen and CO₂. Three per cent CO₂ in air causes the same distribution of ³²P-labelling as 3 per cent CO₂ in N₂. ³²P-labelling is strongly dependent on the pH of the medium. In the absence of CO₂, polyphosphate labelling is highest in the acidic range, whereas organic phosphates and ATP show optimum labelling and the highest percentage of the total ³²P in the alkaline pH range. The effect of CO₂ is strongest between pH 5 and 6, that of oxygen between pH 8 and 9. Apparently the pH of the medium exerts a considerable influence upon the phosphate metabolism inside the cells.Increasing concentration of CO₂ lead to the same change of ³²P-labelling in nitrogen as in air and to saturation at about 1 per cent CO₂ under the conditions used. The curves are in good agreement with those of O₂-evolution at increasing concentrations of CO₂, but they show completely different rates.Young cells respond to CO₂ and O₂ differently from cells in the photosynthetically most active stage. In young cells both gasses are less effective.The effect of CO₂ is explained by a strong increase in noncyclic photophosphorylation which can proceed only slowly in N₂. ATP-consumption connected with high rates of CO₂-fixation may be the reason for the low rates of ³²P-labelling in the polyphosphate fraction when CO₂ is present. The influence of external pH on ³²P-labelling is partly due to the pH-dependence of phosphate uptake, but the different response of several fractions to the pH of the medium suggests that the pH of the cytoplasm and possibly even the pH of the interior of the chloroplasts is affected by the external pH. The effect of O₂ in the absence of CO₂ or at low CO₂-concentrations is explained by the well-known inhibition of photosynthesis by oxygen. Increasing concentrations of CO₂ reverse this inhibition and correspondingly change the distribution of ³²P between the phosphate fractions. The change in sensitivity to CO₂ and O₂ with the cell age is consistent with the change in the rates of maximum photosynthetic CO₂-fixation.

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Herrn Prof. Dr. W. Schumacher zum 70. Geburtstag gewidmet.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

Preparation,purification, and properties of E. coli virus T2

1. A method for the preparation of 8 to 10 liter quantities of T₂ virus lysates, titering 2 to 5 x 10¹¹ infectious units per ml. has been described. 2. Procedures have been developed for the concentration and purification of virus to a high specific infectivity. No fractionation procedure of the several used succeeded in further raising the specific infectivity of these purified preparations. 3. Some of the general properties of the better preparations have been determined. They exhibited titers of 2 x 10¹⁵ infective units per gm. of material or 1.2 x 10¹⁶ per gm. of nitrogen. 4. A study of the distribution of nitrogen among the various fractions of the virus showed that about 6 per cent of the total nitrogen is soluble in 4 per cent trichloracetic acid; that the protein nitrogen is about 40 per cent of the total and the nucleic acid nitrogen is 53 per cent. At least 96 per cent of the total phosphorus is in the nucleic acid fraction. Less than 0.5 per cent quantities of lipid and PNA were found.     

Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons

Mixed micelles of ³²P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2-diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH-cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4′-(trimethylammonium chloride-5′-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells.     

THE BIOSYNTHESIS OF C13 COMPOUNDS : I. THE BIOSYNTHESIS OF C13-LABELED STARCH

1. Starch, containing 7.05 atom per cent C¹³ excess has been produced in the mesophyll cells of bean leaves, starting with C¹³O₂ containing 7.26 atom per cent C¹³ excess. Approximately 67 per cent of the C¹³ taken up by the leaves was determined in the starch fraction. 2. The soluble carbohydrate, containing 6.72 atom per cent C¹³ excess, accounts for approximately 23 per cent of the C¹³ taken up by the leaves. The remainder was principally in the coarse tissue fragments fraction (9.73 per cent of the C¹³ utilized). 3. The apparatus and procedures used in this experiment are described.     

On the chemical structure of the apical droplet from eggs of Culex pipiens

The chemical nature of the apical droplet from eggs of Culex pipiens was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12β-hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14β-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with β-OH myristic acid.Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive ³²PO₄^?3 and ³⁵SO₄^?2 showed no significant incorporation of phosphorus, but a sulphur-containing anionic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as mono-and diglycerides. The structural evidence explains in part the surfactant properties of the apical drop.     

Effects of Various Concentrations of Carbon Dioxide on Respiration and Potassium Uptake in Barley Roots

Barley roots contain a CO₂ sensitive respiratory fraction which is inhibited in 50 per cent CO₂ and is partially restored upon subsequent exposure to air. The residual O₂ consumption occurring at CO₂ concentrations between 50 per cent and 95 per cent amounts to about 40 per cent of the O₂ uptake in air and can support K⁺ uptake for a limited time at a rate equal to or higher than occurs in air. Above 95 per cent CO₂ both O₂ and K⁺ uptakes decrease rapidly. 2,4-dinitrophenol (DNP), in the range of 10^?6 to 10^?5M, stimulates O₂ uptake by the roots in air. The stimulation is absent when roots are treated with DNP in 80 per cent CO₂, presumably because of the reduced demand for inorganic phosphate and phosphate acceptor at the lower respiratory level in high CO₂. In either air or CO₂, K⁺ uptake is strongly inhibited by DNP. A comparison of the respiratory and K⁺ uptake data indicates that O₂ consumption is a necessary requirement for the uptake process in high CO₂. Protoplasmic streaming in the root cells is rapidly stopped by high CO₂ although K⁺ uptake and O₂ consumption continue. The cation uptake mechanism in high CO₂ appears to be limited to the stationary cytoplasm. It is also possible that a similar mechanism may be involved in cation uptake in air.     

A chemical and biological approach towards the definition of calcareous soils

Summary Several agricultural problems are associated with the presence of certain levels of CaCO₃ in soils. The level of CaCO₃ at which the phosphate fixation becomes an apparent agricultural problem, is thought to be an appropriate margine at which the soil can be considered calcareous. Thus, a set of soil mixtures, ranging in CaCO₃ content from 1 to 96% was prepared and used in a column study to determine the level at which the CaCO₃ fraction becomes a dominant factor controlling. P³² movement and distribution.Increasing the percentage of oolitic sand, in the soil mixture, from 1 to 10% caused a sharp drop in P³² movement with soil solution and any increase in CaCO₃ content above 10% did not show any further drop in P³² movement. The amount of P³² removed with the soil solution was generally low compared to that retained in soil columns. Studying the distribution of P³² in soil columns, after five displacements, has indicated that the migration of P³² from the top soil increased by increasing CaCO₃ from 1 and 2 to 6%. The amount of P³² removed was however retained in lower sections. A very sharp decrease in P³² migration from the top soil was observed when CaCO₃ content was raised from 8 to 10%.A similar picture was shown when the CaCO₃ material used was in clay size fraction. However the sharp increase in phosphate retention in top soil sections took place at CaCO₃ content of 8% rather than at 10%. A limit of 8 to 10% CaCO₃ was proposed as an appropriate margine for defining calcareous soils.     

Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway

Aluminum (Al³⁺) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al³⁺ using Coffea arabica suspension cells. We pre-labeled cells with [³²P]orthophosphate (³²Pi) and assayed for ³²P-PA formation in response to Al³⁺. Treating cells for 15 min with either AlCl₃ or Al(NO₃)₃ inhibited the formation of PA. In order to test how Al³⁺ affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al³⁺ inhibited mas-7 induction of PA response, both before and after Al³⁺ incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with ³²Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl₃ suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl₃ did. PLD in vivo activation by mas-7 was reduced by Al³⁺. These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.     

Effect of Feeding Millet (Sorghum vulgarie) Protein at Various Protein Levels on Adrenal Lipids of Rats

A significant increase in adrenal weight, total lipids, cholesterol phospholipids and glycerides (mono-and triglycerides) was observed in rats fed millet at 5, 10 and 15 % protein levels respectively for a period of six weeks as compared to rats fed casein at 10 per cent level. Increases in cholesterol were in both its free and esterified fraction. Adrenal phosphatidyl etha-nolamine was increased in all millet fed rats whereas phosphatidyl choline increased in M–15 % and decreased in M–5 % groups. Other phospholipid fractions viz. monophosphatidyl inositol, lysophosphatidyl ethanolamine, sphingomyeline, phosphatidic acid and polyglycerophosphatide also showed significant alterations in rats fed millet protein as compared to control. Incorporation of acetate–l–¹⁴C into adrenal lipids was lower and that of glucose–U–¹⁴C, palmitate–l–¹⁴C and NaH₂³²PO₄ was higher than the control.     

Changes in the pattern of phospholipid synthesis during the induction by cytokinin of cell division in soybean suspension cultures

A method is described for preparing fully viable, cytokinin-starved soybean (Glycine max (L.) Merr. cv. Acme) cells from a suspension-culture of callus tissue. The cells respond to kinetin treatment by re-initiating cell division. We present evidence, from the pattern of incorporation of ³²P-labelled inorganic phosphate into individual phospholipids during the first hour of this response, that the synthesis of phosphatidylinositol (PI) and of phosphatidic-acid head-groups is affected within 15 min. The polyphosphoinositide phosphatidylinositol 4-phosphate, but not phosphatidylinositol 4,5-bisphosphate, was detected in the tissue. The characteristics of cytokinin-induced PI synthesis in cytokinin-starved soybean cells appear to resemble the PI response of animal cells.Abbreviations DPG diphosphatidylglycerol - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PS phosphatidylserine - Pi inorganic phosphate - TLC thin-layer chromatography     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Changes in 32P-phosphorus compounds during translocation in Laminaria digitata (L.) lamouroux

³²P was applied to a Laminaria digitata thallus and the pattern of ³²P phosphorylated compounds was studied, as a function of time, in the different tissues involved in translocation, i.e. source, pathway and sinks. The results showed that, 3 hours after absorption by the uptake region (lamina), the bulk of the radioactivity was incorporated into organic compounds (70 to 80% of total ³²P taken up), hexose monophosphates being the heaviest labelled. Further change in that region was marked by an accumulation of ³²P in the inorganic pool (65 to 70% after 13 days). Conversely, the ³²P pattern in the medulla of the stipe, which initially showed a similar pattern to the uptake region, did not vary during translocation. The pattern of ³²P distribution into sinks (growing stipe peripheral tissue or hapteron) leads to accumulation of the radioactive element in inorganic and acid-insoluble fractions. These results are discussed in terms of comparative distribution of ³²P in the different parts of the thallus and suggest that phosphate moves as Pi in that alga.Abbreviations TCA trichloroacetic acid - Po organic phosphate - Po sol acid-soluble organic phosphate fraction - Po insol acidinsoluble organic phosphate fraction - Pi morganic phosphate fraction - P lip lipidic phosphate - Np protein nitrogen - ATP adenosine triphosphate - ADP adenosine diphosphate - PEP phosphoenolpyruvic acid - PGA phosphoglyceric acid - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - UDPG uridine diphosphoglucose     

Solubilisation et caractérisation d'une glycoprotéine : fucosyl-transférase cérébrale

1. A glycoprotein: fucosyl-transferase activity was demonstrated in sheep cerebral cortex, using desialylated fetuin as exogenous acceptor and detergent Trition X 100 for solubilization.

2. Addition of Triton X 100 to the membrane suspension gave first an activation then a solubilization of the cerebral fucosyl-transferase.

3. Hydrophobic chromatography was investigated for purification of the enzyme. Binding was effective using alkyl-agarose chromatographic columns with two or more than two atoms of carbon, but elution was only possible with ethyl and butyl-agarose.

4. Combination of subcellular fractionation and hydrophobic chromatography on ethylagarose led to a 30 fold purification.

5. After ethyl-agarose chromatography, some properties of fucosyl-transferase were studied: the optimal temperature was 25°C. The optimum pH was about neutrality. Light activation was observed with Mn²⁺ concentration below 1 mM.

6. Homogeneity was tested using Ultrogel chromatography, polyacrylamide gel electrophoresis and ultracentrifugation.

7. It was concluded that ethyl-agarose hydrophobic chromatography easily bind a few solubilized proteins (about 20 per cent of the ST supernatant). When elution was performed, these proteins, including fucosyl-transferase, were released from ethyl-agarose columns as a stable aggregate, only dissociated with lower ionic strength.

Ethyl-agarose fraction (eluted with KCl 120 mM) showed homogeneity:

— with Ultrogel AcA 22 chromatography (Mw = 300.000).

— with polyacrylamide gel electrophoresis without S.D.S.

— with the analytical ultracentrifugo giving Mw = 280.000; S₂₀ = 10.

But after dialysis overnight against a buffer without KCl, ultracentrifugation technics showed no homogeneity. Futhermore, SDS gel electrophoresis gave more than four bands.     

Aktive und inaktive Phosphataufnahme in Blattzellen von Elodea densa bei hohen Phosphat-Außenkonzentrationen

Summary The phosphate uptake in the leaf cells of Elodea densa shows multiple isotherms in the range [S]>1 mmole P/l to 100 mmoles P/l. In the dark the uptake isotherms contain three distinct parts (II/1, II/2 and II/3); the first two obey Michaelis-Menten kinetics, whereas the third is exponential. In the light the phosphate uptake curve consists only of two parts (II/1 and II/2) agreeing with Michaelis-Menten kinetics, the exponential part being absent.Cellular phosphate content was found to be 45 mmoles/l. Data concerning the membrane potential E for Elodea densa were obtained from Jeschke (1970). In accordance with the Nernst equation a change from the hyperbolic curve to an exponential one was expected at a concentration of about 60 mmoles P/l in the dark and at above 100 mmoles P/l in the light. The results obtained agree with these theoretical calculations: in the dark, the change from the hyperbolic to the exponential curve was observed at [S]=50 mmoles P/l, which is in electro-chemical equilibrium with the cellular orthophosphate content of about 35 mmoles/l (inorganic P content amounting to 80 per cent of total phosphate). In the light no change towards an exponential curve was noticed.The effect of the uncoupler CCCP in the light and in the dark was examined in order to elucidate its influenc on ³²P incorporation into the fractions of inorganic, organic and acid-insoluble phosphates, the inorganic fraction representing phosphate uptake. The inhibition of the uptake into the inorganic part decreases with an increasing inactive component of total uptake, while the fixation in the organic fraction is severely curtailed at all concentrations tested. The acid-insoluble fraction remains unaffected.

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Abkürzungen und Symbole CCCP Carbonylcyanid m-Chlorphenylhydrazon - Du Dunkel FG Frischgewicht - GP Gesamtphosphat - [H₂PO₄ ^-]_i Innenkonzentration - [H₂PO₄ ^-]_o Außenkonzentration - Ko Kontrolle - Li Licht - P Phosphat - P_a anorganisches TCE-lösliches Phosphat - P_o organisches TCE-lösliches Phosphat - P_u TCE-unlösliches Phosphat - P_gl TCE-gesamtlösliches Phosphat - [S] Außenkonzentration des H₂PO₄ ^--Ions - TCE Trichloressigsäure     

THE ENZYMIC HYDROLYSIS OF PHOSPHATIDYL INOSITOL BY GUINEA PIG BRAIN:

Abstract—

• 1 Phosphatidylinositol hydrolase activity of homogenates of guinea pig brain was studied by using [2-³H]inositol labelled substrate and measuring the release of radioactivity into the acid soluble fraction.
• 2 Inositol phosphate and diglyceride were found to be the main hydrolysis products. The principal enzyme involved, therefore, is a phosphatidylinositol inositolphosphohydrolase.
• 3 Most of the enzymic activity (61 per cent) was found in the soluble fraction. Osmotic shock of the high speed particulate fraction resulted in release of an additional 23 1 per cent into the soluble fraction. However, as contrasted to lactate dehydrogenase, significant activity remained particulate bound.

     

Improved two-step method for the assay of adenylate and guanylate cyclase.

A two-step assay for adenylate and guanylate cyclase is described utilizing α-³²P-labeled ATP or GTP as substrate and involving purification of the resulting ³²P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mm HCl for cGMP and 10 mm HClO₄ for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the ³²P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.     

Effectiveness of some iron phosphates as sources of phosphorus for plants

Summary Two well-characterized crystalline ferric phosphates, two colloidal ferric phosphates, and fluorapatite were tested under greenhouse conditions as sources of phosphorus for corn over a 3-cropping period. The selected compounds are representative of those expected to form in soils as reaction products from more soluble phosphate fertilizers.Strengite, FePO₄·2H₂O, was completely unavailable in acid soils and gave only a slight phosphorus response on soils limed to pH 7.6. Uptake of P from hydrogen ammonium ferric phosphate, H₈NH₄Fe₃(PO₄)₈·6H₂O, was approximately 70 per cent that from MCP, and increased with cropping.The colloidal ferric phosphates were approximately 78 per cent as available as MCP and became more available with liming and cropping. In the soil limed to pH 6.5, their effectiveness increased from 47 per cent that of MCP in the first crop to 100 per cent as effective by the third crop.Fluorapatite, included as an insoluble calcium phosphate source, was completely unavailable.