Alkaline <i>O</i>→<i>N</i>-transacylation. A new method for the quantitative deacylation of phospholipids

Clarke, N.; Dawson, R. M. C.

doi:10.1042/bj1950301

Cited by 322 publications

(161 citation statements)

References 16 publications

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“…An aliquot of the chloroform phases obtained from the purified membranes was used for the determination of PtdIns by HPTLC (see below). The remains of these lipid extracts were deacylated according to the method of [21]. The chloroform phase was mixed with 1 ml monomethylamine reagent (2596, v/v) and incubated in a sealed glass tube at 53OC for 40 min.…”

Section: Extraction and Deacylation Of Phospholipidsmentioning

confidence: 99%

Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle

1994

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The total membrane concentrations of PtdIns, PtdIns4P, and PtdIns(4,5)P, contribute to the functional capacity of the Ins(1,4,5)Pj signalling system which is operating in skeletal muscle but the function of which is still unknown. Total amounts of these phosphoinositides have been determined in purified membranes of transverse tubules (lT) and terminal cistemae (TC) of the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. PtdIns and PtdIns4P have been detected in both membrane systems whereas PtdIns(4,5)P, (290 pol/mol phospholipid) is confined only to 'FT. A much greater pool of PtdIns(4,5)P, seems, however, to be located in the sarcolemma away from the triadic junction.Key wor&: Phosphoinositide; PtdIns(4,5)P,; Phospholipase C, Inositol 1,4,56sphosphate;Triad; Skeletal muscle IntruduetIonThe concept that inositol 1,4,5-triphosphate (Ins-(1,4,5)PJ acts as an intracellular chemical transmitter in the fast excitation-contraction coupling process of skeletal muscle has been put forward by several groups [l-3]. There are, however, severe objections to this idea which have been worked out in detail by [4]. A second concept favours a messenger action of Ins(1,4,5)P, on a larger time scale aiming at the excitation-contraction coupling mechanism and/or the energy metabolism of the skeletal muscle fibre [5]. A key event of the Ins( 1,4,5)P,-signalling pathway is the cleavage of Ins(1,4,5)P, from phosphatidylinositol4,5-biphosphate (PtdIns(4,5)Pb by phospholipase C (PLC) [6] that has to fit in quite different time frames depending on which of the concepts is realised in skeletal muscle. It is well established that the activity of skeletal muscle PLC is strongly regulated by myoplasmic Ca2+ in a concentration range that is reached during muscle activation [7,8]. Up to now it is unknown whether this Ca2'-sensitivity, which is common to all known PLC isoforms [9], represents the primary event for PLC-activation or whether a different membrane borne process switches on the enzyme Ca2+-independently. A putative assignment of the activation mechanism to one of the established classes (G protein activated for PLC-B; receptor tyrosine kinase activated for PLC-y [6,9]) is not possible because a PLC isoform of skeletal muscle has not yet been identitied by protein or cDNA sequencing. There is, however, evidence that two PLC, i.e. a cytosolic and a membrane associated form, coexist in skeletal muscle [7,8] and that the myoplasmic enzyme might be different from the known PLC families [lo]. The membrane associated enzyme is localised mainly at the TT [7,8]. Membranes of the SR seem either to be devoid of this enzyme [7] or contain marginal PLC activity [8]. Compared to the knowledge accumulated on skeletal muscle PLC only little is known about membrane localisation and content of its substrate, PtdIns(4,5)P,. In the present work we have studied in detail the total concentration and localisation of phosphatidylinositol (Ptdlns), phosphatidylinositol4-phosphate (PtdI&P), and PtdIns-(4,5)P, in TT membranes and in membran...

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Section: Extraction and Deacylation Of Phospholipidsmentioning

confidence: 99%

Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle

1994

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“…Electrophoresis was carried out at 4 kV for 15 miri in 0.06 M sodium oxalate buffer (pH 1.54). After electrophoresis, the dried paper was sprayed with Clark/Dawson spray reagent (Clark et al, 1981) to locate the inositol phosphate:,. R F values of inositol phosphates formed in the reaction and those of other inositol phosphates are as follows: Ir6, 1.0; 1-1,2,3,5,6-0.81; I-1,2,3-P3, 0.72; I-1,2,6-P3, 0.68.…”

Section: Hve Analysis Of Inositol Phosphatesmentioning

confidence: 99%

Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase from Lily Pollen

1994

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Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I-1,2,3,4,5,6-P,). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. l h e specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum 1.) pollen is described. Structures of the intermediate inositol phosphates and the final product were established by a variety of nuclear magnetic resonance techniques ('H-, 31P-, and 3'P-'H-detected multiple quantum coherence spectroscopy, and total correlation spectroscopy). O n the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D-5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. l h e physiological significance of alkaline phytase and the biological role of I-1,2,3-P3 remain to be identified.Phytic acid, myo-inositol hexakisphosphate, is a major constituent of seeds and pollen grains (1-5% of dry weight) (Loewus, 1990;Raboy, 1990). In mature lily (Lilium longiflorum L.) pollen and seeds, phytic acid is localized in membrane-bound phytic-rich granules. The presence of phytic acid in plant cells has been known for some time (Loewus, 1990;Raboy, 1990). However, it was only recently recognized that phytic acid is present in virtually all mammalian cells in concentrations higher than most other inositol phosphates (Menniti et al., 1993) and may function as a neurotransmitter (Vallejo et al., 1988). The discovery that I-1,4,5-P3 plays a crucial role in calcium cellular signaling (Bemdge et al., 1989) has greatly increased interest in the structure, metabolism, and biological role of inositol phosphates, including phytic acid.The primary enzymes responsible for the degradation of phytic acid are phytases. Phytases are a special class of phosphatases that catalyze the sequential hydrolysis of phytic acid to inositol phosphates and, in some cases, to inositol. Phytases occur in a variety of organisms including plants, fungi, and animals (reviewed by Cosgrove, 1980a). A variety of phytases differing in pH optima, substrate specificity, and * Corresponding author; fax 1-906-487-2061. specificity of hydrolysis have been identified in plants and fungi (Cosgrove, 1980a(Cosgrove, , 1980b. Acid phytases from wheat bran and Aspergilli have been extensively studied and the stereospecificity of hydrolysis has been well established (Cosgrove, 1980b). Based on the specificity of initial hydrolysis, two classes of acid phytases are recognized by the Intemational Union of Pure a...

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“…The phospholipids were separated on a 4.5 x 250 mm amino-NP column (5 ,m spherical silica with n-propylamine bonded phase, III Supplies Co, Wallingford, CT) with a 5-cm guard column. The column was washed for 2 (4). Monomethylamine reagent was prepared by gently heating a mixture of dry methylamine hydrochloride and sodium hydroxide, and then allowing the resulting gas to bubble through 40 mL of ice-cold methanol:water:n-butanol (4:3:1 v/v/v) until the volume had increased to 65 mL.…”

Section: Separation Of Inositol Phospholipids By Hplcmentioning

confidence: 99%

“…To further characterize the S. saman phospholipids, HPLCpurified lipids were subjected to mild alkaline hydrolysis by the method of Clarke and Dawson (4). This procedure removes the fatty acyl chains from phospholipids with minimal secondary reactions.…”

Section: Deacylation Of S Saman Phospholipidsmentioning

confidence: 99%

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman

Coté

DePass²,

Quarmby³

et al. 1989

Plant Physiol.

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To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an altemative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphafidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [3H]

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Alkaline O→N-transacylation. A new method for the quantitative deacylation of phospholipids

Cited by 322 publications

References 16 publications

Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle

Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle

Specificity of Hydrolysis of Phytic Acid by Alkaline Phytase from Lily Pollen

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman

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