Localization and isozyme patterns of phosphatase in <i>Fibricola seoulensis</i>

, Kim, H J; , Kim, C W

doi:1993.31.4.353

Korean J Parasito > Volume 31(4):1993 > Article

Original Article


Korean J Parasitol. 1993 Dec;31(4):353-361. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1993.31.4.353
Copyright © 1993 by The Korean Society for Parasitology


Localization and isozyme patterns of phosphatase in Fibricola seoulensis
H J Kim and C W Kim^*
Department of Biology, Gyeongsang National University, Chinju 660-701, Korea.

Received August 17, 1993; Accepted September 20, 1993.
Abstract
The present study was carried out to investigate the localization and isozyme patterns of acid phosphatase and alkaline phosphatase in metacercariae and in adults of F. seoulensis by enzyme-histochemistry method and electrophoresis. Acid phosphatase showed a strong activity at pH 5 in the intestinal caecum of adults, but showed no reactions in the non-substrate control and in the inhibitor-treated control. Alkaline phosphatase showed a strong activity at pH 8 in the intestinal caecum and the tribocytic organ of adults, and in the intestinal caecum and in the genital anlagen of metacercariae. In non-denature PAGE, ten bands of protein fraction from the extracts of metacercariae and twenty-two bands from adults were detected. In denature PAGE, two protein bands having molecular weights of 192 kDa and 123 kDa were detected in the metacercariae, but absent from adult stage. In adults, protein fractions of 27.5 kDa, 24.5 kDa, 21.4 kDa, 18 kDa, 16 kDa and 15 kDa were detected. In non-denature PAGE, isozymes of acid phosphatase showed the most strong activity at pH 5, whereas no activity was shown at pH 2 and pH 7. One isozyme band (62 kDa) of AcPase was recognized in metacercariae and 4 isozyme bands (95 kDa, 85 kDa, 73 kDa and 62 kDa) in adults.

Figures

Fig. 1
Histochemical localization of acid phosphatase in F. seoulensis treated with substrate, Naphthol AS-BI phosphate.

1~2, Treated at pH 2: 1. (A:adult) 40 ×, 2. (M: metacercaria) 40 ×, 3~4, Treated at pH 4:3. (A) 40 ×, 4. (M) 100 ×, 5~6, Treated at pH 5:5. (A) 40 ×, 6. (M) 100 ×. 7~8, Treated at pH 7:7. (A) 40 ×, 8. (M) 100 ×. OS: oral sucker, P: pharynx, VS: ventral sucker, IC: intestinal caecum, TO: tribocytic organ, TG: tribocytic gland


	Fig. 2 Histochemical localization of acid phosphatase in F. seoulensis. 9~10, Treated without substrate: 7. (A:adult) 40 ×, 8. (M: metacercaria) 100 ×. 11~12, Treated with inhibitor (Oxalic acid): 9. (A) 40 ×, 10. (M) 100 ×.


	Fig. 3 Histochemical localization of alkaline phosphatase in F. seoulensis treated with substrate, Naphthol AS-MX phosphate 13~14, Treated at pH 6 (A:40 ×, M: 40 ×), 15~16, Treated at pH 7 (A: 40 ×, M: 100 ×), 17~18, Treated at pH 8 (A: 40 ×, M 100 ×), 19~20, Treated at pH 9 (A:40 ×, M: 40 ×), IC: intestinal caecum, GA: genital anlagen TO: tribocytic organ EP: excretory pore


	Fig. 4 Histochemical localization of alkaline phosphatase in F. seoulensis treated with substrate. 21~22, Treated at pH 7 (A: 40 ×, M: 100 ×), 23~24, Treated at pH 8 (A: 40 ×, M 100 ×), TG: tribocytic gland


	Fig. 5 Protein fractions in extracts of F. seoulensis by non-denature PAGE and denature PAGE. NA: non-denature adults, NM: non-denature metacercariae, DA: denature adult, DM: denature metacercariae, MW: molecular weight, Ma: marker protein, TD: tracking dye


	Fig. 6 Isozyme patterns of acid phosphatase and general protein patterns in extracts of F. seoulensis by non-denature PAGE. NA: non-denature adults, NM: non-denature metacercariae, MW: molecular weight, Ma: marker protein, 5: pH 5

Tables


	Table 1 The activity and localization of acid phosphatase in adults and metacercariae of F. seoulensis in various pH valule


	Table 2 The activity and localization of alkaline phosphatase in adult and metacerariae of F. seoulensis in various pH valule

References


1.	Anderson PJ, et al. Proc 4th Int Congr Neuropathol 1962;1:75.

2.	Barka T. J Histochem Cytochem 1961;9:542.

3.	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.

4.	Cho SY, et al. Chung-Ang J Med 1983;8(1):13–27.

5.	Fujino T, et al. Jpn J Parasitol 1983;32(1):1–12.

7.	Hong ST. Studies On Intestinal Trematodes In Korea: VII. Growth, Development And Recovery Of Fibricola Seoulensis From Experimentally Infected Rats And Mice. Korean J Parasitol 1982;20(2):112–121.

8.	Hong SJ, Lee SH, Seo BS, Hong ST, Chai JY. Studies On Intestinal Trematodes In Korea: IX. Recovery Rate And Development Of Fibricola Seoulensis In Experimental Animals. Korean J Parasitol 1983;21(2):224–233.

9.	Huh S, Lee SH, Seo BS. Histochemical findings of the tribocytic organ and tegument of Fibricola seoulensis. Korean J Parasitol 1990;28(3):155–160.

*10.*	Kang CG, et al. Korean Biochem J 1990;23(3):356–362.

*11.*	Kim CH, et al. Korean J Zool 1989;32(4):429–440.

*12.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*15.*	Moore MN, Halton DW. Fasciola hepatica: histochemical observations on juveniles and adults and the cytopathological changes induced in infected mouse liver. Exp Parasitol 1976;40(2):212–224.

*16.*	Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981;117(2):307–310.

*17.*	Neumann H. Substrate selectivity in the action of alkaline and acid phosphatases. J Biol Chem 1968;243(18):4671–4676.

*18.*	Ohashi O. Jpn J Parasitol 1971;20:399–405.

*19.*	Ohashi O. Jpn J Parasitol 1972;21:16–27.

*20.*	Ohman C. Parasitology 1965;55:481–502.

*21.*	Seo BS, Rim HJ, Lee CW. Studies on the parasitic helmiths of Korea: I. Trematodes of rodents. Korean J Parasitol 1964;2(1):20–26.

*22.*	Seo BS, Cho SY, Hong ST, Hong SJ, Lee SH. Studies On Parasitic Helminths Of Korea 5.Survey On Intestinal Trematodes Of House Rats. Korean J Parasitol 1981;19(2):131–136.

*23.*	Seo BS, Lee SH, Hong ST, Hong SJ, Kim CY, Lee HY. Studies On Intestinal Trematodes In Korea: V. A Human Case Infected By Fibricola Seoulensis (Trematoda: Diplostomatidae). Korean J Parasitol 1982;20(2):93–99.

*25.*	Thorpe E. Comparative enzyme histochemistry of immature and mature stages of Fasciola hepatica. Exp Parasitol 1968;22(2):150–159.