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Molecular cloning of a gene encoding acid alpha-glucosidase from Tetrahymena pyriformis.

Lysosomal acid alpha-glucosidase is essential for the degradation of glycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena pyriformis secretes acid alpha-glucosidase into its culture medium. We have earlier reported the purification and characterization of acid alpha-glucosidase from T. pyriformis. The exact molecular mechanism of secretion of this enzyme has not yet been clarified. In the present study we have isolated a full length cDNA clone encoding acid alpha-glucosidase from T. pyriformis. The isolated clone (3019 bp) contained an open reading frame encoding 923 amino acids, and has an estimated molecular mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8-kb mRNA transcript. N-terminal amino acids after the first methionine fulfilled the requirement of a signal peptide. The deduced amino acid sequence contains the amino acid sequences determined of several peptides derived from the purified enzyme, and was found to have 34% identity and 45% similarity with that of human lysosomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.

Amino Acid Sequence↗

PubMedDate not supplied

Enzymatic characterization of phospholipase D of protozoan Tetrahymena cells.

Phospholipase D (PLD), which is present in plant, bacterial, and mammalian cells, has been proposed to be involved in a number of cellular processes including transmembrane signaling and membrane deterioration. We demonstrated the existence of evolutionally related PLD activity in the unicellular eukaryotic protozoan Tetrahymena. The partial characterization of this enzyme showed that PLD in Tetrahymena cells was a neutral phospholipase, which catalyzed both transphosphatidylation and hydrolysis reac tions. The activity was markedly stimulated by phosphatidylinositol 4, 5-bisphosphate (PIP2) but was insensitive to phorbol 12-myristate 13-acetate (PMA) and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), suggesting that it is a PIP2-dependent PLD and that protein kinase C (PKC) and GTP-binding proteins are not implicated in the regulation of this enzyme. For its maximal activity Ca2+ was not required. This enzyme was also capable of hydrolyzing phosphatidylcholine (PC) but not phosphatidylethanolamine (PE), implying that PC was a preferred substrate. Subcellular fractionation showed that PLD-like activity localized mainly to the membrane fraction, especially microsomes. As an initial step to explore the functions of PLD in Tetrahymena, the PLD-like activity was determined during the different culture phases, and it was found to be significantly and transiently elevated in the early logarithmic phase, indicating its possible role in the development of Tetrahymena.

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