[국내논문]SDS-겔로부터 효율적인 단백질 분리방법과 31 kDa 연접후치밀질 단백질의 동정 An efficient and reliable electroelution method from SDS-PAGE: Identification of a 31 kDa protein in the postsynaptic density fraction as adenine nucleotide translocator 1원문보기
연접후치밀질(PSD)의 분자조성은 아직 대체로 알려져 있지 않다. 본 연구에서는 다양한 크기의 연접후치밀질 단백질들을 효율적으로 SDS-겔에서부터 추출하는 방법을 제시하고, 이를 이용하여 연접후치밀질의 1% n-octyl glucoside에 용해되지 않는 분핵에 존재하는 31kDa크기의 단백직(PSD31로 명명)을 SDS-겔로부터 분리하고, trypsin 처리하여 생성되는 펩티드의 아미노산 서열을 결정하였다. PSD31의 아미노산 서열은 adenine nucleotide translocator 1(ANT1)과 매우 상동성이 높았다. 연접후치밀질에 ANT1이 존재함은 신경조직의 세포외 공간에 adenosine nucleotide를 유리할 수 있는 기구가 연접에 존재함을 시사한다.
연접후치밀질(PSD)의 분자조성은 아직 대체로 알려져 있지 않다. 본 연구에서는 다양한 크기의 연접후치밀질 단백질들을 효율적으로 SDS-겔에서부터 추출하는 방법을 제시하고, 이를 이용하여 연접후치밀질의 1% n-octyl glucoside에 용해되지 않는 분핵에 존재하는 31kDa크기의 단백직(PSD31로 명명)을 SDS-겔로부터 분리하고, trypsin 처리하여 생성되는 펩티드의 아미노산 서열을 결정하였다. PSD31의 아미노산 서열은 adenine nucleotide translocator 1(ANT1)과 매우 상동성이 높았다. 연접후치밀질에 ANT1이 존재함은 신경조직의 세포외 공간에 adenosine nucleotide를 유리할 수 있는 기구가 연접에 존재함을 시사한다.
The molecular composition of the postsynaptic density (PSD) is largely hon. In this report, an electroelution protocol was demonstrated to be used for efficient isolation of PSD proteins with diverse molecular sizes. Using this protocol, a 31 kDa protein in the 1% n-octyl glucoside-insoluble PSD fra...
The molecular composition of the postsynaptic density (PSD) is largely hon. In this report, an electroelution protocol was demonstrated to be used for efficient isolation of PSD proteins with diverse molecular sizes. Using this protocol, a 31 kDa protein in the 1% n-octyl glucoside-insoluble PSD fraction (termed as PSD31) was purified from SDS-gels, and internal peptides were determined for amino acid sequences. The amino acid sequences of the PSD31 were highly homologous with the adenine nucleotide translocator 1 (ANTI). The association of ANTl with PSD suggests presence of a mechanism in synapses for releasing adenosine nucleotides into the extracellular space.
The molecular composition of the postsynaptic density (PSD) is largely hon. In this report, an electroelution protocol was demonstrated to be used for efficient isolation of PSD proteins with diverse molecular sizes. Using this protocol, a 31 kDa protein in the 1% n-octyl glucoside-insoluble PSD fraction (termed as PSD31) was purified from SDS-gels, and internal peptides were determined for amino acid sequences. The amino acid sequences of the PSD31 were highly homologous with the adenine nucleotide translocator 1 (ANTI). The association of ANTl with PSD suggests presence of a mechanism in synapses for releasing adenosine nucleotides into the extracellular space.
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제안 방법
Efficient elution of PSD proteins from SDS-gels. Proteins in the n-octyl glucoside (OG)-pellet (OG-P) fraction were separated in 6% preparative SDS- polyacrylamide gels and each protein band was electroeluted as described in detail under Materials and Methods. Equal relative volumes of eluant, corresponding to sixty micrograms of the OG-P fraction (lane P) were electrophoresed in a 6% SDS-gel, stained with Coomassie dye and destained as usual.
peptides was verified by the presence of methiones (M) at the -1 positions of the CNBr-cleaved peptides and In this study, we described an efficient and reliable electroelution protocol, showed that it could beapplicable for extracting proteins with diverse molecular sizes from SDS-gels, and succeeded in obtaining internal amino acid sequences of a 31 kDa proteins in the CXe-insoluble PSD fraction identifying it as ANTI, or its homologue.
성능/효과
The protoc이 employed in this study is based on the assumption that proteins stained with Coomassie dye in a buffer that lacks fixatives could be visualized unambiguously and also the stained proteins could be efficiently eluted since they are not fixed to the gel matrix. As expected, the present protocol resulted in a good resolution that allowed unambiguous identification of major protein bands (approximately over 1% of the total fraction), and elution efficiencies were reasonably high (60-80% for most of the PSD proteins), irrespective of their molecular sizes. It should be also noted that the calculated elution efficiency must be underevaluated, since the background smear in the PSD lane was much higher than in sample lanes.
First, the ANTI is an abundant protein representin응 ~3% of the total NOG-P proteins. Second, the ANTI is resistant to extraction by Triton X-100 during One-Triton PSD preparation and by NOG, indicating strong association of the ANTI with PSD. The function of the ANTI in PSD is highly speculative at present.
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