• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.604-610
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• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.2
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• pp.129-135
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• 2000

Previously, we have reported that ${\rho}-chlorophenylalanine$ (PCPA), a serotonin depletor, profoundly increased pancreatic fluid and bicarbonate secretion but remarkably inhibited pancreatic amylase secretion in anesthetized rats. The present study was performed to verify the detailed effects of PCPA on pancreatic amylase synthesis that is directly related to amylase exocrine secretion. PCPA significantly decreased pancreatic RNA and protein contents as well as the amylase activity. However, pancreatic DNA content, trypsin and chymotrypsin activities were not influenced by the treatment of PCPA. The rate of pancreatic amylase synthesis, which was assessed by the amount of incorporated $[^{35}S]-methionine$ into amylase for 1 h, was also significantly decreased by 44% in PCPA-treated rats. In order to determine whether the PCPA-induced decrease of amylase synthesis resulted from change in the level of amylase mRNA, Northern blot analysis was performed. The mRNA expression level of amylase was also decreased by 48% in the PCPA-treated rats, indicating that the inhibitory effect of PCPA on the synthesis of pancreatic amylase was mainly regulated at a step prior to translation. It was also revealed in SDS-polyacrylamide gel electrophoresis that the qualitative change of amylase was induced by PCPA. The 54 KDa amylase band seems to be degraded into small molecular weight protein bands in PCPA-treated rats, suggesting that the PCPA- induced decrease of amylase may be partly attributed to the degradation of synthesized amylase.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.2
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• pp.161-166
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• 2001

The storage proteins of the mulberry longicorn beetle, Apriona germari Hope, were purified and characterized. Three kinds of storage protein (SP1, SP2 and Sp3) were purified from the last instar larval hemolymph of A. germari by the FPLC techniques, anion exchange chromatography and gel permeation chromatography. The SP1, SP2 and SP3 have a native molecular weight of 480, 440 and 420 kDa, respectively. In the SDS-polyacrylamide gel electrophoresis analysis, these storage proteins are composed of a single protein subunit with molecular weight of 90, 85 and 80 kDa, respectively. This result showed that the storage proteins are hexameric protein. The SP1 and SP2 were stained with Schiffs reagent, but SP3 was not stained. It can be assumed that SP1 and SP2 are glycoprotein. Western blot analyses using the each of polyclonal antiserum against purified SP1, SP2 and SP3 showed that the three antibodies reacted with the each of SP bands, respectively. Also, antibodies against SP1 and SP3 cross-reacted with the SP3 and SP1, respectively. However, SP2 was not cross-reacted with these two antibodies. Also, antiserum against SP2 did not cross-reacted with the SP1 and SP3.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.2
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• pp.167-172
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• 2001

The vitellin of firefly, Pyrocoelia rufa, is composed of three polypeptides, designated Vn1 (175 kDa), Vn2 (160 kDa) and Vn3 (45 kDa) in SDS-polyacrylamide gel electrophoresis. Three subunits of vitellin were presented in the female adult hemolymph, ovary and egg extracts, but not observed in the male. This vitellin was purified from the eggs of P. rufa by the FPLC techniques, anion exchange chromatography and gel permeation chromatography. In nature, vitellin of P. rufa has molecular weight of 400 kDa. Western blot analysis using polyclonal antiserum against purified vitellin showed that the antiserum was reacted with the three polypeptides, Vnl, Vn2 and Vn3 from the female adult hemolymph, ovary and egg extracts. Amino acid residues at N-terminus of three subunits were sequenced. The N-terminal sequences of large subunits, Vnl and Vn2, were similar to each other, But, the N-terminal sequences of small subunits Vn3, did not have any signnificant homology with large subunits.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.281-286
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• 2010

This study was to evaluate the protein profile of seminal plasma using 2-DE in Hanwoo. Seminal plasma was harvested from five mature Hanwoo, and seminal plasma protein was extracted by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $300\;{\mu}l$. Immobilized pH gradient (IPG) strip was used 18 cm and 3~11 NL. SDS-PAGE was used 12% acrylamide gel. Each gels were visualized by comassie brilliant blue and silver staining. These spots were analyzed by MALDI-TOF MS and searched on NCBInr. The result, 20 proteins of 36 protein spots were searched through peptide sequencing on the NCBInr. 8 proteins profiled by 2-DE were proved through previous bovine studies and the name of each protein was albumin, nucleobindin, clusterin, TIMP-2, spermadhesin Z13, spermadhesin-1 and BSP proteins (BSP 30 kDa and BSP A1/A2). 12 new proteins were ATP synthase, protein MAK16 homolog, Transmembrane protein 214, E3 ubiquitin-protein ligase BRE1A, dual serine/threonine and tyrosine protein kinase, tissue factor pathway inhibitor 2, alpha-actinin-4, RUN domain-containing protein 3B, catenin alpha-1, protein-glutamine gamma-glutamyltransferase 2, plakophilin-1 and inter-alpha-trypsin inhibitor heavy chain H1 has not been previously described in the bovine seminal plasma study. These proteins may be contribute to define the type of proteins affecting fertility of male and improve the fertilizing ability of semen in Hanwoo.

• Journal of Life Science
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• v.8 no.2
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• pp.137-144
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• 1998

These studies were undertaken to investigate changes of major components occuring during germination of sesame (Sesamum indicum L.) seeds, Changes of total lipid and protein contents, and fatty acid composition were determined. Also, the correponding values of various components in cotyledons, hypocotyls and roots were measured according to germination stage. The results were summarized as follows; During germination, total lipid and protein contents decreased. In particular, protein contents rapidly decreased to the 3 days after gemination(DAG), and then total lipid contents rapidly decreased. In changes of total lipid and protein of cotyledons, hypocotyles and roots detected at the 10, 15 and 20 DAG, some variations were determined. The contents of lipid and protein in hypocotyls rapidly decreased, but since than no changes were observed. In contract, in roots similar changes patterns were observed, while since 15 DAG a rapidly increase was wxamined. In fatty acid composition of total lipid ,saturatedmfatty acids such as palmitic acid increased during the germination. On the other hand, unsaturated fatty acid such as olic acid and linoleic acid decreased during the same periods. In changes of fatty acid composition of total lipid of cotyledons, hypocotlys and roots, saturated fatty acids such as palmitic acid and stearic acid increased during the germination. However, linoleic acid decreased during the same germination suggesting that this may be due to the rapid degradation. However, linoleic acid decreased during the same periods. According to SDS-PAGE analysis, there was no detectible polypeptide bands on the gel before seed germination suggesting that this may be due to the rapid degradation of the storage peotein in the mature seed by hydrolytic enzymes during the stag. As germination continued polypeptide bands, one with 40KD, two with 32∼34Kd and one with 24KD, were detected on the gel.

• Journal of Ginseng Research
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• v.30 no.3
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• pp.106-111
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• 2006

Adverse changes in individual's biochemistry under heavy metal stress are directly linked with its metabolic activity and health status. The present investigation highlights the differences in protecting role of Panax ginseng extract against mercuric chloride induced alterations in serum proteins. The assessment was based on dividing fifty albino rats into two sets, one for acute and the other for sub-acute study. All the sets had five groups with five albino rats in each i.e. control group, mercuric chloride treated group, Panax ginseng extract treated group, mercuric chloride followed by Panax ginseng extract treated group and Panax ginseng extract followed by mercuric chloride treated group. Mercuric chloride was given orally 0.926 mg/kg body weight for acute set and 0.044 mg/kg body weight for sub-acute set after LD50 (9.26 mg/kg body weight) determination by probitt analysis. 10 mg/kg body weight Panax ginseng extract was given in both acute and sub-acute sets after incorporating safety trials. The control group received tween-20 and distilled water only. The result exhibited significantly reduction (P<0.01) in serum protein, albumin and globulin following mercuric chloride intoxication whereas significant (P<0.01) enhancement in other groups with Panax ginseng extract as an ingredient confirming its protective role. All serum samples were also electrophoresed in 10% SDS with standard marker using discontinuous buffering system. Gradual disappearance of alpha-2 and beta-1 globulin bands from electrophoretic pattern was observed, while a single sharp band was observed between beta-2 and gamma globulin in serum protein pattern of acutely mercuric chloride treated rats. However, this band could not be visualized in sub-acute studies. Panax ginseng extract exhibits a better protection after acute intoxication.

• Fisheries and Aquatic Sciences
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• v.8 no.4
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• pp.213-219
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• 2005

Vitellogenin (VTG) was purified from the blood plasma of estradiol-17$\beta$ ($E_2$)-treated male marbled sole (Limanda yokohamae) using gel filtration and anion exchange chromatography. The purity of the marbled sole VTG (msVTG) was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing. The purified msVTG was used to produce monoclonal and polyclonal antibodies in mice and rabbits, respectively, and the specificity of the polyclonal antisera for msVTG was confirmed by Western blot analysis. The antibodies cross­reacted with a protein of molecular mass approximately 160 kDa in the plasma samples of mature female marbled sole. No cross-reactivity was observed with the plasma of male fish. A direct non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed using the monoclonal anti-msVTG and polyclonal anti-msVTG antibodies, with purified msVTG as the standard protein. The values of the intra- and inter-assay variations were within the ranges of $8.l-9.8\%$ and $8.5-12.2\%$, respectively. The sensitivity was about 0.3 ng/mL. Serial dilutions of plasma from mature female sole reacted with the msVTG-antibodies in the sandwich ELISA, whereas the plasma from male fish did not. The results indicate that the maturation status of female marbled sole can be identified using a sandwich ELISA for msVTG.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.2
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• pp.285-291
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• 1999

To confirm the food quality of Urechis unicinctus which have been favored as a special raw seafood in southern area in Korea, the chemical composition of Urechis unicinctus and those glycoprotein were studied. Freeze dried Urechis unicinctus was composed of more than 70% of crude protein and 15% of total carbohydrate. The amino acids such as glycine(18.69%), glutamic acid(12.50%) and aspartic acid(9.37%) were noted as major components of total amino acids. The predominant free amino acids were alanine(32.98%), glycine(27.50%), asparagine(19.65%) and taurine(8.29%), and the sum of them were more than 88% to total free amino acids, so they may cause unique taste of Urechis unicinctus with sweetness. As the basis of sugar composition analysis, 56.35% of glucose and 30.49% of N acetylglucosamine were contained respectively, and they might also play an important role in a sweet taste. The leading carbohydrate moiety of glycoprotein from Urechis unicinctus was identified as glucose and N acetylglucosamine similarly to raw muscle, and they occupied more than 50% of total sugar content. Fucose(18.32%) and D glucuronic acid(12.31%) also detected in higher levels com pared to raw muscle. The total amino acid profile of glycoprotein showed a similar trend to raw muscle protein but glycine was detected a significantly lower than that in raw muscle. The glycoprotein from Urechis unicinctus was composed of 4 kinds of subunits, i.e. 25kDa, 20kDa, 18kDa and 12.5kDa of molecular weights through the SDS polyacrylamide gel electrophoresis. On the basis of the IR spectrum of absorptions appeared in 1,240cm-1 and 850cm-1, the glycoprotein had sulfate groups.

• Journal of the Korean Society of Food Science and Nutrition
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• v.18 no.3
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• pp.287-299
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• 1989

This research was conducted in order to investigate thermal stability and nutritional value of corn lipids and proteins during thermal processing. The lipids of raw and popped corn were fractionated and analyzed by column and gas chromatography. The effect of thermal processing on corn proteins was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. There was no remarkable change in proximate compositions during thermal processing. The lipid fractions obtained by silicic acid column chromatography were composed of neutral lipid(93.5%), glycolipid(3.8%), and phospholipid(2.7%), Although the thermal processing showed the increase in the ratio of unsaturated/saturated fatty acid, there was no significant differences in the lipid composition between raw and popped corn. Most of each protein fractions had lower molecular weight than 25,000 dalton and albumin fractions were distributed in the molecular weight range 11,500-94,000 daltons. Popped corn proteins did not show marked differences in their electrophoretic migrations when compared with raw corn proteins.