Vie di ossidazione della frazione extramitocondriale dei coenzimi piridinici: I. - Sull'EfficienzaIn Vivodel sistema ascorbato/deidroascorbato

Abstract

Abstract OXIDATION PATHWAYS OF EXTRAMITOCHONDRIAL PIRIDINE COENZYMES. I. - ON THE « IN VIVO » EFFICIENCY OF THE ASCORBATE-DEYHDROASCORBATE SYSTEM. — An evaluation of the efficiency in vivo of the AA-DHA couple as an electron carrier system has been attempted, by measuring after short time of anaerobiosis the rate of the increase of AA and of the dicrease of DHA in etiolated pea internode segments and in potato tuber disks. The changes of reduced glutathione (GSH) contents as induced by anaerobiosis or by the addition of DHA to the incubation medium were also followed. In the pea segments anaerobiosis induced a significant increase of AA and a corresponding decrease of DHA. These changes were almost completed after 10 minutes from starting anaerobiosis. The value (extrapolated to 0 time) of the initial rate of DHA desappearance under anaerobiosis was taken as representing the rate of DHA reduction to AA « in vivo », under aerobic conditions. As this rate — in a steady state situation — corresponds to that of the inverse process of oxidation of AA to DHA, this value should give and indication on the « in vivo » efficiency of the AA-DHA system as an electron carrier in respiration. As some AA was probably reoxidized to DHA in the very short period required to kill the tissue, the value of the AA DHA turnover thus calculated is probably somewhat lower than the real one. According to the present work, the oxidative turnover of the AA-DHA system would results of 0,7 micromoles/g. fr. weght/h. for the pea internode tissues and of 0,9 micromoles/g. fr. weght−h for the potato tuber (aged disks). These values would account for 5% of total oxygen uptake, in the former, and for 3% in the latter material. The very high AA/DHA ratio usually prevailing in living cells suggests that the contents in DHA (and thus the activity of the AA oxidizing systems) is a limiting factor for the efficiency of the AA-DHA system as an electron carrier. This view is supported also by experiments in which DHA (at pH 5) was fed to pea internode segments and to potato tuber disks : as the presence of DHA into the medium induced — under anaerobiotic conditions — a rapid increase of the level of AA in both types of materials. In aerobiosis uptake and reduction of DHA to AA was evident in the potato tuber tissue, while it appeared very scarce in the pea internodes. As an interpretation of this behaviour it is suggested that, in aerobiosis, the very active and probably surface localized ascorbic acid oxidase of the pea tissue re-oxidises the AA formed from reduction of the DHA fed; an accumulation of DHA into the cells would follow, and this excess of DHA would inhibit the enzyme GSH-DHA reductase. This enzyme, in fact, appears, from « in vitro » experiments, to be strongly inhibited by DHA when the DHA/GSH ratio becomes higher than 1. On the other hand, the same hypothesis is also supported by the finding that the addition of DHA to the medium induces a significant drop in the GSH level (probably due to its oxidation to GSSG) only under those conditions in which DHA is absorbed and reduced to AA; that is, in the pea internodes, under anaerobiosis, and in the potato disks, under both anaerobiosis and aerobiosis. These results are also taken as confirming the indication from the enzymatic data that GSH is acting, in vivo as a reducing agent for DHA. The results of this investigation are thus interpred as showing that a comparatively small, but by no means negligeable fraction of respiration is mediated, in higher plant tissues such as those of the pea stem and the potato tuber, by and electron transfer system including glutathione and the ascorbate-dehydroascorbate couple. The efficiency of this system in the materials investigated appears to account for 3–5% of the total 02 uptake (minimum value). As enzyme systems transferring electrons from TPNH to ox. glutathione are widely distributed and generally very active in higher plant tissues, it is suggested that the sequence TPNH-GSH-AA/DHA - O2 is probably of considerable importance in mediating the reoxidation of extramitochondrial trophosphoridine nucleotide and thus in permitting the operation of the TPN requiring pentose phosphate pathway of respiration.