Summary The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.

(26)(27)(28). In addition, recent work has demonstrated a further isoenzyme of malate dehydrogenase localized in plant microbodies (22,23,29). Although the isoenzymes of malate dehydrogenase in higher plant tissues have been the subject of many investigations (11,(21)(22)(23)(28)(29)(30), little is known about the distribution and function of these isoenzymes in the algal cell. When Euglena cell organelles were separated by sucrose density gradient centrifugation, malate dehydrogenase activity was recorded in several fractions (17). Besides malate dehydrogenase activity in the mitochondrial fraction, activity was present in the particulate glycolate oxidoreductase fraction, the supernatant, but not in the chloroplast fraction (17). When grown phototrophically on an appropriate light-dark regime, cultures of Euglena gracilis divide synchronously, an appropriate doubling of cell number occurring in each dark period within certain limits of cell concentration (7). By using division synchronized cultures, the development of malate dehydrogenase activity in relation to other cellular activities can be investigated. In the present paper, we report on the characterization of malate dehydrogenase isoenzymes in cell fractions and their expression over the division cycle in Euglena cultures. MATERIALS AND METHODSGrowth, Synchronization Regime, and Sampling of Culture. Division synchronized cultures of E. gracilis Klebs strain Z were obtained exactly as described previously (4). As before, samples were removed at ts, t12, t17, and ti,, this referring to the hour of sampling after commencement of the 24-hr cycle in which the culture was used. Asynchronous cultures were grown at 25 C in the photoautotrophic growth medium of Cramer and Myers (9) at a continuous light intensity of 6000 lux provided by banks of fluorescent tubes (Osram white) and gassed with air at a rate of 71/hr. Cells were harvested in middle exponential phase of growth by centrifugation at 10OOg for 4 min.CeUl

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The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1, 5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1, 5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and D-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by D-threo-chloramphenicol but not by L-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by D-threo-chloramphenicol but not by L-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1, 5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.Although exogenous glycolate can support the photoheterotrophic growth of a few algae (23,26), in most instances, the glycolate metabolized by the constituent reactions of the glycolate pathway is derived from an intermediate of the Calvin cycle. In Euglena during 14CO2 photosynthesis at certain stages of the cell cycle, glycine, serine, and glycerate are rapidly labeled; the glycerate formed is uniformly labeled even after short periods of photosynthesis, suggesting that it is derived from uniformly labeled glycolate via the reactions of the glycolate pathway (7,8). Conditions that favor the expression of Calvin cycle enzymes and resultant high photosynthetic rates are accompanied by the synthesis of glycolate pathway enzymes. The regreening of bleached Euglena cells in the light gives a concomitant increase in P-glycolate phosphatase and glycolate dehydrogenase activities (6). Similarly, the rapid increase in photosynthetic rate that results on transfer of Chla-*nydomonas mundana from an acetate-containing medium to autotrophic conditions was accompanied by a striking increase in glycolate dehydrogenase activity (21). In cells already capable of high photosynthetic rates, conditions unfavorable for glycolate biosynthesis such as high CO2 concentration result in the repression of P-glycolate phosphatase (27) and glycolate dehydrogenase (9,27).One possible explanation of these results ...

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