Characterization of three rabbit liver lysosomal proteinases with fructose 1,6-bisphosphatase converting enzyme activity

Abstract

Four distinct enzymes that catalyze the limited proteolysis of rabbit liver fructose 1,6-bisphosphatase (Fru-P 2ase, EC 3.1.3.11) have been purified from rabbit liver lysosomes by gel filtration and ion-exchange chromatography. They convert “neutral” Fry-P 2ase to a form with alkaline pH optimum and are referred to as “converting enzymes”. On the basis of size, these converting enzymes have been characterized as CE-I ( M r ≊ 150,000 ), CE-II ( M r ≊ 70,000 ), and CE-III ( M r ≊ 30,000 ). The activity in CE-III has been identified as a mixture of cathepsins B and L. Only this fraction was inhibited by leupeptin. None of the activities was inhibited by pepstatin and all were activated by cysteine. Incubation of Fru-P 2ase with each of the converting enzyme fractions results in a similar increase in its catalytic activity measured at alkaline pH and a decrease in its sensitivity to AMP, measured at neutral pH. A significant fraction of the total Converting Enzyme II activity (30–40%) remains associated with the membrane fraction after the lysosomes are disrupted by freezing and thawing. All of the soluble converting enzymes showed maximum activity at pH 5.0 and became unstable above pH 6.5. In contrast, the membrane-bound form of Converting Enzyme II showed maximum activity at pH 6.5 and increased stability at neutral pH.