Purification and Properties of Chlorophyllase from Ailanthus altissima (Tree-of-Heaven)

Abstract

Chlorophyllase from Ailanthus alissima leaves has been purified 63-fold by a combination of heat treatment, ultracentrifugation, gel filtration, and chromatography on diethylaminoethyl cellulose. While the enzyme is inhibited to some degree by Triton X-100, a modification of the assay procedure of Klein and Vishniac has been shown to be far superior to the use of aqueous acetone systems. The enzyme was found to have a pH optimum on pheophytin a of 4.5. Chlorophylls a and b, pheophytins a and b, and pyropheophytin a were hydrolyzed by the enzyme while protochlorophyll a and 4-vinyl proto. chlorophyll a were not hydrolyzed but were competitive inhibitors. p-Nitrophenyl acetate was not hydrolyzed. The enzyme does not appear to contain an essential sulfhydryl group since sodium tetrathionate and p-chloromercuribenzoate did not affect its activity.Since the discovery of chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) by Willstitter and Stoll in 1913 (36), progress in our understanding of both the physiological role and of the physical, chemical, and catalytic properties of the enzyme has been quite limited. There is biological evidence consistent with both synthetic (5,7,14,15,26,30,33,34) and degradative (20,24,27,38) functions for chlorophyllase, but the data are not conclusive for either or both of these possibilities. Acquisition of definitive experimental data for this enzyme has been hindered because both the enzyme and its known substrates are insoluble in aqueous buffers. In the past decade, procedures have been developed in several laboratories (1,5,7,13,18,22,30) for solubilizing chlorophyllase from a number of plant tissues. Solubilization of substrate has been accomplished in nearly all cases by using a buffer containing from 40 to 80% acetone. Interpretation of enzyme kinetics for reactions done under these conditions is difficult. However, a more immediate problem is that substrates are only partially soluble even at high acetone concentrations (1, 3 difficult and means that quantitative assays of enzymatic activity are subject to large error. Therefore, most of the quantitative data for chlorophyllase in the literature and interpretations based upon these data are of doubtful validity.More information about the physical, chemical, and catalytic properties of chlorophyllase is needed before experiments to show the physiological function of the enzyme are likely to be successful. As a beginning to this process, we have partially purified chlorophyllase from Ailanthus altissima leaves and studied the effect of detergents, acetone concentration, pH, and ionic strength on the activity of the enzyme. Data on the effect of pH and acetone concentration on stability of the enzyme are also given. Kinetic parameters for some substrates and inhibitors of chlorophyllase have been obtained. MATERIAIS AND METHODSChlorophyllase was prepared from young leaves of wild Ailanthus altissima trees. Cellex-D (DEAE'-cellulose) from Bio-Rad Laboratories was prepared for chromatography according to th...

Properties of Chlorophyllase from Capsicum annuum L. Fruits

“…We can deduce that the active site may contain at least one sulfhydryl group partici pating in the formation of the substrate-enzyme complex, which was inhibited by p-HMB. The re sults agree with those of Kuroki et al (1981) for chlorophyllase from tea leaves, although McFeeters et al (1971) concluded the absence of Table II these functional groups in chlorophyllase from A ilanthus altissima.…”

“…Chlorophyllase (Chlase) (Chlorophyll-chlorophyllide hydrolase, EC 3.1.1.14), an intrinsic membrane-bound enzyme located in photosynthetic systems of higher plants and algae (Fernändez-Lö-pez et al, 1992;Shioi and Sasa, 1986;McFeeters et al, 1971), has been extensively studied since its discovery (W illstätter and Stoll, 1913). For long time there has been a controversy about the main function of the enzyme.…”

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley

“…7-Hydroxy-Chl a, Chl b, and Chl a were used as educts in a combined enzymatic and chemical procedure (21,32). In the first step, the phytol chain was removed by the chlorophyllase reaction (40). In the second step, the double bond in ring D of the macrocycle was reestablished by chemical dehydrogenation with 2,3-dichloro-5,6-dicyanobenzoquinone (21,32).…”

Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus

“…The reactions were stopped by adding 500 l of acetone. After centrifugation for 30 min at 12,000 ϫ g, Pchlide a and Chlide a in the supernatant were spectrophotometrically quantified by using an extinction coefficient of ⑀ 626 ϭ 30.4 mM Ϫ1 cm Ϫ1 for Pchlide a (16) and ⑀ 665 ϭ 74.9 mM Ϫ1 cm Ϫ1 for Chlide a (37). For the analysis of DPOR substrate recognition, the Pchlide reduction assay was performed in the presence of substrate analogs (see Table 1).…”