Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena

Abstract

Pab. II,SUMMARY The purification and characterization of a novel phosphodiesterase (PDE) is presented. The activity was detected in the extracellular medium of Tetrahymena thermophila cultures, by the release of p-nitrophenol from p-nitrophenylphosphocholine (PNPPC) with an acidic pH optimum In cell homogenates, it is sedimentable, shows a latency similar to that of acid phosphatase and is co-secreted with this enzyme, indicating that it is a lysosomal hydrolase. PNPPC-PDE was purified to homogeneity from the extracellular medium, yielding a single band of 58 kD on SDS polyacrylamide gel electrophoresis. It catalyzed the release of glycerol from glycerophosphoeholine (GPC) and GPC competitively inhibits degradation of PNPPC. We present further evidence indicating that the natural substrate for PNPPC-PDE is GPC. Thus, Tetrahymena becomes the first eukaryote in which a lysosomal GPC-PDE is observed. This finding provides a new pathway for the complete breakdown of phosphatidylcholine in a lysosomal medium.

Purification and Characterization of Lysophospholipase C from Pig Brain

“…In the following step, the fractions (No. [18][19][20][21][22][23][24][25][26] from Concanavalin A chromatography was pooled and subjected to DEAE Sephacel rechromatography (NaCl gradient, 0-0.1 M), which gave rise to an enzyme peak (No. 42-53) with a specific activity of 1,996 lmole /h/milligram protein (Fig.…”

“…Fractions (No. [18][19][20][21][22][23][24][25][26], expressing high lyso-PLC activity, were pooled, and dialyzed overnight against 20 mM Tris buffer, pH 8.0 containing 2 lM ZnCl 2 twice.…”

Analysis of Expressed Sequence Tags (ESTs) in the Ciliated Protozoan Tetrahymena thermophila

“…EST 141 matches acid phosphatase (Table 2). In Tetrahymena an acid phosphatase has been identified and genetically mapped (Florin‐Christensen and Florin‐Christensen 1999) but the protein has not yet been purified nor the gene cloned. Ly‐sosomal acid phosphatase is in all likelihood associated with phagocytosis, endocytosis and/or autophagy.…”

Μελέτη των φωσφοϊνοσιτιδίων του μονοκύτταρου ευκαρυωτικού οργανισμού tetrahymena

“…Σε συνθήκες ανάπτυξης η έκκριση συνοδεύεται από βιοσύνθεση ενζύμων με αποτέλεσμα η συνολική δραστικότητα να αυξάνεται, ενώ δεν συμβαίνει το ίδιο σε συνθήκες ασιτίας [Muller, 1972, Banno et al, 1987]. Η έκκριση τέτοιων ενζύμων περιλαμβάνει την έκκριση όξινων φωσφατασών, RNΑσών, πρωτεασών, γλυκοζιδασών, λιπασών, φωσφολιπασών και φωσφοδιεστερασών [Muller, 1972, Florin-Christensen et al, 1986α, 1986β, Banno et al, 1987, Florin-Christensen and Florin-Christensen, 1999.…”