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Korean J Parasitol > Volume 34(1):1996 > Article

Original Article
Korean J Parasitol. 1996 Mar;34(1):49-57. English.
Published online Mar 20, 1996.  http://dx.doi.org/10.3347/kjp.1996.34.1.49
Copyright © 1996 by The Korean Society for Parasitology
Characterization of the partially purified proteinase from Trichomonas vaginalis
Duk-Young Min,*Jae-Sook Ryu and Keun-Hee Hyun
Department of Parasitology, College of Medicine, Hanyang University, Seoul 133-791, Korea.
Received December 21, 1995; Accepted January 31, 1996.

Abstract

Characterization of a purified proteinase from Trichomonas vaginalis was carried out using bacitracin-sepharose affinity chromatography. Trichomonas vaginalis KT-9 isolate was used as a source of enzyme study. Proteinase activity was determined using Bz-Pro-Phe-Arg-Nan as the substrate. Optimum pH for the purified proteinase activity was 7.0 and 6.0, 9.0 with DTT. Optimum temperature was 37℃ and isoelectric point was 7.2. Activity of this proteinase was inhibited by E-64, antipain, leupeptin, Hg2+ and Zn2+ and activated by DTT and cysteine. Activity of the purified proteinase was visualized by gelatin SDS-PAGE. The gelatinolytic activity of the purified proteinase was inhibited by E-64, antipain, leupeptin, and IAA, but not by PMSF and EDTA. On SDS-PAGE, the molecular weight of the purified proteinase was 60,000 daltons. Sera of rabbits infected with T. vaginalis reacted specifically in immunoblots with this proteinase. These results indicate that 60 kDa of purified proteinase was cysteine proteinase with antigenicity.

Figures


Fig. 1
Bacitracin-Sepharose affinity chromatography of proteinases from T. vaginalis cell lysates supernatants. Proteinase activity was determined using Bz-Pro-Phe-Arg-Nan. Activity peaks were pooled, concentrated, desalted and assayed. Absorbance at 280 nm (●), proteinase activity (–■–).


Fig. 2
SDS-PAGE profile of the purified proteinase from T. vaginalis cell lysate supernatants. Lane A,T. vaginalis cell lysate supernatants; Lane B, purified proteinase; M, molecular weight marker.


Fig. 3
Effect of pH on purified proteinases activity of T. vaginalis cell lysate supernatants. Assays were carried out without DTT (●) or with 10 mM DTT (–■–).


Fig. 4
Effect of incubation temperature on purified proteinases activity of T. vaginalis cell lysate supernatants.


Fig. 5
Gelatin-containing SDS-PAGE analysis of the purified proteinases from T. vaginalis cell lysates supernatants. Each sample was loaded into 7.5% acrylamide gel. After electrophoresis and Triton X-100 treatment, the gel was incubated at pH 6.0 phosphate buffer with 5 mM DTT. A: Lane 1. purified proteinase 10 µg; Lane 2. T. vaginalis cell lysates supernatant 10 µg. B: Lane 1. purified proteinase 5 µg; Lane 2. purified proteinase 5 µg + E-64; Lane 3. purified proteinase 5 µg + IAA; Lane 4. purified proteinase 5 µg + leupeptin; Lane 5. purified proteinase 5 µg + antipain; Lane 6. purified proteinase 5 µg + EDTA; Lane 7. purified proteinase 5 µg + PMSF; Arrows indicate molecular weight of marker.


Fig. 6
Isoelectric focusing analysis (pH range 3-10) of the purified proteinase. Isoelectric point markers included the following proteins: amyloglucosidase (3.6), trypsin inhibitor (4.6), β-lactoglobulin A (5.1), carbonic anhydrase II (5.9), carbonic anhydrase I (6.6), myoglobulin (6.8, 7.2), lentil lectin (8.2, 8.6, 8.8), trypsinogen (9.3). Lane A, Markers; Lane B, purified proteinase.


Fig. 7
Immunoblot analysis of purified proteinase under denaturing conditions.

Lane 1,T. vaginalis cell lysate supernatant; Lane 2, purified proteinase (arrow). (IS, immunized rabbit serum; NS, normal rabbit serum)


Tables


Table 1
Proteinase purification from T. vaginalis lysate by bacitracin-sepharose affinity chromatography


Table 2
Activity of purified proteinase towards various synthetic substratesa)


Table 3
Effects of inhibitors on purified proteinase of T. vaginalis


Table 4
Effects of metal ions on purified proteinase of T. vaginalis

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