Graduate Thesis Or Dissertation
 

Effects of washing and aging on the metabolism of red beet root slices

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  • The rate of the C¹⁴0₂ release from glucose, specifically labeled with C¹⁴ at 1, 2, 3+4, and positions, was used to study the relative participation of pathways of glucose catabolism in red beet slices. Evidence was obtained which indicated that in mature red beets glucose was catabolized mainly via glycolysis-tricarboxylic acid cycle (EMP-TCAC). Approximately 15-22 percent of the total glucose catabolized in fresh slices appeared to be mediated via the pentose phosphate pathway (PPP). Older beet roots were found to have more PPP activity than young beet root slices. When red beet slices were washed in either demineralized distilled water or 0.01M KH₂PO₄ solution by shaking in a gyratory shaker, the relative participation of the PPP increased with the duration of washing. Increase in the relative participation of the PPP was more in the slices washed in KH₂PO₄ solution than in water alone. However, changes in the relative participation of pathways were less marked with red beet slices than with other storage tissues as reported by other workers, The TCAC was active in both fresh and washed beet slices. C¹⁴ release from specifically labeled glucose by fresh beet slices was very strongly inhibited by 0.05M malonate, whereas much less inhibition was found inwashed slices. This strongly suggests TCAC participation in the fresh slices. Enzyme assays with crude extracts prepared from red beet slices indicated that the following enzyme activity changes occurred during a 24-hour washing period. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase (TPN) slightly increased in activity; TPN reduction and malic dehydrogenase (TPN) activities slightly decreased; TPNH oxidizing power increased significantly; 6-phosphogluconate dehydrogenase activity increased 3-5 fold. The increase in TPNH oxidizing power may play a role in increasing the relative participation of PPP, but the most marked increase was found in 6-phosphogluconate dehydrogenase activity and the rate of gluconate utilization. Washing periods longer than two days caused a marked decrease in glucose-6-phosphate dehydrogenase and triose phosphate dehydrogenase activities. Experiments with glucose specifically labeled with H³ indicated that the TPNH produced by the PPP was utilized for biosynthetic purposes rather than as a respiratory substrate. Metabolic changes of slices aged under moist conditions in petri dishes were also examined. The rate of oxygen uptake increased 3-5 times in 20-24 hours. O₂ uptake by fresh slices was inhibited 60-70 percent by 8 x 10⁻⁵M HCN. The sensitivity of respiration to HCN decreased rather rapidly during aging and resulted in a cyanide stimulation (about 30 percent) of respiration in about 10-12 hours. After about 36 hours of aging, cyanide sensitivity began to reappear. The rates of C¹⁴ release from specifically labeled glucose by fresh and aged red beet slices showed that both the PPP and the EMPTCAC were stimulated by aging. Preferential increase in PPP participation was indicated to be very small, if any. Release of C¹⁴ O₂ from specifically labeled acetate, succinate, aspartate and glutonate showed TCAC activity in both fresh and aged slices. Glucose-6-phosphate dehydrogenase activity doubled and 6-phosphogluconate dehydrogenase activity increased by about 300-400 percent as a result of aging for 36 hours, The rate of gluconate utilization increased 10-20 times by aging over that of fresh slices. The rate of uptake and utilization of organic acids, amino acids and glucose increased several fold due to 24-hours of aging. Biosynthetic activities such as protein synthesis of these tissues were also indicated to be increased during aging. From these studies, it does not seem justifiable to explain age induced respiration as being due to a release of inhibition on TCAO activity or as preferential increase in PPP activity, but it seems to be a stimulation of the activity of the total metabolic machinery probably caused by removal of a metabolic block which is still not understood.
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