Isolation and characterization of one soluble and two membrane-associated forms of phosphoinositide-specific phospholipase C from human platelets

Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69,000 and 63,000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57,000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lower than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that are distinct from at least one cytosolic form (PLC-II) of the enzyme.     

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Partial purification and characterization of membrane-bound and cytosolic phosphatidylinositol-specific phospholipases C from murine thymocytes

Membrane-bound and cytosolic phosphatidylinositol (PI)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographies on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolic enzyme increased more than 1,800- and 1,400-fold, respectively, compared with those of microsomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70,000 by gel filtration. These purified enzymes also hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2 in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphatidylcholine enhanced the activity of the membrane-bound enzyme, while phosphatidylcholine (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.     

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.     

Purification and characterization of a maturation-activated myelin basic protein kinase from sea star oocytes

A meiosis-activated myelin basic protein (MBP) kinase was purified approximately 8700-fold from soluble post-germinal vesicle breakdown extracts from maturing oocytes of the sea star Pisaster ochraceus. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE-cellulose, hydroxylapatite, phosphocellulose, phenyl-Sepharose, heparin-Sepharose, polylysine-Sepharose, and Mono-Q. The final product exhibited an apparent molecular mass of approximately 42 kDa by both native gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this precisely correlated with the chromatographic behavior of the recovered MBP kinase activity on a Superose 6/12 column. The kinase utilized the MBP as the major substrate with little or no phosphorylation of histones (H1, H2A, or H2B), casein, phosvitin, protamine, or 40 S ribosomal proteins. The purified enzyme was relatively insensitive to high concentrations of beta-glycerol phosphate, calmodulin, EGTA, NaCl, sodium fluoride, dithiothreitol, spermine, and heparin but was quite sensitive to inhibition by metal ions such as Mn2+, Zn2+, and Ca2+. The true Km values for ATP and myelin basic protein were determined to be 58 and 25 microM, respectively, using double-reciprocal plots. The purified enzyme was unable to utilize GTP in place of ATP. The enzyme was shown to rapidly undergo autophosphorylation. The autophosphorylation was sensitive to alkali treatment implying that phosphate was incorporated on serine/threonine residues. The properties of this MBP kinase are reminiscent of a protein kinase that is also activated in a cyclic fashion at M-phase during the early cell divisions of sea star and sea urchin embryos (Pelech, S. L., Tombe, R., Meijer, L., and Krebs, E. G. (1988) Dev. Biol. 130, 26-36).     

Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Purification and characterization of PLC-beta m, a muscarinic cholinergic regulated phospholipase C from rabbit brain membrane

Two isozymes of phosphoinositide-specific phospholipase C were isolated and purified from salt-washed rabbit brain membranes. The membranes were extensively washed with isotonic, hypertonic and hypotonic buffers prior to solubilization with sodium cholate. Two isozymes (PLC-IV and PLC-beta m) were purified by a combination of DEAE-Sephacel, AH-Sepharose, heparin-Sepharose, AcA-34 gel filtration and mono-Q FPLC chromatographies. The major activity (PLC-beta m) was purified to homogeneity and had an estimated molecular weight of 155,000 on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This isozyme was immunologically identified as PLC-beta, an isozyme previously characterized in bovine brain cytosol and 2 M KCl membrane extracts. A second isozyme, PLC-IV, was immunologically distinct from PLC-beta and PLC-gamma and was purified to a stage where three protein bands (Mr 66,000, 61,000 and 54,000) on SDS-PAGE correlated with enzyme activity. The catalytic properties of the isozymes were studied and found to be very similar. The specific activities for PIP2 were greater than those obtained when PI was used. Both PLC-IV and PLC-beta m were Ca2(+)-dependent; near maximal stimulation for PI and PIP2 hydrolysis was observed at 0.5 microM free Ca2+. Sodium pyrophosphate and sodium fluoride stimulated phospholipase C activity of both isozymes. Polyclonal antibodies raised against PLC-beta m were able to inhibit carbachol and GTP gamma S stimulated phospholipase C activity in 2 M KCl washed rabbit cortical membranes. This suggests that in rabbit brain muscarinic cholinergic stimulation regulates PLC-beta m.     

Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000-fold purification was achieved by a combination of heparin-Sepharose, DEAE-cellulose and gel-permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5-bisphosphate as substrate the enzyme expresses a specific activity of greater than 100 mumol min-1 mg-1. The phospholipase C is activated by Ca2+ (0.1-10 microM). The behaviour of this particulate enzyme is discussed in the context of a agonist-induced phosphatidylinositol hydrolysis.     

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.     

Demonstration and purification of multiple bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes.

Calmodulin (CaM)-stimulated phosphatase in bovine brain or bovine lung CaM-binding protein fractions were fractionated on a heparin-Sepharose column into three activity peaks, designated in order of column three activity peaks, designated in order of column elution as the brain peak I (BPI), peak II (BPII), and peak III (BPIII) or the lung peak I (LPI), peak II (LPII), and peak III (LPIII) phosphatases, respectively. The pooled individual peak fractions were further purified on a fast protein liquid chromatography Superose 12 column. Analysis of the purified samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that they all contained major peptides corresponding to alpha and beta subunits of the brain CaM-stimulated phosphatase. The phosphatases had similar specific activities and were similarly stimulated by Ni2+, Mn2+, Mg2+ + Ca2+, and CaM. They showed differential reactivity on immunotransblots with an alpha subunit-specific monoclonal antibody VJ6, which reacted strongly toward BPI and weakly toward BPIII and LPI, but showed no reactivity toward BPII, LPII, and LPIII. Each of the alpha subunits of the purified phosphatases had a distinct V8 protease and chymotrypsin peptide map. The results suggest that both bovine brain and bovine lung contain multiple CaM-stimulated phosphatase isozymes. The suggestion of three mammalian brain CaM-stimulated phosphatase isozymes is in agreement with the results of recent molecular cloning studies (Kuno, T., Takeda, T., Hirai, M., Ito, A., Mukai, H., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun. 165, 1352-1358; Guerini, D., and Klee, C.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9183-9187; da Cruz e Silva, E. F., and Cohen, P. T. W. (1989) Biochim. Biophys. Acta 1009, 293-296). The successful purification of the individual isozymes may facilitate the elucidation of molecular basis and physiological significance of the isozymes.     

Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D.

1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.     

Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor)

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, I. Igaue [1992] Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.     

Characterization of phosphoinositide-specific phospholipase C from human platelets.

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.     

Beta-lactamase inhibitors. The inhibition of serine beta-lactamases by specific boronic acids.

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].     

Flavanone-7-O-glucosyltransferase activity from Petunia hybrida

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 microM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavanoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.     

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)