Purification and properties of chlorophyllase from greened rye seedlings.
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.
D001429:Bacteriochlorophylls / Q000302:isolation & purification; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002523:Edible Grain / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D010447:Peptide Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis; D012247:Rhodospirillum rubrum / Q000032:analysis; D012434:Secale / Q000201:enzymology*
D001429:Bacteriochlorophylls; D010940:Plant Proteins; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase; D010447:Peptide Hydrolases