Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.     

Studies on the isolation and purification of chloroplasts from Euglena gracilis

Summary A method has been developed for the isolation of chloroplasts from Euglena gracilis grown under mixotrophic conditions. This method utilizes sucrose density-gradient centrifugation in the AXII zonal rotor and allows the rapid preparation of large amounts of chloroplasts free from contaminating whole cells and other cytoplasmic materials. The majority of the isolated chloroplasts appear intact in phase contrast and electron micrographs. The purified chloroplast fraction contains DNA, the major species of which has a density of 1.682 g/cm³. The species of DNA having a density of 1.707 g/cm³ seemed to result from the presence of contaminating nuclear fragments which could be removed by isopycnic flotation.This research was supported in part by a grant from USPHS No. AM-07189 (to J. M. E.), American Cancer Society Fellowship No. PF-443 (to J.F.P.) and grant No. 2972E06 from the Office of Sponsored Research, University of Florida (to J.F.P.). Univ. of Fla. Agr. Exp. Stat. No. 4538.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

Comparison of two fatty acid synthetases from Euglena gracilis variety bacillaris

Two enzyme systems from Euglena gracilis var. bacillaris which catalyze the de novo biosynthesis of fatty acids have been compared. One is a multienzyme complex of high molecular weight which is independent of ACP for activity in vitro, and the other is an ACP-dependent system of discrete enzymes (M. L. Ernst-Fonberg, (1973) Biochemistry12, 2449–2455). The latter activity is present in small amounts in etiolated cells and increases upon exposure of dark-grown cells to light, while multienzyme complex fatty acid synthetase activity decreases by about one-half after 24 hr of exposure to light. Results from the greening of dark-grown cells in the presence of cycloheximide, chloramphenicol, or spectinomycin suggests that the chloroplast ribosomes are involved in the appearance of the ACP-dependent activity; alternatively, the cytoplasmic ribosomes appear to be the site of biosynthesis of the multienzyme complex fatty acid synthetase (or a protein responsible for its activation). The fatty acid synthetase activities from several chloroplast mutants were measured. The ACP-dependent activity was reduced or not present depending on the degree of impairment of chloroplast development, while the multienzyme complex activity in all instances continued to respond to light or darkness.Antibodies against the purified multienzyme complex extensively inhibited its activity whereas the activity of the ACP-dependent system was consistently stimulated. The two enzyme systems are immunologically cross reactive but not identical.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

Tryptophan synthetase in Euglena gracilis strain G

The five enzyme activities in the synthesis of l-tryptophan have been obtained in extracts of Euglena gracilis. One of these, tryptophan synthetase, has been studied in detail. The general catalytic properties of tryptophan synthetase, including the range of reactions catalyzed and its substrate and cofactor affinities, are similar to those reported for other organisms. The Euglena enzyme has two properties never previously observed for tryptophan synthetase. First, the rate of catalysis of the conversion of indole-glycerol phosphate to l-tryptophan remained at its maximal value and was unaffected by the ionic environment up to 0.3 m KCl. In contrast, the conversion of indole to tryptophan showed a sharp maximum at 0.08 m KCl. Second, the enzyme is a component of a complex that includes every enzyme in the pathway committed to tryptophan biosynthesis with the exception of anthranilate synthetase, the regulatory enzyme.     

Purification of the chloroplastic valyl-tRNA synthetase from Euglena gracilis.

Euglena gracilis chloroplast valyl-tRNA synthetase was purified 990 fold to a specific activity of about 1100 units/mg protein, by a series of steps including ammonium sulfate precipitation and chromatography on hydroxyapatite, DEAE-cellulose, Blue Dextran — Sepharose and Sephadex G200. The enzyme gives a single band upon polyacrylamide gel electrophoresis, appears to be a monomer with a molecular weight of 126,000 daltons and has Km values of 1.5 × 10^?5 M for L-valine, 5 × 10^?5 M for ATP, and 6 × 10^?8 for tRNA^Val.     

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO₂ fixation were isolated from cells grown on low-vitamin-B₁₂ medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or l-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll