Compartmental Utilization of Carboxyl-¹⁴C-Tripalmitin by Tissue Homogenate of Pine Seeds

Abstract

A tissue homogenate of megagemetophyte of germinating seeds of Jeffrey pine (Pinus Jefferii Grev. and Balf.) was incubated with sonication-dispersed and albumin-carried 14C-tripalmitin in order to elucidate the sequential and quantitative role of cellular organelles in utilizing lipid reserve in seeds. After 5 minutes at 30 C, 25 % of the tracer was localized in the fat bodv fraction, 9% in the pellet containing niitochondria and glyoxysomes, 14% in the supernatant, and 2 % was found as C02. Radioactivity increased with time of incubation in the latter three fractions indicating the forward direction of utilization. Fat bodies contained mainly lipases and hydrolyzed the tracer to palmitate with diglyceride and monoglyceride as intermediates. About two-thirds of the palmitate had left the fat bodies in 5 minutes and entered the pellet fraction within which the tracer was distributed 1 :2 in mitochondria and glyoxysomes, respectively. Longer incubation reduced the ratio to 1 :3 while both organelles acquired more radioactive intermediates. Labeled acetyl-CoA and intermediate of p-oxidation were found in both organelle-containing fractions. The supernatant fraction contained radioactive diglycerides, monoglycerides, palmitate, sterol esters, and phospholipids, indicating lipase activity and direct utilization of fatty acid for the synthesis of sterol esters and polar lipids.Germinating fatty seeds hydrolyze reserve triglycerides to fatty acids and glycerol by lipases associated with fat bodies or spherosomes (4,13,15,16,23). Where and how the fatty acids are subsequently utilized have never been traced even though enzymes of the fl-oxidation sequence were found to be mainly localized in glyoxysomes of castor bean endosperm (7,11) similar to what was observed in ponderosa pine seeds (6). Ultrastructurally, changes were found identical to those which occurred in germinating ponderosa pine (6). These changes included an increase in the number of mitochondria and glyoxysomes, a decrease in the size and number of fat bodies, and a fragmentation and solubilization of protein bodies. The chemical and structural changes indicated an active lipolysis and gluconeogenesis in this species, but the seeds are three to four times larger than ponderosa pine seeds and less time consuming in obtaining experimental material. Therefore, three lots of Jeffrey pine seeds with high germinability were chosen for the intended study.Seeds were soaked in water for 2 hr, stratified or chilled at 4 C for 3 weeks to break their dormancy, and then germinated for 10 days at a daily cycle of 30 C for 8 hr in light and 20 C for 16 hr in the dark. Megagametophytes were dissected from the germinated seedlings, washed with 1 % sodiuLm hypochlorite, and chilled at 4 C. Subsequent procedures were conducted at 0 to 4 C unless specified. Glassware was dry-sterilized at 150 C for 30 min. All solutions were filtered through a nitrocellulose membrane (0.45 ,u in pore size, Brinkmann Instruments, Inc.). Polyethylene tubes and utensils were disinfecte...

Comparative Studies of Glyoxysomes from Various Fatty Seedlings

“…In this experiment, the gradient was linear and the supernatant fraction obtained after centrifugation of the crude extract at 270g for 10 min was put directly onto the gradient. As a result, a cleaner separation of organelles was obtained than that described previously where resuspended particulate fraction was layered on a stepped gradient (5,6). Yet, there was still some contamination of proplastids in the glyoxysomal region.…”

“…Broken glyoxysomes appear at a lighter density in sucrose gradient centrifugation (14, present work) and may contaminate the mitochondrial fraction, especially in a stepped gradient. The possibility that incubation of the pine extract at 30 C might break a substantial amount of the fragile glyoxysomes is supported by the comparatively low recovery of isocitrate lyase in the glyoxysomal fraction in the gradient after incubation (6). These facts should be taken into consideration of the conclusion that in pine megagametophyte, mitochondria can oxidize fatty acids at rate one-half of that in the glyoxysomes (6).…”

“…From these data (6), it was suggested that in pine megagametophyte, the mitochondria and the glyoxysomes oxidize the fatty acids in vivo one-third and two-thirds, respectively. In that work, the mitochondria were not checked for the presence of the necessary enzymes for the oxidation of the fatty acids and only the easily solubilized isocitrate lyase, but not an enzyme that is associated with the glyoxysomal membrane, was checked for possible contamination of the mitochondrial band by broken glyoxysomes.…”

“…In a previous report (6), the crude extract of pine megagametophyte was incubated with radioactive triglycerides and subsequent separation of the particulate fraction by stepped sucrose gradient centrifugation revealed that the mitochondrial band and the glyoxysomal band contained one-third and twothirds, respectively, of the radioactive breakdown products of triglycerides in the gradient. From these data (6), it was suggested that in pine megagametophyte, the mitochondria and the glyoxysomes oxidize the fatty acids in vivo one-third and two-thirds, respectively.…”

“…'This work was begun at the University of California, Santa Cruz, with support from Grant GB 35376 x 1 from the National Sciences Foundation to H. Beevers. lt was shown that in castor bean endosperm, the glyoxysomes house most if not all of the enzymes of the /3-oxidation pathway and that only the glyoxysomes, and not the mitochondria, can degrade fatty acids (8). It was suggested recently that in the fat-containing megagametophyte of germinating pine seeds, mitochondria and glyoxysomes oxidize the fatty acids inl vivo one-third and two-thirds respectively (6 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).…”