Comparative Studies of Glyoxysomes from Various Fatty Seedlings

Abstract

The separation of various organelles from cotton cotyledon (Gossy-pium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperni (Ricinius cornmunis L.). Eqttilibrium densities were 1.12 to 1.13 g,'cm3 for endoplasmic reticulum, 1.17 to 1.19 g/cm' for mitochondria, and 1.25 g/cm' for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from differeiit fattv seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to suborganelle localization and behavior in the presence of KCI and Triton X-100. In pine megagametophyte, the glyoxvsomes and not the mitochondria are the initracellular site for the breakdouwn of stored lipid. Glyoxysomes were first discovered and then studied intensively in the endosperm of germinating castor bean as discrete organelles housing a sequence of gluconeogenesis enzymes including alkaline lipase (18), fatty acid-activating enzyme (9), enzymes of the /3-oxidation pathway (8, 15) and of the glyoxylate cycle (3), and the characteristic microbody marker enzymes (4, 19). It was found that glyoxysomes are also present in many other fatty seedlings (1, 5, 7, 11. 16, 19, 20).In the studies of glyoxysomes, there are some discrepancies as to their possession of nucleic acids and protein-synthesizing ability (5,10), their interrelationship with the leaf peroxisomes in greening cucurbit cotyledons (11,16,20), and the association of certain enzymes to the glyoxysomal membrane (14,21 Isolation of Organelles. The tissues were chopped in grinding medium with a razor blade followed by grinding in a mortar and pestle as described prev,iously (14). The supernatant fraction obtained after centrifugation at 270,g for 10 min was layered onto a sucrose gradient which was composed of 10 ml of 20% (w/w) sucrose on top of 40 ml of a 30 to 60% linear sucrose gradient. The gradient was centrifuged at 21,000 rpm for 4 hr in a Beckman L2-65B ultracentrifuge with Spinco rotor 25.2. Sufficient extract from each tissue was put onto the gradient so that 5 to 6 mg of glyoxysomal protein were obtained in the region of density 1.25 g/cm' in the gradient after centrifugation.Assays. The assays of catalase, glycolate oxidase. Cyt oxidase, malate synthetase, citrate synthetase, isocitrate lyase, malate dehydrogenase, and fatty acyl CoA dehydrogenase (12-14) were as described previously. Alkaline lipase was assayed using N-methylindoxylmyristate as an artificial substrate (18). The assay of NADH-Cyt c reductase followed that described (17) using NADH to initiate the reaction. Triose phosphate isomeras...

Partial Purification and Characterization of the Mitochondrial and Peroxisomal Isozymes of Enoyl-Coenzyme A Hydratase from Germinating Pea Seedlings

“…The activities of the ,-oxidation enzymes were approximately equally distributed in both organellar fractions (Table I). This result is in marked contrast to reports from other workers on the localization of the $-oxidation enzymes in higher plant cells (6,12,15,27), but is consistent with previous publications from this laboratory (21,30,32,34,35). It has been noted that the substrates of the ,8-oxidation enzymes do not easily cross the intact mitochondrial inner membrane, thus this membrane must be disrupted before detecting enzyme activity (34).…”

Reactivation by a bacterial acetate: Enzyme ligase of plant glyoxysomal isocitrate lyase

“…Glyoxysomes were prepared from homogenates of cotyledons of S-day old cucumber seedlings (7 mU isocitrate lyase/mg protein) by isopycnic density gradient centrifugation [6,7]. The matrix enzymes, liberated by osmotic shock, were subjected to isoelectric focussing using the range pH 5-8 [S] .…”

Isolation of Plasma Membranes from Corn Roots by Sucrose Density Gradient Centrifugation

“…NADH-Cyt c reductase activity in this area of the gradient was not sensitive to I ,UM antimycin A, a concentration which strongly inhibited the activity of this enzyme in mitochondria. Endoplasmic reticulum from endosperm and cotyledon of a variety of fatty seedlings equilibrates at a density of about 1.12 g/cc in sucrose gradients (13,20).…”

“…In addition, the 13,000g pellet and the 13,000 to 80,000g pellet from the same homogenate were centrifuged on identical nonlinear sucrose gradients, and membranes were removed from the 34 to 45% (1.15-1.20 g/cc) interface. Based on the results for oat roots, this interface should be rich in mitochondria when the 13,000g pellet is layered on the gradient and in plasma membranes when the 13,000 to 80,000g pellet is the overlay. ATPase, Cyt c oxidase, and total protein were also determined for these "mitochondrial" and "plasma membrane" fractions (Tables I andII).…”