Purification and Properties of Chlorophyllase from Greened Rye Seedlings1

Abstract

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.

Properties of Chlorophyllase from Capsicum annuum L. Fruits

“…1) were used to calcu late kinetic param eters, K m and Vmax. The ob tained values were similar to those found by other authors using other plants and algae; for instance, K m is 10 [.im for Chi a in tea leaves (Kuroki et al, 1981), 12 |.i m in rye seedlings (Tanaka et al, 1982), and 7.0 |i m in Chlorella regularis (Nishiyama et al, 1994).…”

“…Action of the enzyme on substrates (Schoch and Ihl, 1998;Fiedor et al, 1992;Tanaka et al, 1982). The enzyme shows no depen dence on the metal ion (Vezitskii and Sherbakov, 1987), whereas Chi synthase exhibits a high depen dence on the metal chelated by the macrocycle (Helfrich and Rüdiger, 1992;Benz and Rüdiger, 1981).…”

Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

“…The concentrations of Bchlide a and Chlide a in acetone were determined as described previously (22,32; also see below). The pigments were dried and dissolved in dimethyl sulfoxide (DMSO).…”

“…The level of Bchl a, which had been collected in n-hexane, was calculated with an extinction coefficient of 83.9 mM Ϫ1 cm Ϫ1 at 771 nm (32). (32).…”

Dynamic change in photosynthetic pigments and chlorophyll degradation elicited by cereal aphid feeding

“…We have modified the procedure reported by Janave (1997) based on Tanaka et al (1982). The enzyme solution was incubated in 1.0 ml of reaction mixture containing 0.35 ml of 0.1 M potassium phosphate buffer (pH 7.0), 0.288 ml of acetone (to make 30% in final concentration), 0.01 ml of 0.1 M ascorbate, and 12 µl chlorophyll (for final concentration of 10 µM).…”