Summiilary. Enzymes involved in malic acid production via a pathway with 2 carboxylation reactions and in malic acid conversion via total oxidation have been demonstrated in mitochondria of Bryophyllurn tutbiflorumn Harv. Activation of the mitochondria by Tween 40 was necessary to reveal part of the enzyme activities. The temperature behaavior of the enzymes has been investigated, revealing optimal activity of acid-producing enzymes at 350. Even at 530 the optimum for acid-converting enzymes was not yet reached. From the simultaneous action of acid-producing and acid-converting enzyme systems the overall result at different temperatures was estajblished. Up to 150 the net result was a malic acid prodduction. Mloderate temperattures brought about a decrease in this accumulation, which was partly accompanied by a shift to isocitrate production, while at higher temperatures total oxidation of the acids exceeded the production. (21,22,24) and P-enol(pyrtuvate carboxylase has been stuggested as catalyzing the CO., fixatioin. The oxaloacetic acid produiced can be converted to malic acid by malate dehydrogenase, which was also demonstrable in the leaf extracts. A second carboxylation reaction in the acid-synthesizing pathway was proposed on the basis of the constant labeling of malic acid, produced by leaves in 14CO9 (6). The ratio of radioactivity fotund in carbon-4 to that in carbon-I was constantly 2:1 tunder a great variety of conditions, while the labeling of carbon-2 and carbon-3 could never be detected. This was explained by assuming an acid synthesis via carboxylation of ribulose 1,5-di,P to glvcerate 3-P, followed by conversion of glycerate 3-P to P-enolpyruvate, a second carboxylation (resulting in oxaloaceti;c acid) and reduction to malic acid. This pathway implies participation of the oxidative pentose phosphate pathway, so that the following reaction sequence for acid production can be proposed: starch glucose 6-P --ribulose 1,5-diP glycer.ate 3-P P-enolpyruvate -* malate.For acid conversion a role is attributed to the malic enzyme (22,23), which converts malate to pyruvate, followed by total oxidation of this product by the tricarboxylic acid cycle (7,14).That the acid accutmulation is a result of the dominance o-f acid-synthesizing enzymes at low temperatures, while the acid decrease is a consequence of the fact that conversion exceecds production at higher temperatures was demonstrated by Bennet-Clark (3)

Malic enzyme from leaves of the Crassulacean species Bryophyllum tubiflorum was purified more than 1200-fold by a rather simple procedure involving repeated fractionation with ammonium sulphate and gel-filtration. A specific activity (international standards) of 68 was obtained. The molecular weight of the enzyme, measured by the gel-filtration method, was 237000.Thiol activation is required for catalytic activity. The activity is completely dependent on divalent cations; greatest activity was obtained with Mn2+, an equimolar amount of Mg2+ was 86O/, as effective. Maximum activity was obtained at about pH 7.2. At 30 "C and pH 7.2 the K , for Mn2+ was 0.46 mM, that for NADP was 14 pM, and that for malate amounted to 0.37 mM. The K , for malate was temperature-dependent : it remained constant in the temperature range from 17 tco 39 "C, but below 17 "C and beyond 39 "C the affinity for malate decreased and a Kmvalue of 0.94 mM was recorded. The enzyme was completely NADP-specific and was stable at temperatures up to 55 "C; maximum activity was recorded at 50 "C.No effects of monovalent cations such as Naf, K+, Li+, or NH,+ could be recorded. The enzyme was inhibited by coenzyme A and thiamine pyrophosphate, but not by acetyl-coenzyme A. The effects of temperature as well as the inhibition by coenzyme A and thiamine pyrophosphate fit the physiology of Crassulacean acid-metabolism.Crassulacean leaves show a diurnal fluctuation in acid content, i.e. the amount of (predominantly) malic acid rises at night and decreases in daytime. This phenomenon has been called Crassulacean acidmetabolism [ 11. To phosphoenolpyruvate carboxylase a role was attributed in the acidification reactions, while malic enzyme was active in the deacidification reactions [ 2 ] . This kind of malate metabo h m in Crassulaceae may be a variant of the C,-dicarboxylic-acid pathway of GO,-fixation in (efficient) photosynthesis [3].

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