Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

High-performance liquid chromatographic analysis of acylated lipids containing pyrene fatty acids

A high-performance liquid chromatographic method for the separation and quantification of acylated lipids containing pyrene fatty acids is described. The method is adapted from a procedure originally developed for the analysis of tissue lipids (Christie, W. W. (1985) J. Lipid Res. 26, 507-512). Pyrenyl lipid analogs ranging in polarity from cholesteryl ester to lysophosphatidylcholine are completely resolved on a silica column in 50 min by gradient elution with a ternary solvent system. Furthermore, pyrene-labeled triglycerides are resolved according to the number of pyrene fatty acid residues incorporated. Pyrenyl lipids are detected at levels of 10(-13) mol by high-sensitivity fluorescence detection. Accurate quantification of pyrenyl lipids is obtained by correcting peak areas for mobile-phase quenching effects. The close correspondence between chromatograms obtained for the separation of labeled lipids extracted from Hep-G2 cells incubated with either 12-(1-pyrenyl)dodecanoic acid (fluorescence detection) or [1-14C]oleic acid (radioactivity detection) indicates that this HPLC method is equally suitable for analysis of native lipids.     

Nutritional properties of trans fatty acids

The role of trans fatty acids (TFA) present in partially hydrogenated fats widely consumed in food and their link with coronary heart disease has been examined in this review. Most of the studies carried out have been on the effects of TFA on blood-lipid profile. The perceived effects of TFA intake depend on the fat or oil with which they are compared and appears to be in between that of dietary saturated fats and monounsaturated fatty acids. When compared to saturated fat, TFA intake shows lower levels of total and LDL-cholesterol in blood. But when both TFA and saturated fatty acids are compared with cis fatty acids or native unhydrogenated oil, increase in total and LDL-cholesterol are noted. The effects of TFA on HDL-cholesterol and Lp(a) are not clearly established. The undesirable effects of TFA can be overcome by inclusion of essential fatty acids at a minimum of 2 energy per cent level in the diet. The link between trans fatty acid intake and coronary heart disease (CHD) are not unequivocally established.     

Preparation and physical properties of chitin fatty acids esters

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 °C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T_g) and the tensile modulus (E′ at 25 °C) are highest for chitin acetate (218 °C and 5.8 GPa), and lowest for chitin octanoate (182 °C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T_g and the E′ decrease with an increase in the chain length of the acyl constituent.     

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I. Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Aspergillus niger was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34 mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the ortho-position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.     

Purification and properties of the fatty acids synthetase complex from Neurospora crassa, and the nature of the fas-mutation.

A procedure is described for the purification of the fatty acid synthetase complex (FAS) from Neurospora crassa. The enzyme complex has a molecular weight of 2.3 times 10(6), contains 6 mol of 4'-phosphopantetheine per mol, and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a single band, or a closely spaced doublet, which comigrates with standard myosin (molecular weight, 2 times 10(5)). Since the slightly retarded component in the doublet accounts for all protein-bound 4'-phosphopantetheine, the complex appears to be made up of 11 to 12 equally sized subunits, 6 of which carry the acyl carrier protein function. In this unusual arrangement, notably the lack of the low-molecular-weight acyl carrier protein component seen in other FAS systems, as well as in its enzymatic properties, the Neurospora FAS complex is quite similar to the yeast enzyme. The FAS complex of a saturated fatty acid-requiring mutant, previously disignated cel-, contains less than 2% of the 4'-phosphopantetheine prosthetic groups found in the wild-type complex. The leaky phenotype of this mutant, here designated fas-, is accounted for by a residual fatty acid synthesizing activity in its FAS complex, which is several-fold higher than expected from its residual content of 4'-phosphopanthetheine.     

Purification and properties of AMP-deaminase from human uterine smooth muscle. Regulation by adenylate energy charge and activated fatty acids

At pH 7.0 and physiological concentrations of the main regulatory ligands (ATP, ADP, orthophosphate), human uterine muscle AMP-deaminase follows a hyperbolic type of saturation kinetics with S0.5 parameter value about 2 mM. The enzyme is regulated by adenylate energy charge (AEC) variations, being the most active at the AEC value 0.5-0.6 or 0.5-0.7, depending on the size of the total adenine nucleotide pool. Long-chain acyl-CoA strongly inhibit activity of the enzyme, influencing mainly the maximum velocity of the reaction.     

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA; AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema, the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and alpha-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their in vivo properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs; HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of the HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.     

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA;AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema. the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and α-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs;HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of teh HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.     

Purification and characterization of C28-55 fatty acids from Mycobacterium smegmatis

The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components. The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the alpha-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.     

The formation of mutagenic derivatives of benzo[a]pyrene by peroxidising fatty acids

Benzo[a]pyrene (B[a]P) when incubated in the presence of peroxidising polyunsaturated fatty acids such as linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5) or docosahexaenoic acid (C22:6) was converted to oxidised products. Between 7% and 9% of the B[a]P was oxidised in one hour when incubated with arachidonic acid and docosahexaenoic acid. 1,6- 3,6- and 6,12-Quinone derivatives of B[a]P were identified by HPLC. The products of B[a]P oxidation were shown to be mutagenic when tested using Sister chromatid exchange (SCE) technique and the occurrence of SCEs in CHV79 cells was increased significantly. Lipid peroxides also induced SCEs in the absence of B[a]P and there was a positive correlation between the frequency of SCEs and the extent of lipid peroxidation. The results indicate that the oxidation of B[a]P mediated by the non-enzymic peroxidation of polyunsaturated fatty acids is likely to play a role in mutagenesis and, possibly, also in carcinogenesis.     

Studies on the metabolism of unsaturated fatty acids. XV. Purification and properties of 2,4-dienoyl-CoA reductase from rat liver peroxisomes

Peroxisomal 2,4-dienoyl-CoA reductase was purified from rat liver to homogeneity. The subunit molecular weight of 33,000 was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The native molecular weight close to 120,000 was estimated by gel filtration on Sephacryl S-300 Superfine. trans-2, trans-4-Decadienoyl-CoA was the most active substrate among the dienoyl-CoA's of various chain lengths. The total activity of peroxisomal 2,4-dienoyl-CoA reductase exceeded that of the mitochondrial one even in the livers of rats fed with a standard diet. Furthermore both reductases were remarkably and coordinately induced in the livers of clofibrate-treated rats.     

Correlation between medium C-chain fatty acids and polyunsaturated fatty acids

The medium C-chain fatty acids increased in the muscle, lungs, pancreas and adipose tissue (and not in the liver) of the rats injected with CCl4 or nourished with "balanced" diet for the lipids. When CCl4 and balanced diet are furnished together, these acids decrease strongly: the discussion of the results is difficult.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.