• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.23-34
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• 2004

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2'(3')-O-( N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6${\times}$10$\^$6/ M$\^$-1/ sec$\^$-1/ whereas the second nucleotide binds at a slower rate of 4.7${\times}$10$\^$5/ M$\^$-1/ sec$\^$-1/ at 18$^{\circ}C$, RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.

• Molecules and Cells
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• 제23권2호
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• pp.252-257
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• 2007

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.

• Biomolecules & Therapeutics
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• 제18권4호
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• pp.375-385
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• 2010

Conventional cancer chemotherapy is seriously limited by tumor cells exhibiting multidrug resistance (MDR), which is caused by changes in the levels or activity of membrane transporters that mediate energy-dependent drug efflux and of proteins that affect drug metabolism and/or drug action. Cancer scientists and oncologists have worked together for some time to understand anticancer drug resistance and develop pharmacological strategies to overcome such resistance. Much focus has been on the reversal of the MDR phenotype by inhibition of ATP-binding cassette (ABC) drug transporters. ABC transporters are a family of transporter proteins that mediate drug resistance and low drug bioavailability by pumping various drugs out of cells at the expense of ATP hydrolysis. Many inhibitors of MDR transporters have been identified, and though some are currently undergoing clinical trials, none are in clinical use. Herein, we briefly review the status of MDR in human cancer, explore the pathways of MDR in chemotherapy, and outline recent advances in the design and development of MDR modulators.

• BMB Reports
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• 제38권3호
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• pp.266-274
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• 2005

High temperature requirement A (HtrA) and its homologues constitute the HtrA familiy proteins, a group of heat shock-induced serine proteases. Bacterial HtrA proteins perform crucial functions with regard to protein quality control in the periplasmic space, functioning as both molecular chaperones and proteases. In contrast to other bacterial quality control proteins, including ClpXP, ClpAP, and HslUV, HtrA proteins contain no regulatory components or ATP binding domains. Thus, they are commonly referred to as ATP-independent chaperone proteases. Whereas the function of ATP-dependent chaperone-proteases is regulated by ATP hydrolysis, HtrA exhibits a PDZ domain and a temperature-dependent switch mechanism, which effects the change in its function from molecular chaperone to protease. This mechanism is also related to substrate recognition and the fine control of its function. Structural and biochemical analyses of the three HtrA proteins, DegP, DegQ, and DegS, have provided us with clues as to the functional regulation of HtrA proteins, as well as their roles in protein quality control at atomic scales. The objective of this brief review is to discuss some of the recent studies which have been conducted regarding the structure and function of these HtrA proteins, and to compare their roles in the context of protein quality control.

• The Korean Journal of Physiology
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• 제9권1호
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• pp.1-11
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• 1975

The present experiments were carried out to investigate the active center of sodium and potassium ion activated adenosine triphosphatase. An ATPase, activated by sodium ion Plus potassium ion in the presence of magnesium ion, and inhibited by ouabain, has been obtained from rabbit red cell ghosts. The ATPase activity was measured by inorganie phosphate released from ATP. From this values of the measured inorganic phosphate, the activity of ATPase was calculated. The following results were observed. 1. The activity of $(Na^++K^+)-ATPase$ is inhibited by ouabain. This effect may not be due to an effect on sulfhydryl groups, amino groups, carboxyl groups, imidazole groups and hydroxyl groups. 2. The $(Na^++K^+)$-activated enzyme system is inhibited by p-chloromercuribenzoate and by d nitroflurobenzene, and this effect may be due to an effect on sulfhydryl groups. These results indicate that the sulfhydryl groups is attached to sodium-potassium dependent adenosine triphosphate, an aspect of the pump. 3. The $(Na^++K^+)-activated$ enzyme system is inhibited by maleic anhydride and this inhibition is reversed by lysine. This Seems to indicate that the active center of this enzyme is the amino groups. 4. The $(Na^++K^+)$-activated enzyme system is inhibited by iodoacetamide and this inhibition is reversed by the simultaneous present of cysteine and aspartic acid in the suspension medium. This result indicates that this enzyme contains sulfhydryl groups and carboxyl groups. 5. The $(Na^++K^+)-ATPase$ activity is accelerated by adrenaline and this effect is abolished by aspartic acid. This effect of aspartic acid indicate that carboxyl group might be involved in the hydrolysis of ATP by the enzyme system. On the hydrolysis of ATP by the enzyme system. On the basis of these experiments it f·as suggested that the active center of $(Na^++K^+)-activated$ ATPase contains sulfhydryl groups, amino groups and carboxyl groups.

• 한국수산과학회지
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• 제35권3호
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• pp.297-301
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• 2002

베도라치액젓을 옥외의 자연조건 ($25\pm5^{\circ}C$)으로 18개월 동안 숙성 시키면서 2$\~$3개월 간격으로 성분변화에 대하여 조사하였다. 베도라치육의 가수분해도는 숙성 6개월까지는 $75.6\%$로 큰 폭의 증가를 보였으나, 그 이후에는 분해속도가 둔화되어 숙성 18개월 후에는 약 $89\%$의 질소화합물이 육으로부터 액으로 이행되었다. 액젓중의 총질소 및 아미노산성질소함량, 그리고 ATP 관련물질 총량은 숙성기간에 비례하여 일정하게 증가하였으며, 숙성 2개월부터 ATP 관련물질은 거의 대부분 (80.1$\~$$90.5\%$)이 Hx과 요산이었다. HxR+HX 함량과 요산량이 교차되는 숙성 6.8개월 부근은 $76.5\%$의 높은 분해율을 보여 경제적인 출하시점으로 판단된다. 18개월간 숙성시킨 베도라치액젓의 유리아미노산 총량은 6,096.9mg/100mL으로 원료육 아미노산 총량 (7,555.6mg/100g)의 약 $81\%$ 정도였으며, 주요 아미노산은 glutamic acid ($16.3\%$), alanine ($10.9\%$), Iysine ($10.8\%$), valine ($9.9\%$), leucine ($9.4\%$) 등의 순이었다.

• Parasites, Hosts and Diseases
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• 제27권3호
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• pp.161-170
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• 1989

Toxeplasma의 추출액을 3H-casein을 기질로 반응시켰을 때, pH 6.0과 PH 8.5에서 casein을 분해하였으며, pH 6.0에서는 cysteinyl protease의 억제제 인 iodoacetamide(rAh)에 의해 억제되 었고, 활성제 인 dithiothreitol (DTT)에 의해 환성이 증가하였다. 또 pH 8.5에서는 serine protease의 억제제인 phenylmethylsulfonil fluoride (PMSF)에 의해 활성이 억제되었으며, ATP를 첨가할 때 그 활성이 증가하여 ATP 의존성 효소임을 알 수 있었다. 위의 단백질 분해 효소를 부분 정제하기 위해 여러 chromatography를 실시하였는데, 먼저 DE52 (2.Sfx40 cm)에 통과시켰을 때, 0.05M-0.IM NaCl에 의해 유출되는 분획이 pH 6.0에서 황성을 나타내었으며, 0.25V- 0.3M에서 유출되는 분획이 pH 8.5에서 황성을 나타내었다. 이 분회들을 각각 Sephadex G-200 ($2.50{\phi}{\times}40cm$) 에 통과시켜 pH 6.0에서 활성을 나타내는 분획은 exclusion limit내에서, pH 8.5의 분획은 exclusion limit 외에서 분획을 얻었다. 이들을 각각 hydroxylapatite ($2.50{\phi}{\times}10cm$과 $2.5{\phi}{\times}20cm$)를 통과시켜 각각을 0.05M Phosphate로 유출되는 분회에서 높은 환성을 얻었다. 부분 정제된 분획들의 특성을 검토하기 위하여 억제제를 농도별로 처리하였을 때, pH 0.0에서의 분해 효소는 10-3M IAA에 의해 활성이 반감되어 cysteinyl acid protease임을 알 수 있었다. pH 8.5에서의 분해 효소는 10-5M PMSF에 의해 활성이 반감되었고, ATP에 의해 활성이 증가(ATP의 농도가 2.0mM 이상에서는 억제)하여 ATP-dependent neutral serine protease임을 알 수 있었다.

• The Korean Journal of Physiology
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• 제17권2호
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.818-825
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• 2013

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

• 한국수산과학회지
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• 제32권6호
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• pp.693-698
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• 1999

재래식방법으로 까나리액젓 숙성 중에 성분변화를 실험하기 위하여 안면도산 까나리를 구입하여 일광(日光)하 ($25{\pm}5^{\circ}C$)에서 8개월 동안 숙성시키면서 2$\~$3개월 간격으로 성분변화를 조사하였다. 까나리육의 가수분해도는 숙성 6개월까지는 $71.5\%$로 큰 폭으로 증가하였으나, 그 이후에는 분해속도가 둔화되어 숙성 18개월 후의 가수분해도는 $83.2\%$이었다. 수분함량은 숙성기간에 따라서 약간씩 감소하는 반면, 조단백질함량과 VBN함량은 증가하였으며, 회분함량과 pH 및 염분함량은 숙성기간에 따라서 큰 변화가 없었다. 총질소함량은 숙성 8개월까지는 그 이후에는 증가속도 가둔화되어 후에는 숙성 18개월 후에는 1,825mg/100ml이었다. 아미노산성질소함량은 총질소함량보다 증가속도가 더 큰 것으로 나타났는데, 이것은 육의 단백질이 액화되어 액젓으로 이행되는 속도보다 저분자펩티드 및 아미노산이 생성되는 속도가 더 빠르기 때문으로 생각된다. 액젓 중의 ATP 관련물질은 ATP$\~$IMP는 극미량, HxR는 약간 검출되었고, 거의 대부분 ($83.1\~92.9\%$)이 Hx과 산이었다. ATP 관련물질 총량의 증가는 주로 요산량의 증가에 의한 것이며, 숙성기간에 따라서 일정하게 증가하였다. 숙성 8개월 전까지는 HxR+Hx 함량이 요산량보다 높았다가, 그 이후에는 요산량이 HxR+Hx 함량보다 높게 나타났으며, HxR+Hx 함량과 요산량이 교차하는 숙성 8개월 부근은 가수분해도 $74.7\%$, 가용화율 $86.4\%$로 나타나 높은 분해율을 보이는 지점이었고, 맛과 냄새면에서도 좋은 것으로 나타났다. 색도는 숙성기간에 따라서 a값, E값 및 분광광도계 453 nm에서 측정한 흡광도는 증가하였고, L값 및 b값은 감소하였다. 까나리육의 총아미노산함량은 19,741mg/100ml로 나타났으며, 조성은 cystine이 $15.50\%$로 가장 높았고, 다음이 Iysine ($10.23\%$), aspartic acid ($10.06\%$), valine ($8.75\%$), glutamic acid($7.34\%$), leucine($7.23\%$) 등의 순이었다. 18개월간 숙성시킨 까나리액젓의 유리아미노산 총량은 7,911.3mg/100ml로 원료육의 총아미노산함량의 약 $40\%$정도였으며, 조성비는 glutamic acid가 $15.23\%$로 가장 많았고, 다음이 alanine($12.61\%$), lysine ($10.11\%$), leucine ($7.90\%$), isoleucine ($7.18\%$ ), valine ($6.99\%$), aspartic acid($6.70\%$) 등의 순으로 나타나, 원료육의 아미노산 조성과 큰 차이를 보였다.