Properties of Purified Malic Enzyme in Relation to Crassulacean Acid Metabolism

Abstract

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].

Isolation and characterization of cytoplasmic NADP+‐malic enzyme from Lupinus luteus leaves

“…Malic enzyme has been obtained in a highly purified form from several other plant tissues, Brandon and Van Boeckel-Mol (1973) purified the enzyme frotn Crassulaeean plants 1200-fold, Davies and Patil (1975) from potato tubers 2 500-fold, Persanov et al, (1976a) from corn leaves 680-fold, Spettoli et al, (1980) from grape berries 700-fold, The enzytne purified 122-foid in the present study was stable. When stored at 4°C in Tris-HCl buffer, pH 8,4, with 5 mM EDTA and 1 mM 2-mercaptoethanol, the enzyme retained all of its activity for at least 18 h. For kinetic investigation samples not older than 24 h were used.…”

Properties of NADP+‐malic enzyme from pod walls of chickpea (Cicer arietinum)

“…The pH optimum of NADP*-malic enzyme from several plant sources has been reported to range from 7.2-8.0 (Johnson and Hatch 1970, Krishnamurtby and Patwardhan 1971, Andrews et al 1971, Huber and Edwards 1975, Asami et al 1979. Several workers have noted tbat tbe optimum pH for enzymic activity is dependent on the concentration of malate (Walker 1960, Johnson and Hatch 1970, Brandon and Van Boekel-Mol 1973, Dubery and Schabort 1981, Dhillon et al 1985. However, no effect on pH optimum of the enzyme from pod walls was discerned when the concentration of malate in the assay mixture was varied from 1 to 10 mM.…”

Regulation of ‘malic’ enzyme of Solanum tuberosum by metabolites

“…(iii) Effect of concentration of substrates. Previous work on 'malic' enzyme purified from higher plants has shown normal Michaelis-Menten kinetics (Harary et al, 1953;Walker, 1960;Dilley, 1966;Johnson & Hatch, 1970;Coombs et al, 1973;Brandon & van Boekel-Mol, 1973). Since allosteric properties are frequently lost during purification, the kinetic properties of the enzyme were examined at all stages of purification.…”