scholarly journals Characterization of phosphoinositide-specific phospholipase C from human platelets

1987 ◽  
Vol 243 (3) ◽  
pp. 763-771 ◽  
Author(s):  
V Manne ◽  
H F Kung
Keyword(s):  
Phospholipase C ◽  
Specific Activity ◽  
Human Platelet ◽  
Gel Filtration ◽  
Sodium Deoxycholate ◽  
Human Platelets ◽  
Ras Proteins ◽  
Ph Optimum ◽  
Pi Hydrolysis

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.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

scholarly journals Characterization of partially purified phospholipase C from human platelet membranes

1987 ◽  
Vol 248 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Y Banno ◽  
Y Nozawa
Keyword(s):  
Phospholipase C ◽  
Membrane Fraction ◽  
Specific Activity ◽  
Human Platelet ◽  
Gel Filtration ◽  
Total Activity ◽  
Hydrolytic Activity ◽  
Platelet Membrane ◽  
Dependent Manner

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.

scholarly journals Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets

1986 ◽  
Vol 237 (1) ◽  
pp. 139-145 ◽  
Author(s):  
M G Low ◽  
R C Carroll ◽  
A C Cox
Keyword(s):  
Substrate Specificity ◽  
Phospholipase C ◽  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Platelets ◽  
Physiological Significance ◽  
Multiple Forms

The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599 ◽  
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

Abstract A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

scholarly journals Purification and Characterization of Human Platelet Factor 3.

1977 ◽  
Author(s):  
H. Sandberg ◽  
L-O. Andersson ◽  
S. Hoglund
Keyword(s):  
Membrane Filtration ◽  
Human Platelet ◽  
Gel Filtration ◽  
Human Platelets ◽  
Purification And Characterization ◽  
Filtration Fraction ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Main Components

In the purification of platelet factor 3 fresh human platelets prepared by centrifugation were used as starting material. Release material having platelet factor 3 activity, as measured by the Stypven time, was obtained by treating the platelet suspension with collagen or thrombin followed by removal of the platelets by centrifugation. Residual platelets left in supernatant were removed by membrane filtration. Purification of platelet factor 3 from the other components in the release material was accomplished by affinity chromathography followed by gel filtration on Sepharose 2B. The gel filtration fraction having platelet factor 3 activity was studied with respect to chemical composition and appearance in electron mincroscopy. Phospholipids and cholesterol were the main components but small amounts of protein could also be detected. The electron microscopy revealed the presence of numerous membranous particles having diameters between 50-150 Å. The purified fraction was used for immunization of rabbits and an antiserum was obtained which inhibited the platelet factor 3 activity.

scholarly journals The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

1990 ◽  
Vol 1 (12) ◽  
pp. 937-950 ◽  
Author(s):  
L M Machesky ◽  
P J Goldschmidt-Clermont ◽  
T D Pollard
Keyword(s):  
Phospholipase C ◽  
Human Platelet ◽  
Gel Filtration ◽  
Human Platelets ◽  
Lower Affinity ◽  
High Affinity ◽  
Inositol Phospholipids ◽  
Cytoskeletal Dynamics ◽  
Inositol Phospholipid ◽  
Phosphatidylinositol 4

In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2.

scholarly journals Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets

2010 ◽  
Vol 5 (2) ◽  
pp. 1934578X1000500
Author(s):  
Jie Hua ◽  
Ke-long Huang
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Barley Malt

Two 5′- phosphodiesterases (5′-PDE-a and 5′-PDE-b) were isolated from barley malt rootlets, and further purified by gel filtration on Sephadex G-25 and Sephadex G-75. 5′-PDE-a had a pH optimum of 5.0, temperature optimum of 70oC, and specific activity of 0.0143 mM ·mg−1-min−1. 5′-PDE –b had a pH optimum of 6.0, temperature optimum of 65°C and specific activity of 0.0125 mM ·mg−1·min−1. Both enzymes can be used to hydrolyze RNA to form 5′-nucleotides. The enzymes were quite stable at 70oC for 420 minutes. The Km was 0.24 mM for 5′-PDE-a and 0.16 mM for 5′-PDE-b with t-RNA (yeast) as substrate.

scholarly journals Purification and characterization of a collagenase extracted from rabbit tumours

1975 ◽  
Vol 152 (1) ◽  
pp. 131-142 ◽  
Author(s):  
P A McCroskery ◽  
J F Richards ◽  
E D Harris
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Chelating Resin ◽  
Rabbit Muscle ◽  
Ph Optimum ◽  
Chelex 100 ◽  
Cnbr Cleavage ◽  
Total Enzyme

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of α1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as μmol of collagen in solution cleaved/h per mg of enzyme at 35°C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1μM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides α2 and α1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [α1 (I)]2α2 and [α1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [α1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4)2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the α1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.

Partial Characterization of Pyruvate Decarboxylase Isolated From Germinating Pea Seeds

1980 ◽  
Vol 7 (1) ◽  
pp. 35 ◽  
Author(s):  
S Leblova ◽  
J Valik
Keyword(s):  
Sodium Phosphate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pyruvate Decarboxylase ◽  
Temperature Stability ◽  
Hill Coefficient ◽  
Ph Optimum ◽  
The Hill

Pyruvate decarboxylase (EC 4.1.1.1), isolated from 4-day-old germinating peas, was precipitated from a sodium phosphate extract when (NH4)2SO4 was increased from 15 to 30% saturation, desalted on Sephadex G-25 or by dialysis for 24 h, and then chromatographed on a DEAE-cellulose column. This procedure increased the specific activity of the enzyme 120-fold compared with the sodium phosphate extract. The behaviour of the enzyme during gel filtration indicates that it has a high molecular weight. The pea enzyme exhibits a sigmoid dependence on the pyruvate concentration; reaction velocity is half-maximal at a substrate concentration of 1.8 mM and the Hill coefficient is 1.8. Thiamin pyrophosphate (TPP) is the coenzyme, which is relatively firmly bound to the apoenzyme, but can be removed by dialysis for 48 h. The apoenzyme is activated optimally at 2 mM TPP and inhibited by concentrations above 4 mM. The pH optimum for pea pyruvate decarboxylase is 5.8 and maximal temperature stability occurs at 48°C.

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