• Parasites, Hosts and Diseases
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• v.32 no.4
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• pp.243-248
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• 1994

Plasma membrane proteins of a Korean isolate of Trichomonus vofinalis HY-1 were fractionated for antigen analysis. Homogenates of T. vaginalis were fractionated by the differential centrifugation using sucrose step-gradient method. The interface layer from the 25%/45% sucrose was collected as a plasma membrane fraction and its purity was examined by transmission electron microscopy. The antigenicity of plasma membrane fraction was analysed by enzyme-linked immunoelectrotransfer blot technique with immune rabbit serum and compared with surface antigen labelled with N. hydroxysuccinimide-biotin. The fluffy fraction of 25%/45% sucrose interface was homogeneous and membrane particles were present as extended sheet and concentric vesicles showing typical trilamellar appearance under transmission electron microscope. Seven fractions at 40, 50, 60, 110, 130, 140 and 150 kDa were identified as the antigenic membrane proteins in EITB with anti HY-1 rabbit serum. The common band at 60 kDa was detected both in antigenic fractions of plasma membrane and surface protein labelled with NHS-biotin. This result indicates that this protein is considered as a major surface antigen of T. vaginalis. The role of this surface antigen at 60 kDa should be studied further.

• Animal cells and systems
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• v.6 no.3
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• pp.227-232
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• 2002

GroESLx proteins of symbiotic X-bacteria were overproduced in Escherichia coli and their structural characteristics were assayed after simple purification. The GroESx and GroELx were heat-stable at 8$0^{\circ}C$ and 5$0^{\circ}C$, respectively. After heat-treatment, GroESx was purified by DEAE Sephadex A-50 chromatography and GroELx was purified by step- and linear sucrose density gradient ultracentrifugation. Molecular masses of GroESx and GroELx were 50-80 kDa and 800 kDa, respectively, as estimated by sucrose density gradient ultracentrifugation. In chemical cross-linking analysis, subunits of GroESx were mostly cross-linked by incubation for 3 h in 0.4% glutaralde-hyde and GroESx was found to be composed of homo-heptamer subunits. Those of GroELx were cross-linked within 10 min in 0.3% glutaraldehyde and GroELx was in two stacks of homo-heptamer subunits. On the other hand, GroESx and GroELx proteins in a solution could not be cross-linked even after incubation for 3 h in 0.5% glutaraldehyde. GroELx was stable at 4-37$^{\circ}C$. In the presence of both GroESx and ATP, GroELx$_{14}$ was stable at 37$^{\circ}C$ but not at 4$^{\circ}C$ or 24$^{\circ}C$. Thus, we confirmed the oligomeric properties of GroESx$_{7}$ and GroELx$_{14}$ and their stability to heat and in the interaction with GroESx.x.

• The Korean Journal of Food And Nutrition
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• v.8 no.4
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• pp.307-317
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• 1995

The purity of mtDNAs isolated from Archicitrus and Metacitrus leaves was higher in percoll density gradient centrifugation than differential and sucrose density gradient centrifugation. The most clear mtDNAs were obtained from mitochondria included in the Interface band of between 21% and 45% under isomotic, low viscosity conditions in the three step discontinuous percoll density gradient centrifugation. DNase treatment to the crude mitochondrlal suspension still more increased purity of mtDNA by the effective removal of the nuclear and chloroplast DNA and mtDNAs were appeared as a single band at middle position of tube by EtBr /cscl density gradient centrifugation. Agarose gel electrophoresis of mtDNAs resolved a single, broad band containing high molecular weight DNAs In all preparation. Yield of mtDNAs was about 110 and 2 ug Per 2009 in mature and immature leaves respectively. The mtDNA fragment patterns showed by EcoR I treatment were indistinguishable with respect to nom bet and position of bands in Archicitrus and Metacitrus. In the pattern of Hind E restriction, the Metacitrus displayed the unique band between 5.0 and 4.0kb, in addition to four fragments about 5.0, 2.4, 2.15, and 2.0kb, respectively, different from Archicitrus. Also the pattern of total mtDNAs fragment by the treatment of Pst I showed that the distinguishable fragment pat tern was not appeared in Archicitrus(C. iyo Tanaka), but about 6.0, 5.5, 5.0 and 2.Bkb fragments were appeared only in Metacitrus(C. junos Sieb). Therefore it was indicated that two species in intra-subgenus were identical each other, whereas considerable difference was revealed for inter-subgenus.

• BMB Reports
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• v.28 no.2
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• pp.129-137
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• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.