The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Fatty acids of the scleractinian coral Galaxea fascicularis: effect of light and feeding

In order to investigate nutritional interactions in the symbiotic scleractinian coral-zooxanthella association, fatty acids of the coral Galaxea fascicularis were analysed in two groups of cultured microcolonies. The first group was fed with Artemia sp., while the second group was starved. After an initial 1-month period during which both groups were subjected to the same normal light conditions (constant irradiance of 125 E·cm^-2·s^-1 and 14:10 h light:dark), a light cap was used to cover the aquarium and keep all the microcolonies in permanent darkness for 20 days. During the light phase of the experiment it was shown that the nutritional status lead to large variations in the percentage of saturated, mono-unsaturated and polyunsaturated fatty acids. Palmitic acid (C16:0) was the most abundant fatty acid in both groups. Important differences between fed and starved microcolonies occurred during the dark phase of the experiment. In the fed group the dark phase was characterized by a significant increase in polyunsaturated fatty acids. Particularly arachidonic acid (C20:4 n-6) became the most important fatty acid followed by docosatrienoic acid (C22:3 n-3). A slight increase in these two fatty acids was also found in the starved group but the bulk of polyunsaturated fatty acids was significantly decreased. In this group, palmitic acid remained the most important fatty acid while an increased concentration of cis-vaccenic acid (C18:1 n-7) was found at the end of the experiment. The increased concentration of cis-vaccenic acid might indicate that bacteria serve as a source of energy. While the number of zooxanthellae per milligram of protein and the chlorophyll a to protein ratio strongly decreased in the starved microcolonies immediately after the beginning of the dark period, the decrease in fed microcolonies was delayed for about 10 days. Furthermore, after 20 days of dark incubation the chlorophyll a to protein ratio was the same as measured at the beginning of the dark period. This suggests that in the dark the metabolic requirements of the zooxanthellae are in part met from the animal host through a heterotrophic mode of nutrition.Abbreviations CZ cultured zooxanthellae - FAME fatty acid methylester(s) - FDM fed dark microcolonies - FLM fed light microcolonies - MUFA monounsaturated fatty acid(s) - PUFA polyunsaturated fatty acid(s) - SDM starved dark microcolonies - SFA saturated fatty acids - SLM starved-light microcolonies - SW sea water - TFA total fatty acids     

Taxonomy of Pinus species based on the seed oil fatty acid compositions

 The fatty acid compositions of the seed oils from ten pine species have been established by capillary gas-liquid chromatography of the methyl esters. With regard to either normal fatty acids or Δ5-olefinic acids, the general pattern of fatty acids did not differ from that of other pine seed oils reported previously. The main fatty acid was linoleic (9,12–18:2) acid (44.4–57.1%), followed by either oleic (9–18:1) acid (13.4–24.5%) or pinolenic (5,9,12–18:3) acid (1.5–25.2%). When applying multivariate analyses to the chemometric data (13 variables) of 49 pine species (ca. 40% of the living pine species), it was possible to distinguish between several sections: Pinea, Longifolia, Halepensis, Ponderosa-Banksiana, Sylvestris, and Cembra. The latter section was clearly divided into two sub-groups. A few species that presented a low overall content of Δ5-olefinic acids, and that grow in warm-temperate regions, were isolated from the bulk of other pine species. It is hypothesized that Δ5-olefinic acids might be related to cold-acclimation. Received: 5 June 1997 / Accepted: 17 August 1997     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

The role of triiodothyronine in the regulation of synthesis rate and translatable mRNA level of fatty acid synthetase in a preadipocyte cell line

Triiodothyronine (T₃) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob₁₇ preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6–10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 40–42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T₃ included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T₃ effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T₃ would mainly affect fatty acid synthetase as a pretranslational level.     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Acetyl coenzyme A biosynthesis in the chloroplast

Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 M acetate, 10 M bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.Abbreviations ACP acyl carrierprotein - CoASH coenzyme A     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Phospholipid composition and fatty acid desaturation in the roots of rye during acclimatization of low temperature

When the roots of rye plants grown at 20°C were cooled to 8°C the concentration of phospholipid in them more than doubled over a 7 d period in comparison with that in roots remaining at 20°C. The relative abundance of lecithin (PC) declined while that of phosphatidyl ethanolamine (PE) increased; this change was completed after 2 d cooling. Labelling with ³²P suggested that turnover of phospholipids may be inhibited by low temperature. Acyl lipids contained an increased proportion of linolenic acid (18:3) and reduced proportion of linoleic acid (18:2) when roots were cooled at 8°C for 7 d. The ratio of these acids is a relatively more sensitive indicator of desaturation than is the double bond index. Cooling brought about no change in the abundance of the principal saturated acid, palmitic (16:0). In the first 3 days of cooling PC and PE desaturated markedly while there was no change in galactosyl and neutral lipids. Desaturation did not appear to be greatly sensitive to the concentration of dissolved O₂ and was only partly inhibited in 8°C solutions where the oxygen concentration was lowered to 0.5–2.0%. Positional analysis of acyl chains in PC and PE showed that more than 90% of all 16:0 is associated with position I while 65% of the 18:2+18:3 is associated with position II. When roots are cooled the abundance of 18:3 increases in both chains but the relative distribution of saturated and unsaturated fatty acids remains constant in positions I and II. At both 20°C and 8°C there is a high probability that a saturated chain in position I will be paired with the polyunsaturated one in position II.Abbreviations PC Lecithin - PE phosphatidyl ethanolamine - TLC thin layerchromatography - BHT butylatedhydroxytoluene