scholarly journals Analysis of leukotriene B4 metabolism in human promyelocytic HL-60 cells

1991 ◽  
Vol 279 (1) ◽  
pp. 283-288 ◽  
Author(s):  
S Kasimir ◽  
W Schönfeld ◽  
R A Hilger ◽  
W König
Keyword(s):  
Phorbol Myristate Acetate ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Leukotriene B4 ◽  
Dimethyl Sulphoxide ◽  
Lipid Mediator ◽  
Myristate Acetate ◽  
Ph Optimum ◽  
Highly Active ◽  
Undifferentiated State

We previously reported that human alveolar macrophages rapidly metabolize the chemotactic active lipid mediator leukotriene B4 (LTB4) into the dihydro-LTB4 by reduction of one of the conjugated double bonds. We herein report that human HL-60 cells (a myeloid precursor which can be differentiated into granulocyte- as well as monocyte-like cells by dimethyl sulphoxide or phorbol myristate acetate) express a highly active LTB4 reductase in the undifferentiated state. Differentiation by dimethyl sulphoxide (1.3%) along the granulocyte lineage, as confirmed by light microscopy, conversion of NitroBlue Tetrazolium into formazan, failed to induce a substantial capacity for omega-oxidation of LTB4; this reaction is exclusively found in mature granulocytes. Studies with the cell homogenate of undifferentiated HL-60 cells indicated that the activity of the enzyme depends on the presence of NADPH, Ca2+ and Mg2+, with a pH optimum of 7.5 at 37 degrees C. The enzyme was not released into the supernatant after stimulation of HL-60 cells with phorbol myristate acetate (100 ng) or Ca2+ ionophore (7.5 microM). Subcellular fractionation revealed evidence that the LTB4 reductase is located within the membrane fraction. Purification of the enzyme by gel filtration and gel electrophoresis suggests an apparent molecular mass of 40 kDa.

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

2000 ◽  
Vol 347 (2) ◽  
pp. 431-439 ◽  
Author(s):  
Sharon C. A. CHEN ◽  
Lesley C. WRIGHT ◽  
John C GOLDING ◽  
Tania C. SORRELL
Keyword(s):  
Cryptococcus Neoformans ◽  
Virulent Strain ◽  
Pathogenic Fungus ◽  
Gel Filtration ◽  
Peptide Fragments ◽  
Ph Optimum ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Control Value ◽  
Highly Active ◽  
Phospholipase B

Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH4)2SO4 fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent Vmax values for PLB and LPL activities were 12.3 and 870 μmol/min per mg of protein respectively; the corresponding Km values were approx. 185.3 and 92.2 μM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe3+. LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Effects of thrombin, phorbol myristate acetate and prostaglandin D2 on 40-41 kDa protein that is ADP ribosylated by pertussis toxin in platelets

FEBS Letters ◽  
1986 ◽  
Vol 204 (2) ◽  
pp. 341-346 ◽  
Author(s):  
Stephen P. Halenda ◽  
Mario Volpi ◽  
George B. Zavoico ◽  
Ramadan I. Sha'afi ◽  
Maurice B. Feinstein
Keyword(s):  
Phorbol Myristate Acetate ◽  
Pertussis Toxin ◽  
Prostaglandin D2 ◽  
Myristate Acetate
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