Proteases from Trypanosoma brucei brucei. Purification, characterisation and interactions with host regulatory molecules.

African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and alpha2-macroglobulin effectively inhibited trypanopain-Tb, with the Ki values for cystatin C and low-molecular-mass kininogen (approximately 10(-11) M) predicting, that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or (a2-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats displayed no trypanopain-Tb-like activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.

An innovative preceptor program for intravenous home care nursing.

The demands of intravenous home care nursing require a comprehensive orientation of newly hired staff. This article describes a model preceptor development program to promote collaboration in the orientation experience. Nurses are selected by their managers to attend a 3-day training program. An evaluation questionnaire is given to participants at the end of the program. Results indicate that at the conclusion of the program, the attendees feel prepared to assess learning needs, to plan instructional activities, and to evaluate learning outcomes of their future preceptees. This program can easily be adapted for use by other home care agencies providing IV nursing.

The preparation of an enzyme associated with aflatoxin biosynthesis by affinity chromatography.

An affinity matrix for the purification of norsolorinic acid dehydrogenase, an enzyme involved in aflatoxin biosynthesis, was prepared by coupling norsolorinic acid to an agarose gel. This matrix was found to be ineffective in isolating active enzyme, and was therefore modified by methylation, using diazomethane. The methylated matrix produced a one-step purification of the enzyme from a crude homogenate, resulting in a 138-fold purification. The active isolate was found to contain one major and two minor bands upon nondenaturing electrophoresis, and all the norsolorinic acid dehydrogenase activity was associated with the major band. It was concluded that the matrix exhibited true affinity for the enzyme, and that affinity chromatography was a valuable approach to isolating other secondary metabolic enzymes involved in the biosynthesis of the aflatoxins.

Fractionation of the cellulolytic enzymes produced by a species of Monilia; purification and properties of an extracellular beta-D-glucosidase.

Extracellular cellulolytic enzymes produced by a species of Monilia could be fractionated by chromatography on SP-Sephadex, Con A-Sepharose, and cellobiose-Sepharose. These methods did not separate the beta-D-glucosidases (beta-D-glucoside glucohydrolases, EC 3.2.1.21) from the cellulases and xylanases within a single purification step. Fractionation by isoelectric focusing on a flat-bed granulated gel gave all of the beta-D-glucosidase activity in a single zone isoelectric at pH 8-9. The beta-D-glucosidase could be further purified to homogeneity by column isoelectric focusing at pH 8.0-10.5, and gel filtration on Biogel P-100. The purified beta-D-glucosidase showed optimal activity at pH 4-5 and 50 degrees, was isoelectric at pH 8.87, and had a molecular weight of 46,600. SDS-Polyacrylamide-gel electrophoresis demonstrated that the beta-D-glucosidase was not dissociated into subunits and, hence, consisted of a single polypeptide chain. The enzyme is considered a glycoprotein, as it binds to Con A-Sepharose. The beta-D-glucosidase hydrolyzed (1----2)-, (1----4)-, and (1----6)-beta-D-glucosidic linkages but not cellulose. Nitrophenyl beta-D-glucopyranosides and beta-D-xylopyranosides were also degraded. The beta-D-glucosidase was competitively inhibited by D-glucose (Ki 0.67 mM).