• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.3
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• pp.273-279
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• 1993

Hydrolytic patterns of 11S globulin (glycinin), storage protein of soybean, by soymilk-clotting enzymes Iand IIfrom Bacillus sp. K-295G-7, which was the first soymilk-clotting enzyme to be found in a bacteria, was investigated. The clotting time of about 4~5 min is revealed by the Enzymes Iand II(0.025 units at 35$^{\circ}C$) on the acidic subunit. In electrophoresis, acidic subunit (A$_3$, M.W. 45,000) disappeared almost completely within 2 min and new products corresponding to the molecular weight of 16,000 and 20,000 were formed by the action of Enzymes I and II. Furthermore, Enzyme II produced a degradation compound having a molecular weight of about 30,000. In contrast, the hydrolytic patterns of basic subunit (M.W. 20,000) by Enzymes I and II were similar, but Enzyme II produced low molecular weight products slower than that of Enzyme I.

• Applied Biological Chemistry
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• v.31 no.4
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• pp.361-370
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• 1988

To investigate the interaction between whey and soybean protein, thermal changes of component proteins were analyzed by column chromatography and gel electrophoresis. In the Sephadex G-200 chromatography of the mixture treated at above $80^{\circ}C$, the amount of low molecular weight proteins and high molecular aggregates were increased. This implicated that dissociation of 1ls globulin into subunits and the formation of soluble aggregates between these subunits and whey proteins that contain thiol and disulfide groups. These interaction between soy proteins and ${\beta}-lactoglobulin$, ${\alpha}-lactalbumin$, and proteose-peptone 3 were confirmed by gel electrophoresis. Bovine serum albumin, Immunoglobulin-G(H), Lactoferrin, 1ls-subunits(basic and acidic), and subunit of 7s globulin were also considered to interact each other depending on the condition of the salt solutions.

• Applied Biological Chemistry
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• v.29 no.1
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• pp.73-77
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• 1986

Aqueous solutions of 2,4-D, BA or $GA_3(10^{-6},\;10^{-5},\;and\;10^{-4}M,\;respectively)$ were sprayed onto soybean (Glycine max) plants in the flowering stage, and proteins of immature (33days after flowering) and mature (77days after flowering) seeds were analyzed by electrophoresis to elucidate the effects of the growth reguators on protein synthesis or protein accumulation in the seeds. Accumulations of some proteins were altered by 2,4-D or BA at certain concentrations, but no proteins were affected by $GA_3$. The ${\alpha}\;and\;{\alpha}'$ subunits of 7S and acidic subunit of 11S disappeared in mature seeds after treatments at the flowering stage with 2,4-D or BA. The presence of ${\alpha}\;and\;{\alpha}'$ subunits of 7S and acidic subunit of 11S in immature seeds indicated that the absence of the above polypeptides in mature seeds did not result from inhibitions in syntheses of the polypeptides by the growth regulators. Disappearance of the above proteins in mature seeds seemed to be concerned with the action of specific proteolytic enzyme (s) (metalloendopeptidase?), and 2,4-D and BA might promote gene expression or activation of the enzyme.

• Preventive Nutrition and Food Science
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• v.2 no.1
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• pp.77-82
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• 1997

The comparison soy protein coagulation by heat-and enzyme-treatment are summarized. The gelation mechanism of glycinin by heating was mainly due to dissociation and aggregation of the basic subunit of 11S globulin. In case of 7S globulin, macro-soluble aggregates may be formed by noncovalent intraction more than 30min at 8$0^{\circ}C$. Whereas, coagulum occured by the microbial enzyme was more minuter than the other Ca-, HCI-coagulum. Heat treatment attacked the basic subunit of 11S globulin and this results agreed very, how-ever, preferred acidic subunit to basic subunit of 11S globulin and attacked the 7S globulin, that could produce coagulum products within 4~5min at $65^{\circ}C$.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.47 no.2
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• pp.85-94
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• 2002

Antibodies raised against the purified p-subunit of $\beta$-conglycinin were used in immunohistochemical studies to monitor the pattern of $\beta$-conglycinin mobilization in the cotyledons during soybean [Glycine max (L.) Merr.] seed germination. Western blot analysis revealed that the break down of the $\beta$-subunit of $\beta$-conglycinin commenced as early as 2 days after seed imbibition (DAI). Concurrent with the degradation of the $\beta$-subunit of $\beta$-conglycinin, accumulation of 48, 28, and 26 kD proteolytic intermediates was observed from 2 to 6 DAI. Western blot analysis also revealed that the acidic subunit of glycinin was mobilized earlier than the basic subunit. The basic glycinin subunit was subjected to proteolysis within 2 DAI resulting in the appearance of an intermediate product approximately 2 kD smaller than the native basic glycinin subunit. In contrast to the major seed storage proteins, lipoxygenase was subjected to limited proteolysis and was detected even after 8 DAI. The first sign of $\beta$-conglycinin breakdown was observed near the vascular strands and proceeded from the vascular strands towards the epidermis. Protein A-gold localization studies using thin sections of soybean cotyledons and antibodies raised against the $\beta$-subunit of $\beta$-conglycinin revealed intense labeling over protein bodies. A pronounced decrease in the protein A-gold labeling intensity over protein bodies was observed at later stages of seed germination. The protein bodies, which were converted into a large central vacuole by 8 DAI, contained very little 7S protein as evidenced by sparse protein A-gold labeling in the vacuoles.

• Journal of Plant Biology
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• v.32 no.1
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• pp.51-65
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• 1989

Glycinin is the major storage protein in soybean. It has been known that a molecule of glycinin is composed of 6 subunits, each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one (NW 39K and 19K, respectively). To study the molecular origin and the relationship of glycinin subunit polypeptides, antibodies against A-and B-polypeptide were obtained by immunizing rabbits with either of the antigens purified by gel filtration and preparative electrophoresis. Each antibody was not only specific for its own antigen polypeptide in soybeans but also recoginzed the precursor which was synthesized in vivo and in vitro. The polyadenylated mRNAs were isolated from immature seeds and leaves and were translated in vitro using wheat germ extract. One of the seed-specific translation products. MW 60K, was identified to be the precursor of glycinin subunit by immunoprecipitation with antibodies against glycinin A- and B-polypeptide. Mature A- and B-polypeptides were not detected in the translte in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield A- and B-polypeptides which from a glycinin subunit in the cell. Glycinin genes were expressed with the maturation of soybean seeds in a tissue-specific and developmental stage-specific manner.

• Korean Journal of Plant Resources
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• v.12 no.3
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• pp.198-203
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• 1999

7S and 11S globulins are two major storage proteins in soybean seed. For improving the quality of soybean seed protein, an increase of 11S/7S ratio would be a desirable objective because 11S globulin contains much more sulfur-containing amino acids than 7S globulin. In this study, six soybean varieties grown at three locations were used for genetic variation analysis of 7S and 11S globulins. It was possible to screen the soybean genotypes having aberrant subunit compositions of the two globulins by a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). So, heritabilities, genotypic and phenotypic correlations among eight globulin fraction contents of soybean seeds were estimated. The mean value of 7S and 11S globulin fraction contents were 38.9% and 61.1%, respectively, and the ratio of 7S to 11S globulin ranged from 0.58 to 0.74. The high heritability value was found in $\beta$ subunits but the values of acidic and basic subunits were relatively low. Genotypic correlations were higher than the corresponding phenotypic correlations in most of globulin subunit contents. $\beta$ subunits was negatively correlated with $\alpha$ and $\alpha$' subunits among 7S fractions, while no significant correlation between $\alpha$ and $\alpha$' subunits could be found In case of 11S fractions, acidic and basic subunits exhibited no genotypic but negative phenotypic correlation.

• Korean Journal of Food Science and Technology
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• v.32 no.6
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• pp.1221-1226
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• 2000

Specific antibodies were produced to develope the enzyme-linked immunosorbent assay for analysis of soy proteins and the properties of the antibodies were compared. Isolate soy protein(ISP), and ISP heated with SDS and urea (ISP(SU)), acidic subunits(AS) of 11S globulin were immunized to produce polyclonal antibodies. By using competitive indirect ELISA(ciELISA), the reactivities of the antibodies toward soy proteins treated with different methods were investigated and shown as $IC_{50}$. $IC_{50}'s$ of anti-ISP antibodies to ISP, ISP(SU), ISP treated with 2-ME(ISP(ME)), and crude 11S were 20, 30, 36, and $1000\;{\mu}g/mL$, respectively. And the values of anti-ISP(SU) antibodies to the same antigens were 100, 5, 4, and $220\;{\mu}g/mL$ and those of anti-AS antibodies were 20, 2, 2.5, and $200\;{\mu}g/mL$, respectively. Therefore, anti-AS antibodies showed the highest reactivities toward soy proteins among the produced antibodies as determined by ciELISA.

• BMB Reports
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• v.32 no.2
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• pp.181-188
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• 1999

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semialdehyde. Recombinant 4-aminobutyrate aminotransferases were overexpressed as their catalytically active forms in E. coli by coproduction with thioredoxin and their solubilities were also dramatically increased. In order to study the structural and functional aspects of the C-terminal domain of brain 4-aminobutyrate aminotransferase, we have constructed a C-terminal mutant of pig brain 4-aminobutyrate aminotransferase and analyzed the functional and structural roles of C-terminal amino acids residues on the enzyme. The deletion of five amino-acid residues from C-terminus did not interfere with the kinetic parameters and functional properties of the enzyme. Also, the deletion did not affect the dimeric structure of the protein aligned along the subunit interface at neutral pH. However, the deletion of the C-terminal region of the protein changed the stability of its dimeric structure at acidic pH. The dissociation of the enzyme acidic, facilitated by the deletion of five amino acids from C-terminus, abolished the catalytic activity.

• Biomedical Science Letters
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• v.13 no.3
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.