• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Applied Microscopy
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• v.23 no.2
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• pp.53-77
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• 1993

In this study, an attempt was made to investigate the probable organelles participating in the secretion of biligrafin. The animals (ICR male mice, 25-30gm) were divided into normal control and 6 biligrafin injected groups to which 30% biligrafin (0.006ml/gm b.w.) were injected at 10, 20, 40, 80, 160 and 320 min prior to the sampling. The mice of each group were perfused through the heart with ice-cold 2.5% glutaraldehyde buffered with 0.1M Na-cacodylate (pH. 7.4) under the Na-pentobarbital (Nembtal 0.0015mg/gm b.w.) anesthesia and liver tissues were taken from each group. Some specimens were immersed 1 hr in the same solution used in the perfusion. After an overnight rinse in 0.1M Na-cacodylate buffer containing 10% DMSO and 7.6% sucrose, $75{\mu}m$ fronzen sections were made for cytochemical study. The sections were incubated in thiamin pyrophosphatase (TPPase) and inosine diphosphatase (ID Pase) media for 70 min at $37^{\circ}C$ respectively and acid phosphatase (AcPase) medium for 40 min at $37^{\circ}C$. They were postfixed in 1 % $OsO_4$ for 1 hr. The other specimens were immersed for 8 hrs in the fixative consisting of 2.5% glutaraldehyde and 3.0% paraformaldehyde buffered with Na-cacodylate (pH. 7.4). All of the osmificated specimens were processed for electron microscopy. In both normal and biligrafin injected groups, endoplasmic reticulum (ER), vacuoles, Golgi apparatus and lysosomes were seen in the vicinity of bile canaliculus. In the biligrafin injected groups, however, the Golgi apparatus appeared to be decreased and ER and vacuoles were dilated and increased. The rough endoplasmic reticulum (RER) having a few attached ribosomes appeared to be the round saccule, especially at 20 min after biligrafin injection. Smooth endoplasmic reticulum (SER) seemed to be formed by the detachment of ribosomes at the cisternal end of RER. The cistern of SER showed saccules which probably budded off to form the vacuole. The vacuoles were devoid of visible centents. This finding seemed to be in agreement with the biochemical property of the bile constituents. The fusion between the vacuoles and bile canaliculus were frequently seen in the groups injected with biligrafin. The lysosome did not show any changes in the biligrafin injected groups. Accumulation of some material and lipid droplets were seen at the 40 and 80 min after biligrafin injection, especially at the latter. At 160 and 320 min after biligrafin injections, however, they were decreased successively while the RER stack, free ribosomes and polysomes were increased. Although the reactive products of TPPase and IDPase were observed in the ER saccules and vesicles of the normal control and biligrafin injected groups, the fusion between the bile canaliculus and saccules or vesicles could easily be seen in the latter. The AcPase activity, however, was observed in the cistern at the maturing face of Golgi apparatus and lysosomes in both normal and biligrafin groups. The results suggest that the biligrafin is excreted via the vesicles, vacuoles or sacoules probably derived from the SER without the participation of Golgi apparatus and lysosomes, and the excess amount of material is stored as inclusions during the repairing of the organelles being overactive.