• BMB Reports
• /
• 제42권4호
• /
• pp.223-226
• /
• 2009

We measured the pH-induced spectral changes of yeast iso-1-cytochrome c on silver nanoparticle surfaces using surface-enhanced resonance Raman scattering (SERRS) at 457.9 nm. At a pH of ~3, the Met80 ligand in yeast iso-1-cytochrome c is assumed to dissociate, leading to a marked conformational change as evidenced by the vibrational spectral shifts. The Soret band at ~410 nm in the UV-Vis spectrum shifted to ~396 nm at pH~3, indicating a transition from a low spin state to a high spin state from a weak interaction with a water molecule. Thus, SERRS spectroscopy can measure the pH-induced denaturalization of cyt c adsorbed on metal nanoparticle surfaces at a lower concentration with a better sensitivity than ordinary resonance Raman spectroscopy.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 1986년도 추계학술대회
• /
• pp.505.1-505
• /
• 1986

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.

• Archives of Pharmacal Research
• /
• 제12권2호
• /
• pp.79-87
• /
• 1989

Enzymatic methylation of arginine and lysine residues of several cytochrome c and lysine residue of calmodulin always resulted in lowering of their respective isoelectric points (pI). Employing cytochromes c derived from various sources, we examined a possible relationship between the degree of amino acid sequence degeneracy and the magnitude of change in the pI values by enzymatic methylation, and found that there was no correlation between these two parameters. By constructing space-filling models of oligopeptide fragments adjacent to the potential methylation sites, we have noted that not all the methylatable residues are able to form hydrogen bonds prior to the methylation. Two preparations of yeast apocytochrome c, one chemically prepared by removing heme from holocytochrome c and the other by translating yeast iso-1-cytochrome c mRNA in vitro, exhibited slightly higher Stokes radii than the homologous holocytochrome c, indicating relatively 'relaxed or open' conformation of the protein. However, when the in vitro synthesized methylated apocytochrome c was compared with the unmethylated counter-part, the Stokes radius of the latter was found to be larger.

• 한국미생물·생명공학회지
• /
• 제19권4호
• /
• pp.348-355
• /
• 1991

Bacillus subtilis의 Beta-1,4-glucanase 유전자와 Saccharomyces cerevisiae의 alcohol dehydrogenase isoenzyme I 유전자 (ADHI)의 promoter와 mouse $\alpha$-amylase의 분비신호를 연결하여 분비형 플라스미드를 구성하였으며 이를 산업용 알콜생산 효모인 Saccharomyces cerevisiae 54에 도입하여 형질전환시켰다. 한편 $\beta$-glucanase 유전자의 발현정도를 증대시키기 위해 CYC1 유전자의 전사종결신호를 부가한 후 역시 Saccharomyces cerevisiae 54에 도입하였다. 형질전체들은 carboxymethyl cellulose가 함유된 평판배지에서 $\beta$-glucanase를 분비하고 있음을 확인할 수 있었다. 전사종결 신호가 부가된 경우엔 전체역가가 2배 정도 증가하였다. 형질전환체들이 세포밖으로 분비한 효소역가는 전체의 60 정도였다.