The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDWPAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatik characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His, tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 pM.Keywords: cathepsin L; propeptide; expression ; Paramecium tetraurelia.Proteases serve many distinct functions in the cellular metabolism. Intracellularly, most obvious roles are in degradation of ingested food and turnover of cellular proteins. This usually occurs in lysosomes or digestive vacuoles. Other functions exist in the developmental and metabolic control of specific proteins through activation or inactivation by limited proteolysis [ 11. Extracellularly, secreted proteases are used to access solid proteinacous food or to facilitate tissue invasion via collagenolytic and elastinolytic activities as reported for some pathogenic bacteria, parasitic protozoa, and ras-transformed tumor cells [2-41. In the unicellular ciliate Paramecium tetraurelia the formation and processing of digestive vacuoles and lysosomes has been investigated predominantly by electron microscopy [5]. In these studies, 16 hydrolases of presumed lysosomal localization have been superficially identified more than a decade ago. Among those were the proteolytic cathepsins B, C, and D [ 5 ] . Nothing is known about extracellular proteases. During attempts to identify and study protein phosphatases from P. tetraurelia, we noticed the presence of a highly active protease in cell homogenates that hydrolyzed the '*P-labelled phosphorylase a which was used as a substrate for the protein phosphatases, to smaller ['*P]phosphopeptides. These labelled peptides were not acid-precipitable and thus quenched the phosphatase assay. Because hitherto no detailed enzymatic study of any protease from this ciliate had been reported, we decided to examine the nature of this protease activity. After separation from protein phosphatase two prot...

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Objectives-To investigate if sequence alterations of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a contributory factor to the pathogenesis of motor system degeneration. EAAT2 serves as a candidate gene as its reduced expression was reported in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin. Methods-Sequence alterations were screened for in the coding region of EAAT2 in 55 patients with ALS and one family with autosomal dominant HSP (AD-HSP). Results-In ALS, no sequence alteration in the EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation of the EAAT2 gene was detected in two of seven aVected patients with AD-HSP in the same kindred. The absence of cosegregation with the familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with ALS or in 50 non-neurological controls. Conclusion-The allelic variant of the EAAT2 gene in conjunction with the primary gene defect may be a modifying factor for the highly variable AD-HSP phenotype. (J Neurol Neurosurg Psychiatry 1998;65:594-596)

Approximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild-type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin-regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild-type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurinŜ OD1 interaction in the etiology of fALS. ß