Immunoassays for the detection of human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and enzyme-inhibitor complexes.

Immunoassays have been developed for human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and TIMP complexed with both of the active enzymes. Selection of antibodies of defined specificity enabled measurement of both the pro and active forms of the metalloproteinase. Free TIMP was quantified by the selection of a monoclonal antibody which did not recognise TIMP when complexed with metalloproteinases. Detection of enzyme-inhibitor complexes was achieved by capturing the TIMP component of the complex and revealing the metalloenzyme using specific antibodies.

Biochemical and ultrastructural investigation of the effect of Stelazine (trifluoperazine) on Hymenolepis diminuta (Cestoda).

The effects of the phenothiazine, Stelazine, on Hymenolepis diminuta were investigated. The cestode was incubated for 10 min at 37 degrees C with 1 mM trifluoperazine, in the presence and absence of Ca2+. Assay of brush border enzymes showed that drug treatment lowered the activities of alkaline phosphatase, Ca2+-ATP'ase, 5'-nucleotidase and type 1 phosphodiesterase. This occurred in parallel with a significant reduction in tegumental protein. Under these conditions gross changes in ultrastructural appearance and cellular organization were observed. There was a lack of ordered microtriches and the distal cytoplasm was absent. Glycogen granules were scattered throughout the cytoplasm within the subtegumental layer. The connective tissue also appeared to be in some disarray. The effects of Stelazine appeared to be dependent on time and were significantly increased when Ca2+ was included in the incubation medium. Incubation with the less hydrophobic phenothiazine trifluoperazine sulphoxide had minimal effect on the integrity of the cestode. The results reported here support the premise that certain phenothiazines may be considered as potential cestocidal agents.

Effect of phenothiazines on Hymenolepis diminuta with special reference to the brush border Ca2+-dependent ATPase.

Incubation of Hymenolepis diminuta with the calmodulin antagonist trifluoperazine causes lesions in the brush border of the cestode. Exposure to a phenothiazine of lower lipophilicity, trifluoperazine sulphoxide, had little effect. Characterisation of isolated brush border revealed two forms of Ca2+-ATPase which exhibited maximum activity at pH 5.5 and 7.5. Both forms were Ca2+-dependent but only the latter was influenced by calmodulin and trifluoperazine. It is suggested that the Ca2+-ATPase present in the tapeworm brush border may be the site of trifluoperazine toxicity.

Ca2+ inhibition of brush border alkaline phosphatase activity in Hymenolepis diminuta.

The brush border membrane of Hymenolepis diminuta contains several Ca2+-dependent enzymes. Following our isolation of a Ca2+-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca2+ concentrations approaching those in the calcareous corpuscles of H. diminuta.

The effect of trifluoperazine (Stellazine) on Hymenolepis diminuta in vitro.

The influence of the phenothiazine trifluoperazine (Stellazine) on the rat tapeworm Hymenolepis diminuta was examined. The parasite was incubated in glucose-containing Krebs-Ringer media (pH 7.4) at 37 degrees C which included Ca2+ or EGTA and a range of trifluoperazine concentrations (0-2 mM). Release of soluble protein and lactate dehydrogenase activity were taken as measures of release of cytosolic components. The release of lactate dehydrogenase depended on drug concentration, maximum levels occurring at 2 mM trifluoperazine, this corresponded to 2% of the total lactate dehydrogenase present in the cestode. The effect of phenothiazines of differing lipophilicity were compared, and for trifluoperazine sulfoxide only minimal amounts of lactate dehydrogenase activity and protein were released. These values were similar to those obtained when H. diminuta was incubated in drug-free media. Our findings suggest that the integrity of the parasite is related to its calmodulin content. The potential cestocidal properties of trifluoperazine are considered.

Evidence for a Ca2+-dependent activator protein in the rat tapeworm Hymenolepis diminuta.

A low molecular weight, acidic, heat stable protein has been characterised from the rat tapeworm Hymenolepis diminuta. This protein was found to activate cyclic 3', 5'-nucleotide phosphodiesterase in a Ca2+-dependent manner. The activation process was inhibited by the phenothiazine drug trifluoperazine. The biochemical properties of this protein clearly resemble those of ovine brain calmodulin. Our investigation thus concludes that there is a calmodulin-like activator protein in this cestode.