Changes in the Ascorbate System during Seed Development of Vicia faba L

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H₂O₂ produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.     

High-performance liquid chromatographic assay of l-α-glycerophosphorylcholine using a two-step enzymic conversion

A high-performance liquid chromatographic method using an enzymic reactor for determination of l-α-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of l-α-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml l-α-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 μl of injected sample. The precision of the method in the analysis of l-α-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.     

The mechanism of phosphate permeation in purified bean mitochondria

The permeability properties and mechanism of Pi transport wereinvestigated in purified bean mitochondria.

1. Purified bean mitochondria are impermeable to small moleculesand ions. However, Pi, arsenate, acetate and formate can enterthe osmotically active space of bean mitochondria.
2. Nigericinor the association of valinomycin and FCCP cause mitochondrialswelling in isoosmotic potassium phosphate.
3. The SH-blockingreagents mersalyl, pHMB and NEM inhibit variousmitochondrialfunctions dependent on the translocation of Piand arsenateacross the membrane. These include the respirationstimulatedby ADP, Ca²⁺+Pi, and K⁺+valinomycin +Pi; the swellingin ammoniumphosphate medium and, in the presence of nigericin,in potassiumphosphate medium; the energy-linked yalinomycin-inducedswellingand the subsequent CICCP-induced shrinking. The uncoupler-stimulatedrespiration, as well as the other processes when acetate issubstituted for Pi, are not influenced by SH reagents.
4. Mersalyland pHMB cause complete inhibition at about 20 nmoles/mgprotein,whereas, NEM is effective at about 1 µmole/mgprotein.The inhibition by mersalyl and pHMB, but not that byNEM, issigmoidal and reversed by 2-mercaptoethanol. Non-inhibitoryamounts of mersalyl protect the Pi transport from irreversibleinhibition by NEM.
5. We concluded that a carrier-mediated transportsystem for Piis present in bean mitochondria, and that someof its propertiesare similar to the Pi carrier of animal mitochondria.

(Received June 5, 1975; )     

Mitochondrial enzymatic adaptation of skeletal muscle to endurance training.

Some mitochondrial enzymatic activities (succinate dehydrogenase, NADH cytochrome reductase, cytochrome oxidase) were studied in the gastrocnemius and soleus muscle of the rat. The modifications of the enzyme activity, induced by endurance training, were found to be functions of 1) daily work load and 2) total training time. The treatment with an effective dose of vasodilating substances (papaverine, nicergoline, dipyridamole, and bamethan) showed that 1) nicergoline, bamethan, and dipyridamole were differently able to shorten the time of appearance of the increase in the enzymatic activities; 2) however, long-term treatments with these drugs did not prove able to modify the plateau level of the enzymatic activity increase, for a given amount of endurance training; 3) the pharmacodynamic effect on enzymatic activities was in no way related to the vasodilating effect of these drugs, since the effect was not observed with papaverine. The transition from a given level of endurance training to a lower one led to a proportional decrease of the mitochondrial enzymatic activities, thus pointing out the relation between amount of training and enzymatic activity. The drugs studied were unable to modify the decrease of enzymatic activity induced by lower work load.