• 한국식품영양과학회지
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• 제22권6호
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• pp.811-814
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• 1993

Sarcodon aspratus(Berk.) S. Ito에서 분리정제한 단백질가수분해효소의 특성을 조사하였다. 이 효소는 당을 2.1% 함유하고 있었다. Rose bengal, N-bromo succinimide, phenyl methyl sulfonyl fluoride(PMSF)와 같은 화학적 수식 시약에 효소 활성이 저해되었으며, 1차 반응속도론적 불활성 mode를 가지므로 활성 부위는 tryptophan과 serine으로 추정된다. N-말단에서 21번째 잔기까지의 아미노산 배열은 V-T-T-K-Q-T-N-A-P-W-G-L-G-N-I-S-T-T-N-K-L-으로 동정되었다.

• 한국식품영양과학회지
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• 제20권1호
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• pp.35-39
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• 1991

Properties of a protease purified from Neungee[Sarcodon aspratus(Berk, ) S. Ito] have been investigated. The enzyme displays a glycosylated serine protease. The enzyme is able to hydrolyze alanine glycine methionine glutamine and cysteine of N-CBZ and N-t-BOC-L-amino acid derivatibes relatively strongly but splits valine proline and isoleucine derivatives with low affinity which means the enzyme has the broad substrate spectrum toward the amino acids. Interestingly the enzyme was inhibited by bromelain inhibitor. That is the active site environ-ment of the enzyme is believed to be similar to that of bromelain However peptide mapping studies show that the two enzymes have distinct different cleavage sites.

• Biomolecules & Therapeutics
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• 제14권1호
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• pp.30-35
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• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.