Rapid purification of malate synthase from cotyledons of Brassica napus L.

A rapid and efficient method for the purification of malate synthase, an enzyme uniquely confined to glyoxysomes, from cotyledons of Brassica napus L. has been developed. The two step purification procedure is based on the consequent utilization of the tendency of malate synthase to form high molecular weight aggregates. Malate synthase was purified 75-fold to apparent homogeneity with a specific activity of 180 nkat/mg protein. The estimated molecular weight of malate synthase subunits was 63 kDa. Polyclonal antibodies raised against malate synthase in rabbits detect on Western blots only one single polypeptide with an identical molecular weight.

Fatty acid ß-oxidation and glyoxylate cycle enzyme activities of induced glyoxysomes from anise suspension cultures.

Homogenates of dedifferentiated anise (Pimpinella anisum L.) suspension cultures grown in B-5 medium with sucrose as source of carbon show all but 3 glyoxysomal enzyme activities: NAD-dependent oxidation of palmitoyl-CoA, isocitrate lyase, and malate synthase are lacking. Substitution of 20 mmol/l acetate for sucrose leads to the appearance of these enzyme activities. Only then glyoxysomes with a buoyant density of 1.23 kg/l in sucrose gradients are formed showing the enzyme activities for both ß-oxidation of fatty acids and glyoxylate cycle. Quantitatively and qualitatively they resemble glyoxysomes isolated from endosperm of 4 d old anise seedlings. Therefore, the suspension cultures constitute a valuable system for the study of both mechanisms and regulation of glyoxysome formation in anise.

Loss of competence for glyoxysome formation during somatic embryogenesis in anise (Pimpinella anisum L.) suspension cultures.

Somatic embryogenesis in anise (Pimpinella anisum L.) suspension cultures induced by transfer to hormone-free growth medium may be synchronized by previous selection of cell aggregates with diameters between 100-240 µm. Around 80-90% of the embryoids are globular after 2-3 d, heart-shaped after 5-7 d and torpedo-shaped after 9 d. In embryogenic medium without source of carbon or with 20 mmol/l acetate differentiation and growth cease. But like in dedifferentiated cell aggregates the key enzyme activities for glyoxysomes such as isocitrate lyase and malate synthase are induced in globular (3 d old) and heart-shaped (5 d old) embryoids, but not in embryoids at day 7 or later. Similarly, in explants from anise hypocotyl glyoxysomes cannot be derepressed by such treatment. It is concluded that during differentiation of heart-shaped embryoids to torpedo forms the competence of the cells for the yet unknown inducing principle for glyoxysomes is lost.

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings : Ultrastructural evidence.

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3'-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.

The ontogeny of lipid bodies (spherosomes) in plant cells : Ultrastructural evidence.

Maturing embryos of 16 oil plants, anise suspension culture cells, and Neurospora crassa cells were prepared for electron microscopy at different stages during massive lipid accumulation. Lipid-rich structures of certain species were best preserved by dehydration of fixed tissues in ethanol without propylene oxide, embedding in Spurr's Medium, and polymerization at room temperature. In all cells examined, spherical lipid bodies (spherosomes) showed a moderately osmiophilic, amorphous matrix and displayed a delimiting half-unit membrane when sectioned medially. Associations with the endoplasmic reticulum (ER) were viewed at any stage during lipid body development but with different frequency in the different plant species. Plastids of fat-storing cells exhibited conspicuously undulate outer and inner envelope membranes that formed multiple contact sites with each other and protuberances into both cytoplasm and stroma. Some species, e.g., Linum, have plastids with tubular structures that connect the inner membrane to the thylakoid system; in addition, in the stroma vesicles fusing with or apparently passing through the envelope were observed. The outer envelope membrane may be associated with ER-like cytoplasmic membrane structures. In addition, lipid bodies of various sizes were found in contact with the plastid envelope. The ultrastructural observations are interpreted to match the published biochemical evidence, indicating that both plastids and ER may be involved in the synthesis of storage lipids and lipid body production.

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons.

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent KM was 6.5 mmol l(-1), with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l(-1) NaCl and on the presence of at least 0.1 mol l(-1) NaCl in the test mixture. Desoxycholate and up to 0.1 mol l(-1) CaCl2 also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.

Isolation and biochemical properties of two types of microbody from Neurospora crassa cells.

Cells of the Neurospora crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS), and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole carbon source ("acetate medium") induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble isocitrate lyase activity increased rapidly after a lag phase of about 45 minutes. Addition of 0.1 mM cycloheximide to the acetate medium 3 hours after transfer of the cells halted the rise of isocitrate lyase activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 hour. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of "slime" cells by sucrose density gradient centrifugation yielded two major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activites of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of two types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activities were recovered at 1.24 kg/l.

Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons.

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.

Membranous appendices of spherosomes (oleosomes) : Possible role in fat utilization in germinating oil seeds.

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a "half unit membrane" that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these "handles". At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.

Kinetin action on the development of microbody enzymes in sunflower cotyledons in the dark.

Removal of the roots from etiolated sunflower seedlings (Helianthus annuus L.) at various stages of development resulted in a premature or enhanced decline of the activities of catalase (E.C. 1.11.1.6) and isocitrate lyase (E.C. 4.1.3.1) (glyoxysomes), and hydroxypyruvate reductase (E.C. 1.1.1.26) and glycolate oxidase (E.C. 1.1.3.1) (leaf peroxisomes) in the cotyledons. Treatment of the cuttings with kinetin in the dark inhibited the loss of glyoxysomal enzyme activities and, at the same time, induced a three to fivefold increase in leaf-peroxisomal enzyme activities. The decline of glyoxysomal enzyme activities was also suppressed after application of both kinetin and cycloheximide (50 µg/ml). The kinetin-mediated rise of leaf-peroxisomal enzyme activities was strongly curtailed in the presence of cycloheximide. The dose effect curves of kinetin action on the development of glyoxysomal and of leaf-peroxisomal enzyme activities were different, supporting the hypothesis that the mechanisms of kinetin action on the two microbody enzyme systems are different. Nitrogen nutrition of intact seedling effected the development of microbody enzyme activities in a pattern closely resembling that of kinetin action. Presumably endogenous cytokinins produced by the roots are involved in the regulation of microbody enzyme activities in cotyledons of dark-grown sunflower seedlings.

Studies on the Development and Localization of Catalase and H(2)O(2)-generating Oxidases in the Endosperm of Germinating Castor Beans.

During germination of castor bean seeds (Ricinus communis var. Hale), the changes of activity of catalase, uricase, and alpha-hydroxyacid oxidase of the endosperm follow a rise and fall pattern with a peak between day 4 and 5 similar to that observed for the glyoxylate cycle enzymes. After 3 days of germination, most of the activities of these enzymes are recovered from the glyoxysomal fraction separated by isopycnic sucrose density gradient centrifugation.The activities of the oxidases of the glyoxysomal fractions increase somewhat more slowly than those of catalase or isocitrate lyase during the first 3 days of germination. But separation of the oxidative enzyme activities from glyoxysomal isocitrate lyase activity was not achieved by density gradient centrifugation or by density gradient electrophoresis. The sedimentation velocities of these two kinds of enzyme activities were found to be identical. The role and regulation of the glyoxysomal uricase, glycolate oxidase, lactate oxidase, and catalase activity in endosperm cells are discussed.

[Light dependent carotenoid synthesis : VIII. Different action mechanisms of light and mercuribenzoate]. / Untersuchungen über die lichtabhängige Carotinoidsynthese : VIII. Die unterschiedlichen Wirkungsmechanismen von Licht und Mercuribenzoat.

The fungus Fusarium aquaeductuum synthesizes large amounts of carotenoids only after illumination or in the presence of mercuribenzoate (HMB) in the dark. The effect of HMB is abolished entirely by the addition of excess thiols such as cysteine or mercaptoethanol; evidently "active" HMB must be present continuously within the cell. In contrast, photoinduction needs only a short exposure to light to set off pigment production in a subsequent dark period. Illumination of mycelia preincubated with HMB in the dark induces an additional proportion of carotenoid synthesis which shows the same kinetics and quantitative yields as that observed in untreated controls. This demonstrates that HMB, though inhibiting O2-uptake by 50%, does not interfere with light-induced carotenoid production as such. The effects of light and HMB are additive under various experimental conditions tested. After addition of thiols to mycelia treated with HMB in the light, carotenoid production decreased to an extent equal to the amount mediated by HMB in the dark controls. These results provide strong evidence that the mechanisms and the action sites of light and HMB in carotenoid synthesis are different. Incubation with HMB after exposure to light reduces the agent's effectiveness, which declines gradually to about 30% during the first two hours.These findings are interpreted to mean that in Fusarium there are two isoenzyme systems at work in carotenoid production: a constitutive one showing very low net activity is subject to direct stimulation by HMB; light somehow eliminates this enzyme system and, at the same time, induces an isoenzyme set which exhibits high carotenogenic activity and is insensitive to HMB treatment.

Uricase and allantoinase in glyoxysomes.

In fat-degrading tissues of seedlings of seven different plant species examined, uricase activity (urate:O(2) oxidoreductase, EC 1.7.33) was associated with particulate fractions. After equilibrium density centrifugation on sucrose density gradients the enzyme activity was recovered in the glyoxysomal band (density: 1.25 grams per cubic centimeter). Allantoinase is also present in glyoxysomes but, equally, in the proplastid region (density: 1.22 grams per cubic centimeter). Xanthine oxidase, xanthine dehydrogenase, allantoicase, and urease were not detected in glyoxysomes from castor bean endosperm. Uricase in these particles shows its maximal activity at pH 8.9. The apparent K(m) is 7.4 mum. Urate concentrations greater than 120 mum as well as certain other purine compounds inhibit the enzyme. Cyanide at a concentration of 10 mum is a potent inhibitor. 2,6-Dichlorophenolindophenol did not substitute for oxygen as electron acceptor.

[Light dependent carotenoid synthesis : V. Extinction of the photoinduction by reducing substances and substitution of hydrogen peroxide for light]. / Untersuchungen über die lichtabhängige Carotinoidsynthese : V. Aufhebung der Lichtinduktion durch Reduktionsmittel und Ersatz des Lichts durch Wasserstoffperoxid.

The effect of dithionite, hydroxylamine and hydrogen peroxide on the light dependent carotenoid synthesis in Fusarium aquaeductuum has been investigated in order to find indications whether redox reactions are involved in the first steps of photoinduction.Addition of dithionite (5·10(-3) M/l) to the mycelium some time after illumination prevented carotenoid synthesis completely; however, when dithionite was removed after 30 min by washing the mycelium with buffer, Fusarium synthesized nearly the same amounts of carotenoids as it does without dithionite incubation. To prevent this direct effect on biosynthesis of pigments, the mycelium was treated for only 30 min at different times before and after a short illumination with buffered dithionite solution. When dithionite was present during the illumination or was applied up to 21/2 min after the lights had been switched off, no carotenoids were synthesized at all. The inhibitory effect of dithionite gradually decreased during a 171/2 min period following the end of the illumination time. After this period treatment with dithionite showed no irreversible influence whatsoever on the carotenoid synthesis. Essentially the same results were obtained when hydroxylamine (10(-2) M/l, freshly prepared) was used as a reducing agent.On the other hand incubation with buffered hydrogen peroxide solution (10(-2) to 10(-1) M/l) in the dark simulated the effect of illumination in inducing carotenoid synthesis. Both the kinetics of the pigment production and the inhibition by cycloheximide suggest that treatment with hydrogen peroxide in the dark truly substitutes for photoinduction. From these results it is concluded that dithionite and hydroxylamine are capable of reducing as yet unknown "photooxidation products" which are produced during illumination, as proposed by several authors. This oxidative action of light can be simulated by incubation of the mycelium with hydrogen peroxide.Furthermore results are presented which suggest that in Fusarium light acts in two ways: 1. it induces a de novo protein synthesis giving rise to an enhanced carotenoid production ("light dependent synthesis") and 2. it inhibits a carotenoid synthesizing system ("dark synthesis") which functions with low activity in the mycelium in the dark.