The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolases

Abstract

A cell fractionation scheme was developed for studying the distribution of certain hydrolases, especially phosphohydrolases, in a Crithidia sp. (Trypanosomatidae). Between 26-56% of the total cellular hydrolase activities were soluble (probably of flagellar pocket origin), and 5-40% was sedimentable. A particulate fraction obtained after isopycnic density gradient centrifugation (P = 1.187-1.241), designated fraction FA/FB, was enriched in various acid hydrolases (relative specific activities 1.33-6.24) and displayed latent phosphohydrolase activities. The density gradient distributions of these hydrolytic enzymes were compared with each other and with malate dehydrogenase (mitochondrial marker). From the results obtained it appears that the sedimentable acid hydrolases of Crithidia are associated with a heterogeneous population of subcellular particles. Cytochemical observations on the FA/FB fraction supported this finding and revealed the association of acid phosphatase reaction product with subcellular elements resembling multivesicular bodies. A detailed study of the hydrolytic activity toward both ortho- and pyrophosphate substrates by the soluble, as compared to the FA/FB fractions, revealed the existence of distinct phosphohydrolase activities. These differences in properties may be due to the existence of phosphohydrolase isoenzymes and they in turn relate to a differentiation in the cellular digestive functions of Crithidia.