• 생명과학회지
• /
• 제17권6호통권86호
• /
• pp.772-777
• /
• 2007

식물의 루이신 생합성에 관여하는 3-isopropylmalate isomerase (IPMI) (EC 4.2.1.33) 효소의 소단편을 암호화는 Leucine D유전자를 벼로부터 분리하고 OsLeuD유전자로 명명하였다. OsLeuD유전자는 257개의 아미노산을 암호화하고 있으며 cyanobacteria의 IPMI 단백질과는 약58% 그리고 green sulfur bacteria들의 IPMI 단백질과는 약48%의 상동성을 갖고 있었다. 벼의 OsLeuD 유전자는 japonica벼 (Oryza sativa L.)의 2번 염색체의 26.45 Mb의 위치로서 109.3 cM 거리에 좌위하고 있었다. OsLeuD유전자는 잎과 성숙하는 종자에 많이 발현이 되었으므로 대사가 급증하는 발생단계 에 발현이 조절되는 것으로 여겨진다. OsLeuD유전자와 단백질은 균류와 yeast 보다 광합성 박테리아의 유전자와 높은 동질성을 보이는 것으로 보아 OsLeuD유전자는 식물의 엽록체 유전자 genome 에서 기원하여 핵 genome으로 이동 진화된 유전자로 추측된다.

• Applied Biological Chemistry
• /
• 제34권4호
• /
• pp.334-338
• /
• 1991

고상법으로 새로운 반응기구에 의한 bradykinin 및 $(D-Phe7\;-Leu^8)$ bradykinin을 합성하였다. Coupling은 N, N'-dicyclohexylcarbodiimide로 행하였으며 HBr 용액으로 cleavage한 후 조펩티드는 high pressure liquid chromatography로 정제하였다. 이들 펩티드의 순도는 paper chromatography, thin layer chromatography, paper electrophoresis, 융점측정기 및 아미노산기분석기에 의하여 분석하였다. Endopeptidase인 ${\alpha}-chymotrypsin$과 trysin, exopeptidase인 carboxypeptidase A와 leucine aminopeptidase를 사용하여 in vitro 상에서 이들 펩티드의 분해실험을 하였다. ${\alpha}-Chymotrypsine$ 및 carboxypeptidase A에 의하여 이들 펩티드는 빠르게 분해하였으나 leucine aminopeptidase는 N-말단의 2번 위치에 proline의 imino결합 때문에 분해하지 않았다.

• Bulletin of the Korean Chemical Society
• /
• 제31권4호
• /
• pp.874-880
• /
• 2010

A series of 13- and 14-membered cyclic enkephalin analogs based on the moderately $\mu$ selective prototype compound Tyr-C[D-$A_2bu$-Gly-Phe-Leu] 8a were synthesized to investigate the structure-activity relationship. The modifications of sequence were mainly focused on two positions 3 and 5, critical for the selective recognition for $\mu$ and $\delta$ opioid receptors. The substitution of hydrophobic $Leu^5$ with hydrophilic $Asp^5$ derivatives led to Tyr-C[D-$A_2bu$-Gly-Phe-Asp(N-Me)] 7 and Tyr-C[D-Glu-Phe-gPhe-rAsp(O-Me)] 5, the peptides with a large affinity losses at both $\mu$ and $\delta$ receptors. The substitution of $Phe^3$ with $Gly^3$ led to Tyr-C[D-Glu-Gly-gPhe-rLeu] 3 and Tyr-C[D-Glu-Gly-gPhe-D-rLeu] 4, the peptides with large affinity losses at $\mu$ receptors, indicating the critical role of phenyl ring of $Phe^3$ for $\mu$ receptor affinities. One atom reduction of the ring size from 14-membered analogs Tyr-C[D-Glu-Phe-gPhe-(L and D)-rLeu] 6a, 6b to 13-membered analogs Tyr-C[D-Asp-Phe-gPhe-(L and D)-rLeu] 1, 2 reduced the affinity at both $\mu$ and $\delta$ receptors, but increased the potency in the nociceptive assay, indicating the ring constrain is attributed to high nociceptive potency of the analogs. For the influence of D- or L-chirality of $Leu^5$ on the receptor selectivity, regardless of chirality and ring size, all cyclic diastereomers displayed marked $\mu$ selectivity with low potencies at the $\delta$ receptor. The retro-inverso analogs display similar or more active at $\mu$ receptor, but less active at $\delta$ receptor than the parent analogs.

• 약학회지
• /
• 제38권5호
• /
• pp.530-543
• /
• 1994

To study the feasibility of transmucosal delivery of leucine enkephalin (Leu-Enk) and $[D-ala^2]$-leucine enkephalinamide (YAGFL), their degradation extents and pathways in various rabbit mucosa extracts were investigated by high performance liquid chromatography. The degradation of Leu-Enk and YAGFL was observed to follow the first-order kinetics. The degradation half-lives of Leu-Enk in the nasal, rectal and vaginal mucosal extracts were 1.62, 0.37 and 1.12 hrs and those of YAGFL were 30.55, 9.70 and 6.82 hrs, respectively, indicating Leu-Enk was degraded in a more extensive and rapid manner than YAGFL. But the mucosal and serosal extracts of the same mucosa showed the similar degradation rates for both pentapeptides. The degradation was most rapid in the neutral pH and increasing concentrations of substrates retarded the degradation rates. The maior hydrolytic fragments of Leu-Enk were Des-Tyr-Leu-Enk and tyrosine, indicating the enzymatic hydrolysis by aminopeptidases. However, the data also suggested endopeptidases such as dipeptidyl carboxypeptidase and dipeptidyl aminopeptidase could play some role in the degradation of Leu-Enk. On the other hand, the hydrolytic fragments of YAGFL in all the mucosa extracts were mainly Tyr-D-Ala-Gly and Phe-Leu-Amide, demonstrating the hydrolytic breakdown by endopeptidases. The degradation pathways were further explored by concomitantly determining the formation of smaller metabolites of primary hydrolytic fragments of Leu-Enk and YAGFL in the mucosa extracts.

• 한국잡초학회지
• /
• 제15권1호
• /
• pp.73-84
• /
• 1995

종래의 말포민류에 대한 화학구조 연구에 있어서의 난제들이 최신의 각종 기기분석 및 새로운 정제 방법의 적용에 의해 해결되어졌으며, 이 결과 말포민 A로부터 신규의 $A_4$를 포함한 4종의 A동족체들이 단리 구조결정되었다. 본 연구에 의해 말포민 A동족체의 화학 구조는, $A_1$(1)이 cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-Ile, $A_2$(2)가 cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-Val, $A_3$(3)가 cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-Leu, $A_4$(4)가 cyclo-D-Cys-D-Cys-L-Val-D-Ile-L-Val으로서 확정 되었다. 이들 중, 말포민 $A_3$(3)는 말포민 C와 동일한 화합물로 확인되었다. 단리된 말포민 A동족체에 대해서 옥수수 근 부굴곡활성과 녹두 상배축신장활성을 지표로 하여 생물검정을 실시하였다. 옥수수 근부굴곡 활성의 검정으로부터, 모든 A동족체가 활성을 나타내었으며, 특히 분자량 529의 동족체는 분자량 515의 동족체보다 높은 활성을 보여주었다. 그러나, 녹두 상배축신장활성의 검정에서는 분자량에 따른 차이가 인정되지 않았으며, 이들 중 말포민 $A_4$는 활성형이 아닌 것으로 확인되었다. 양검정법에서 모두 최고 수준의 활성을 나타낸 말포민 $A_1$은, $0.25{\mu}m$ 에서 83%의 옥수수 근부굴곡활성을 나타냈으며, 녹두 상배축신장활성의 경우, $0.1{\mu}m$에서 대조구에 비해 165%의 신장촉진을 보였다.

• 한국잡초학회지
• /
• 제15권1호
• /
• pp.85-98
• /
• 1995

말포민 B로부터 신규의 $B_3$, $B_4$ 및 $B_5$를 포함한 6종의 B동족체들이 단리 구조결정되었다. 본 연구에 의해 말포민 B동족체의 화학구조는, $B_{1a}$(1)가 cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-allo-Ile, $B_{1b}$(2)가 cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-Leu, $B_2$(3)가 cyclo-D-Cys-D-Cys-L-V al-D-Val-L-Leu, $B_3$(4)가 cyclo-D-Cys-D-Cys-L-Val-D-Ile-L-Leu, $B_4$(5)가 cyclo-D-Cys-D-Cys-L-Val-D-Ile-L-Ile, $B_5$(6)가 cyclo-D-Cys-D-Cys-L-Val-D-Val-L-Ile으로서 확정되었다. B's, $B_{1b}$ C 이들 중, 말포민 $B_{1b}$(2)의 화학구조는 말포민 $A_3$ 및 말포민 C와 동일한 것으로 판명되었다. 단리된 말포민 B동족체에 대해서 옥수수 근부굴곡활성과 녹두 상배축산장활성을 지표로 하여 생물검정을 실시한 결과, 모든 B동족체가 활성을 나타내었다. 옥수수 근부굴곡활성의 검정으로부터, 분자량 529의 동족체가 분자량 515의 동족체보다 높은 활성을 보여주었으며, 이는 HPLC상의 retention time에서 확인된 바와 같이, 전체 말포민분자의 극성 이 옥수수 근부굴곡활성에 대해서 영향을 마친다는 사실을 시사한다. 또한, 말포민의 구조를 유지시켜주는 -S-S-가교구조와 제4-제5 아미노산 잔기에 측쇄구조가 상이한 leucine과 isoleucine의 조합이 말포민 수용부위와의 결합에 있어서 최적의 형상을 취할 수 있다는 점에서 말포민류의 활성 발현에 중요한 구조적 요소들일 것으로 추정되어진다. 말포민 $B_{1a}$는 $0.25{\mu}m$에서 90%의 옥수수 근부굴곡활성을 나타내었다. 그러나, 녹두 상배축산장활성의 경우 분자량에 따른 활성의 차이는 인정되지 않았으며, 말포민 $B_5$의 처리시 $0.1{\mu}m$에서 대조구에 비해 1.54%의 신장촉진을 보여주었다.

• Fisheries and Aquatic Sciences
• /
• 제18권2호
• /
• pp.131-135
• /
• 2015

Two new bitter-tasting cyclic peptides comprising six amino acids, namely gamakamide C and D, were isolated from cultured oysters Crassostrea gigas. Dimethylaminoazobenzene sulfonyl-amino acid analysis detected Val and Leu in gamakamide C and Ile and Leu in gamakamide D. The molecular formula of gamakamide C was determined as $C_{43}H_{60}N_{7}O_8S$ by high-resolution fast atom bombardment mass spectroscopy (HR FAB-MS) ($[M+H]^+m/z822.4200{\Delta}-2.4mmu$), and that of gamakamide D was determined as $C_{43}H_{62}N_7O_8S$ by HR FAB-MS ($[M+H]^+m/z836.4379{\Delta}-2.0mmu$). Comparison of amino acid analyses and fragment ions by MS/MS among gamakamide C, D, and E (known), the structures of gamakamide C and D were confirmed $as-{\small{L}}-Val-{\small{L}}-Met(SO)-{\small{L}}-NMe-Phe-{\small{L}}-Leu-{\small{D}}-Lys-{\small{L}}-Phe-$ and $-{\small{L}}-Ile-{\small{L}}-Met(SO)-{\small{L}}-NMe-Phe-{\small{L}}-Leu-{\small{D}}-Lys-{\small{L}}-Phe-$, respectively.

• Journal of Pharmaceutical Investigation
• /
• 제26권3호
• /
• pp.175-185
• /
• 1996

To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

• 약학회지
• /
• 제38권5호
• /
• pp.488-495
• /
• 1994

To evaluate the feasibility of transmucosal delivery of leucine enkephalin (Leu-Enk) and its synthetic analog, $[D-Ala^2]$-leucine enkephalinamide (YAGFL), their physicochemical stabilities in aqueous buffered solutions were first investigated using a stability indicating high performance liquid chromatography. The degradation of Leu-Enk and YAGFL followed the pseudo-first-order kinetics. From the pH-rate profiles, it was found that the maximal stability of the two pentapeptides was at the pH of about 5.0. The shelf lives $(t_{90%})$ for the degradation of Leu-Enk and YAGFL at pH 5.0 and $40^{\circ}C$ were found to be 48.13 and 50.9 days, respectively. From the temperature dependence of the degradation, activation energies for Leu-Enk and YAGFL were calculated to be 13.61 and 13.47 kcal/mole, respectively. A higher ionic strength and a higher initial peptide concentration in buffered solution slowed the degradation of the two pentapeptides. The addition of 2-hydroxypropyl-${\beta}$-cyclodextrin into the peptide solution did not affect the stability significantly.

• 한국미생물·생명공학회지
• /
• 제28권4호
• /
• pp.195-201
• /
• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.