• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1647-1652
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• 2010

Autophagy refers to the bulk degradation of cellular proteins and organelles through an autophagosome and plays a pivotal role in the development, cellular differentiation, aging, and elimination of aberrant structures. A failure of autophagy has been implicated in a growing list of mammalian disease states, including cancer and cardiomyopathy. Two ubiquitin-like systems are highly involved in autophagy, especially in the formation of autophagosomes. Here, we purified and characterized Atg7 (an E1-like enzyme), and Atg3 and Atg10 (E2-like enzymes) in order to gain an insight into the role played by ubiquitin-like systems in the formation of autophagosomes. Interestingly, we observed that Atg7 forms a homodimer to construct an active conformation, unlike other E1-like enzymes. Although Atg3 was detected as a monomer under physiological conditions, Atg10 existed in an oligomeric form, indicating that the mechanism by which Atg10 functions may differ from that of Atg3.

• The Korean Journal of Physiology and Pharmacology
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• 제23권1호
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• pp.89-89
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• 2019

• 한국식품과학회지
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• 제26권1호
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• pp.81-87
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• 1994

하늘타리박의 뿌리인 괄루근에서 분리 정제한 trypsin inhibitor는 TRTI-1과 TRTI-2 두 종류였으며 gel-filtration과 SDS-PAGE 전기영동에서 얻은 결과로부터 TRTI-1은 $4,000{\sim}5,000\;Da$의 분자량을 가진 monomer이며 TRTI-2는 $20,000{\sim}24,000\;Da$의 분자량을 가친 homodimer임을 알 수 있었다. 온도의 안정성에 있어서는 TRTI-1은 $100^{\circ}C$에서 2시간 이상 안정하였으며, TRTI-2는 $0{\sim}70^{\circ}C$의 온도에서는 안정하였으나 $80^{\circ}C$에서부터는 안정성이 떨어지면서 $100^{\circ}C$에서는 저해능이 완전히 소실되었다. Trypsin 활성에 대해서 TRTI-1, TRTI-2 및 상품화된 soybean BBI의 농도에 따른 저해능을 보면 TRTI-1, TRTI-2 및 soybean BBI의 각각의 농도가 $0.8{\mu}M,\;6{\mu}M,\;4{\mu}M$일 때 1mg/ml의 trypsin을 50% 저해하였으며, trypsin의 반응속도에 대한 TRTI의 저해 실험에서 TRTI-1와 TRTI-2 모두 trypsin의 가수분해를 경쟁적으로 저해하였으며 $K_{m}$값은 저해제가 없는 경우 $0.37\;{\mu}M$에 대해 각각 0.97, $0.63\;{\mu}M$이었다. 여러 protease에 대한 TRTI-1과 TRTI-2의 저해능 실험 결과 elastase, pepsin, Nagarase, papain, thermolysin, chymotrypsin, thrombin 및 collagenase 등은 저해하지 못하였고 trypsin만을 특이하게 저해하였다.

• Journal of Microbiology and Biotechnology
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• 제6권2호
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• pp.79-85
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• 1996

By using a T7 expression system, a large amount of Bacillus stearothermophilus endo-xylanase A (XynA) could be produced in Escherichia coli cells. The overproduced enzyme formed inclusion bodies, and so the protein could be more easily purified to homogeneity. The molecular weight of the purified enzyme was estimated to be 22 kDa by SDS-polyacrylamide gel electrophoresis and 43 kDa by Sephacryl S-200 gel filtration, suggesting that the native enzyme was a homodimer. The pI value was determined to be 8.4. The Michaelis constants for birchwood xylan and oat spelts xylan were calculated to be 3.83 mg/ml and 5.03 mg/ml, respectively, and the $V_{max}$ max/ values for both xylans were 2.86 $\mu mole$/min. The purified enzyme was most active at $55^{\circ}C$ and pH 8.0, and stable up to $60^{\circ}C$ and in the near neutral pH range. From the zymogram, Bacillus stearothermophilus was found to have at least three xylanases and the purified one was the smallest among them.

• Molecules and Cells
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• 제37권10호
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• pp.753-758
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• 2014

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins and plays a critical role in clathrinmediated endocytosis. It has a Bin-Amphiphysin-Rvs (BAR) domain which can form a crescent-shaped homodimer structure that induces deformation of the plasma membrane. While other BAR-domain containing proteins such as amphiphysin and endophilin have an amphiphatic helix in front of the BAR domain which plays a critical role in membrane penetration, SNX9 does not. Thus, whether and how SNX9 BAR domain could induce the deformation of the plasma membrane is not clear. The present study identified the internal putative amphiphatic stretch in the $1^{st}$ ${\alpha}$-helix of the SNX9 BAR domain and proved that together with the N-terminal helix ($H_0$) region, this internal putative amphiphatic stretch is critical for inducing membrane tubulation. Therefore, our study shows that SNX9 uses a unique mechanism to induce the tubulation of the plasma membrane which mediates proper membrane deformation during clathrinmediated endocytosis.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.727-731
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• 2010

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD, which was active with either Fe(II) or Mn(II) as a cofactor. The ApSOD was highly stable against high temperature. This thermostable ApSOD is expected to be applicable as a useful biocatalyst for medicine and bioindustrial processes.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.206-212
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• 1999

The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• BMB Reports
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• 제31권5호
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• pp.492-497
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• 1998

Cu,Zn-superoxide dismutase (SOD) was purified from horseradish by using Mono Q and Superose 12 FPLC column chromatography. The native molecular mass of the purified enzyme was approximately 33 kDa, as determined by gel filtration. The subunit molecular weight, as estimated by SDS-PAGE, was 16 kDa. These results indicated that the native enzyme is a homodimer. We investigated the free radical-generating function of horseradish Cu,Zn-SOD by using a chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) which reacts with ${\cdot}OH$ radicals to form $ABTS^{+{\cdot}}$ The formation of $ABTS^{+{\cdot}}$ was required for both active Cu, Zn-SOD and $H_2O_2$. The optimal pH for the free radical-generating activity of this enzyme was 6.0-8.0, and it retained about $40^{\circ}C$ of its maximum activity when exposed at $40^{\circ}C$ for 15 min. A neutral scavenger, ethanol, inhibited the $ABTS^{+{\cdot}}$ formation by horseradish Cu, Zn-SOD more effectively than that by the mammalian enzyme. These results suggest that the active channel of horseradish enzyme is slightly larger than that of the mammalian enzyme.

• BMB Reports
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• 제34권4호
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• pp.329-333
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• 2001

Active lipoprotein lipase (LPL) is known as a noncovalent homodimer of identical subunits, and dissociation of the dimer to a monomeric form renders the lipase inactive. In this study, the oligomerization status of LPL in human and rat plasma was investigated. The LPL activity was barely detectable in the control rat and human plasma. After the injection of heparin, the total lipolytic activity of plasma was rapidly increased, and reached its maximum in 30 min. Changes of the LPL protein correlated well with those of lipolytic activity. The LPL protein that is released by heparin into both human and rat plasma was active and dimeric in the sucrose density gradient ultracentrifugation. In control rat plasma, LPL was inactive, and a great fraction was present as an aggregate. However, the inactive LPL protein in the control human plasma retained the dimeric state, indicating that dimerization can be an entity independent of the catalytic activity of LPL. The released LPL is transported as a complex with lipoproteins in plasma. Lipoprotein profiles, determined by NaBr ultracentrifugation, exhibited typical LDL- and HDL-mammal patterns in humans and rats, respectively, with a smaller amount of the LDL fraction observed in rats. The difference in the lipoprotein profiles might influence the fate of the released LPL in plasma.

• 한국동물학회지
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• 제24권4호
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• pp.201-215
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• 1981

척추동물 6종의 젖산탈수소효소의 초자내 종간 및 종내 분자교잡을 실시한 결과 드렁허리 $H_4$ 동위효소와 가물치 $M_4$ 동위효소의 교잡 및 가물치 $M_4$동위효소와 메기 동위효소의 교잡에서 $H_2M_2$ 동위효소라고 생각되는 교잡형 동위효소를 얻었다. 하등 척추동물 젖산탈수소효소의 하부단위체 M과 H의 상호간 조합에 관한 한 3차 구조의 하부단위체는 동일한 구조를 가진 하부단위체와 이량체를 형성하고 이러한 동질이량체가 사량체를 형성한다고 사료된다.