A study of hydrolases and their release from trichomonads

Abstract

The acid hydrolase N-acetyl-Beta-D-glucosaminadase (NAGase) la a high activity lysosomal enzyme in trichomonads. Tritririchomonas foetus (strains F2, KV1 and CA84-2), Trichomonas vaginalis and Trichomonas augusta all contained and released NAGase. Analysis of NAGase using electrophoretic techniques demonstrated in all the Tritririchomonas foetus strains the same four major forms, named NAGase 1-4, whose mobility suggested apparent M,s of 54 000, 89 000, 100 000 and 158 000 respectively. Trichomonas vaginalis and Trichomonas augusta, however, each contained a single form of this enzyme of apparant M,s 138 000 and 107 000 respectively. In all cases the intracellular and extracellular forms of NAGase appeared to be identical. Growth of Tritririchomonas foetus, F2, in the presence of the glycosylation inhibitor, tunicamycin, resulted in an extra NAGase band with higher mobility assumed to be a non-glycosylated but active enzyme form. NAGase 3 mobility was simultaneously diminished. Extra NAGase forms wars also observed after incubating this parasite In serum free media containing the two proteinase inhibitors, leupeptin and Z-Phe-Phe-CHN2, indicating that proteolytic processing may be involved in the production of NAGase forms. NAGase 1, the enzyme with an apparent M, of 54 000, was purified from Tritririchomonas foetus, F2. Preparative Isoelectric focusing was used to separate the four NAGase forms and as the first step in its purification. Total denuturation of NAGase 1 before electrophoresis produced a decrease in mobility from an apparent M, of 54 000 to a doublet of apparent M,s of 68 000 and 70 000. This transformation was found to be complete within 2 min at 50°C but was dependent at this temperature on the presence of both SDS and Beta-mercaptoethanol. This transformation also occured without sample buffer at 100°C and may relate to the breaking of disulphide bridges. Treatment of NAGase 1 with endoglycosidase reduced its apparent M, by 2000, indicating the enzyme to be an N-linked glycoprotein. Trichomonas vaginalis and Tritririchomonas foetus were also found to contain and release acid phosphatase. SDS-PAQE analysis of this hydrolase found one form with an apparent M, of 126 000 In Tritririchomonas foetus, strains F2 and KV1, Trichomonas augusta also had one form of apparent M, 135 000 while Trichomonas vaginalis contained and released two forms with apparent M,s of 155 000 and 160 000. Despite several immunisation attempts purified NAGase 1 was found to be non-antigenic in rabbits and so It was not possible to analyse the release processes In the detail originally proposed. However, although the results have not allowed details of the release mechanisms and physiology, they have confirmed release and provided more Information on the properties of the hydrolases, especially NAGase.