ABSTRACT
The soluble fraction of spinach chloroplast was used for purification and characterization of an ATP-dependent protease. Purification included Q Sepharose Fast Flow, hydroxylapatite and FPLC Superose 6 column chromatography. The isolated enzyme requires ATP and Mg2+ for stimulation and represents a ubiquitin independent serine protease, containing essential sulphydryl group(s). By using fluorogenic peptides a similarity of chloroplast protease to Escherichia coli Ti protease was observed. The chloroplast protease is immunochemically cross-reactive with the bacterial protease Ti.
Subject(s)
Chloroplasts/enzymology , Heat-Shock Proteins/isolation & purification , Heat-Shock Proteins/metabolism , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , Spinacia oleracea/enzymology , ATP-Dependent Proteases , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Chromatography , Chromatography, Gel , Chromatography, Ion Exchange , Cross Reactions , Durapatite , Escherichia coli/enzymology , Kinetics , Magnesium/metabolism , Molecular Sequence Data , Oligopeptides/metabolism , Substrate SpecificityABSTRACT
Homogenous ATP-dependent protease has been isolated for the first time from mitochondria of yeast Saccharomyces cerevisiae. The enzyme molecule consists of six 120 kDa subunits. It is a serine protease with an absolute ATP requirement for its activity. Basic enzymatic characteristics of the yeast protease are similar to those of the corresponding rat mitochondrial enzyme and of the E. coli protease La. The yeast enzyme immunochemically cross-reacts with the bacterial protease La.
