scholarly journals Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

1982 ◽  
Vol 205 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Toru Momoi ◽  
Yoav Ben-Yoseph ◽  
Henry L. Nadler
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Significant Activity ◽  
Acid Ceramidase ◽  
Ph Optimum ◽  
Farber Disease ◽  
Normal Controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C12:0), N-myristoyl- (C14:0) and N-palmitoyl- (C16:0) sphingosine (C18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C18:0) substrate. The activity towards N-linolenoylsphingosine (C18:3/C18:1), N-linoleoylsphingosine (C18:2/C18:1) and N-oleoylsphingosine (C18:1/C18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C18:0/C18:1). The activity towards N-stearoyldihydrosphingosine (C18:0/C18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C6:0/C18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher Km values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.

scholarly journals Chemical characterization of effective and ineffective strains of Rhizobium leguminosarum bv. viciae.

1999 ◽  
Vol 46 (4) ◽  
pp. 1001-1009
Author(s):  
S F Izmailov ◽  
G Y Zhiznevskaya ◽  
L V Kosenko ◽  
G N Troitskaya ◽  
N N Kudryavtseva ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Rhizobium Leguminosarum ◽  
Unsaturated Fatty Acids ◽  
Effective Strain ◽  
Chemical Characterization ◽  
Unsaturation Index ◽  
Fa Composition ◽  
Sds Page ◽  
Lower Molecular Mass

Chemical composition of lipopolysaccharide (LPS) isolated from an effective (97) and ineffective (87) strains of R. l. viciae has been determined. LPS preparations from the two strains contained: glucose, galactose, mannose, fucose, arabinose, heptose, glucosamine, galactosamine, quinovosamine, and 3-N-methyl-3,6-dideoxyhexose, as well as glucuronic, galacturonic and 3-deoxyoctulosonic acid. The following fatty acids were identified: 3-OH 14:0, 3-OH 15:0, 3-OH 16:0, 3-OH 18:0 and 27-OH 28:0. The ratio of 3-OH 14:0 to other major fatty acids in LPS 87 was higher that in LPS 97. SDS/PAGE profiles of LPS indicated that, in lipopolysaccharides, relative content of S form LPS I to that of lower molecular mass (LPS II) was much higher in the effective strain 97 than in 87. All types of polysaccharides exo-, capsular-, lipo, (EPS, CPS, LPS, respectively) examined possessed the ability to bind faba bean lectin. The degree of affinity of the host lectin to LPS 87 was half that to LPS 97. Fatty acids (FA) composition from bacteroids and peribacteroid membrane (PBM) was determined. Palmitic, stearic and hexadecenoic acids were common components found in both strains. There was a high content of unsaturated fatty acids in bacteroids as well as in PBM lipids. The unsaturation index in the PBM formed by strain 87 was lower than in the case of strain 97. Higher ratio of 16:0 to 18:1 fatty acids was characteristic for PMB of the ineffective strain.

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 Characterization of the unique In Vitro effects of unsaturated fatty acids on the formation of amyloid β fibrils

PLoS ONE ◽  
2019 ◽  
Vol 14 (7) ◽  
pp. e0219465 ◽  
Author(s):  
Miki Eto ◽  
Tadafumi Hashimoto ◽  
Takao Shimizu ◽  
Takeshi Iwatsubo
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Amyloid Β ◽  
In Vitro Effects

scholarly journals Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources

2017 ◽  
Vol 2017 ◽  
pp. 1-19 ◽  
Author(s):  
Iwona Gientka ◽  
Marek Kieliszek ◽  
Karolina Jermacz ◽  
Stanisław Błażejak
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fossil Fuels ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Carbon Sources ◽  
Debaryomyces Hansenii ◽  
Industry Waste ◽  
Identification And Characterization

The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified wereCandida inconspicua,Debaryomyces hansenii,Kluyveromyces marxianus,Kazachstania unispora, andZygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L−1.Kazachstania unisporawas able to accumulate the high amount of palmitoleic acid.

A Possible Mechanism for a Light-Driven Regulation of the Fatty Acid Composition in Galactolipids of Chloroplasts

1979 ◽  
Vol 34 (9-10) ◽  
pp. 815-819
Author(s):  
A. Sauer ◽  
K .-P. Heise
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Specific Activity ◽  
Physiological State ◽  
Phospholipid Fraction ◽  
Chromatographic Behavior ◽  
Acyl Acceptor ◽  
Lipid Mixture ◽  
Spinach Leaves

Abstract The occurrence of acylgalactosylglycerol (AGG) in spinach chloroplasts is proposed. In lipid se­parations of whole spinach leaves this fraction is hidden by variable amounts of extraplastidary steryl glucoside (SG) which depended on the physiological state of the leaf material. Beside the phospholipid fraction, this lipid mixture with unique chromatographic behavior, recently termed GL, exhibited the highest specific activity in the lipid extract of infiltrated leaf sections of spinach after short periods of illumination (2 min) under 14C-fixing conditions. The main source of label was found in the fatty acid residues of the lipids described. The decrease of specific activity in GL after a cold chase was accompanied by an increasing 14C-incorporation into the fatty acid moieties of the MGDG-as well as the phospholipid fraction. This effect was significantly reduced if the 14C-pulse was followed by a dark period. This incorporation behavior suggests, that AGG functions as an intermediary acyl acceptor in the light driven fatty acid transfer between the lipids decribed. SG-synthesis, on the contrary, is independent of additional illumination and seems to be localized outside the chloroplast. The last notion was derived from experiments involving the in­ corporation of [UDP-14C] glucose into SG by spinach leaf homogenates. With regard to the increasing level of trienoic fatty acids in AGG and MGDG during the regeneration of dark pretreated spinach leaves in the light, the data are interpreted in terms of a light regulated acylation of AGG with specific unsaturated fatty acids.

scholarly journals Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts

1986 ◽  
Vol 238 (2) ◽  
pp. 589-595 ◽  
Author(s):  
A L Miller ◽  
R Stein ◽  
M Sundsmo ◽  
R Y Yeh
Keyword(s):  
Lysosomal Enzymes ◽  
Specific Activity ◽  
Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Cultured Skin ◽  
Lysosomal Dysfunction ◽  
Chediak Higashi Syndrome ◽  
Normal Controls

Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.

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.

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