Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma

Mammalian plasma contains a phospholipase D, which is specific for the glycosyl-phosphatidylinositol anchor found on many eukaryotic cell surface proteins (Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984; Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem. Biophys. Res. Commun. 150, 476-482). We have purified this phospholipase D to homogeneity by a four-step procedure involving a Mono Q and phenyl-5PW columns, followed by wheat germ lectin affinity chromatography and finally another Mono Q column. A 4,500-fold purification was achieved with a 5% yield. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous enzyme has a Mr of 110,000 and appears to consist of a single polypeptide chain. It exhibits identical substrate specificity as compared with the crude preparation, is active over a broad pH range (4.0-8.5), inhibited by the thiol-blocking agent p-chloromercuriphenylsulfonic acid and by 1,10-phenanthroline, and is partially heat-labile.     

Purification and characterisation of rape seed phospholipase D

Phospholipase D (PLD, phosphatidylcholine:phosphatidohydrolase, EC 3.1.4.4) has been isolated from matured dry winter rape seed (Brassica napus L.), variety Lirajet). Final purification of the soluble enzyme was achieved by two-step ammonium sulphate precipitation, hydrophobic interaction chromatography and native PAGE followed by electroelution. The specific activity of the final electrophoretically homogeneous preparation was increased about 700 times during the purification process with an overall yield of 4.6%. The activity of purified soluble PLD depends strictly on the presence of Ca²⁺ (120 mM). The pH optimum of rape seed PLD was in the range 5.5–6. The K_m value for phosphatidylcholine depends on the ratio between SDS and substrate concentration. No polymorphism of PLD was detected by SDS-PAGE and size exclusion chromatography of the purified enzyme. The purified enzyme was a monomer with a molecular mass of 105 000 Da determined by SDS-PAGE and of 90 000–100 000 Da assessed by size exclusion chromatography. The amino acid composition of PLD was also determined. Similar intensities of immunochemical cross reactions were demonstrated between PLD extracted from rape seed, soybean, castor bean and sunflower using immunoabsorption technique with the immune serum previously prepared against partially purified rape seed PLD. Data obtained in this study and those gathered from the literature indicate close similarities in molecular, enzymatic and antigenic characteristics between PLDs of oil seeds of different species.     

Purification and applications of a phospholipase D from a new strain of Streptomyces

An enzyme with phospholipase D activity was purified to homogeneity from a new strain of Streptomyces. The molecular mass, assessed by electrospray mass spectrometry, was 52672 Da and the isoelectric point 9.2. The enzyme, which had pH optimum between 4 and 7, showed satisfactory stability and transphosphatidylation activity.     

Purification of phospholipase D from Dacus carota by three-phase partitioning and its characterization

Phospholipase D from Dacus carota (carrot) was purified by subjecting it to three-phase partitioning. The single step of three phase partitioning led to 13-fold purification with an activity recovery of 72%. SDS-PAGE analysis showed a single band with minimum molecular weight corresponding to nearly 60 kDa. The purified enzyme had a pH optimum in the range of 6.0--6.5 and was unstable above 30 degrees C. Kinetic studies showed a K(m) value of 9.5 mM and a V(max) of 0.35 mL min(-1). The enzyme purified by three-phase partitioning was found to resolve into two isoenzymes on a DEAE-cellulose column.     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

Carotenoids synthesized in citrus callus of different genotypes

Eight carotenoids, such as phytoene, α-carotene, violaxanthin, etc., synthesized in citrus callus of 31 genotypes were identified and determined. Though varied with genotypes, the carotenoids composition of callus derived from a certain genotype was stable, while carotenoids contents altered between sub-cultures. Some specific carotenoids were produced in calluses of limited genotypes: β-citraurin was only synthesized in calluses of Nianju tangerine (Citrus reticulata Blanco) and Page tangelo (C. reticulata × C. paradisi); while 9-Z-violaxanthin was only detected in Nianju tangerine and Skaggs Bonanza navel orange (C. sinensis L. Osbeck). Notably, the only carotenoid detected in calluses of Natsudaidai (C. aurantium L.) and other two sweet oranges (C. sinensis L. Osbeck) was phytoene. It implied that citrus calluses could be employed to produce specific carotenoids in the future. To further elucidate the characters of callus carotenoids profile, comparisons of carotenoids profiles was made among calluses, fruit tissues and leaves of four selected citrus genotypes. Results showed that lycopene was not detected in leaves and calluses; nevertheless, both citrus fruits and calluses accumulated phytoene, whereas leaves did not except those of Cara Cara navel orange. It is postulated that citrus callus featured its carotenoids profile different from fruit tissues and leaves. In conclusion, the advantages of using citrus callus as an alternative model research system in understanding the regulation of carotenogenesis have been discussed.     

Purification of polymeric phospholipase Cs from human platelets

Two types of cytosolic phospholipase C specific for phosphoinositides were purified from human platelets. The molecular masses of the purified enzymes were 440 and 290 kDa. These enzymes were concluded to be respectively a trimer and a dimer of homologous 146 kDa polypeptides. The 146 kDa polypeptide may be an immunologically novel isozyme among the 140-150 kDa PLC isozymes. Both enzymes hydrolyzed phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in a Ca2(+)-dependent manner.     

Distribution of glycosylphosphatidylinositol-specific phospholipase D mRNA in bovine tissue sections

It has been reported that mammalian serum, and to a lower extent mammalian liver, brain, pancreas, udder, and milk, contain glycosylphosphatidylinositolspecific phospholipase D activity. However, the sites of synthesis have not been determined. In order to study in which cells(s) of the organism synthesis of glycosylphosphatidylinositol-specific phospholipase D takes place, we undertook a systematic screening of 12 different bovine tissues. In situ hybridization experiments with a specific anti-sense RNA probe, derived from a bovine liver cDNA, revealed that glycosylphosphatidylinositol-specific phospholipase D mRNA is present in mast cells of the adrenal gland, lung, and liver. On the other hand, our specific probe detected no mRNA in bovine pancreas, brain, and udder, although enzyme activity has been reported in these tissues. Northern blot analysis of total bovine liver RNA demonstrated two distinct glycosylphosphatidylinositol-specific phospholipse D mRNAs of approximately 3.3 kb and 4 kb length suggesting that two forms of the enzyme may exist.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Purification and characterization of an anionic isoperoxidase from scented-geranium callus

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.     

Purification of a phospholipase B inhibitor from Saccharomyces cerevisiae

The phospholipase B activity of plasma membrane vesicles from Saccharomyces cerevisiae is inhibited by the 100 000 X g supernatant of mechanically disrupted yeast cells. A 1850-fold purification of the inhibitor activity was achieved by gel filtration, ion exchange chromatography with DEAE-cellulose and hydrophobic interaction chromatography with Octyl-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified inhibitor revealed two main bands with an apparent Mr of 60 000 and 26 500. The phospholipase B activity was strongly reduced but not completely blocked by this preparation, while the lysophospholipase and transacylase reactions, which are catalyzed by the same membrane-bound enzymes (Witt, W. et al. (1984) Biochim. Biophys. Acta 795, 108-116), were not affected.     

Purification and characterization of phospholipase B from Candida utilis

Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90-110 kDa by SDS-PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A(2) (cPLA(2)), found in mammalian tissues.     

Purification of a phosphoinositide-specific phospholipase C from bovine brain

A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization.     

Four caffeoyl glycosides from callus tissue of Rehmannia glutinosa

From the methanolic extract of callus tissue of Rehmannia glutinosa four phenolic glycosides and one aliphatic glycoside were isolated. Two of the phenolic glycosides were identified as acteoside and forsythiaside and the structures of the other two were elucidated as 3,4-dihydroxy-β-phenethyl-0-β-D-glucopyranosyl-(1 → 3) -4-0-calfeoyl-β-D-glucopyranoside and 3,4-dihydroxy-β-phenethyl-0-β-D-glucopyranosyl-(1 → 3) -0-α-L-rhamnopyranosyl-(1 → 6)-4-0-caffeoyl-β-D-glucopyranoside.     

A saponin from callus tissue of Stauntonia hexaphylla.

A new 30-nortriterpenoid saponin was isolated from the callus tissues of Stauntonia hexaphylla. The structure of the saponin (tentatively named mubenoside A) was elucidated as 3 beta,20 alpha-dihydroxy-30-nor-olean-12-en-28-oic acid 3-O-[beta-D-xylopyranosyl(1----2)-alpha-L-arabinopyranosyl(1----3) ]-beta-D- glucopyranoside by means of spectral experiments.