#TITLE_ALTERNATIVE#

• Main Author: Herasari , Dian
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/7616
• Institution: Institut Teknologi Bandung

Protein disulphide isomerase (PDI; E.C.5.4.3.1) is a folding catalyst of disulphide-bonded proteins. PDI in yeast Saccharomyces cerevisiae is encoded by essential gene PDII. In order to understand function of domain and/or residues of amino acid responsible for biological function of PDI, several PDI mutants have been generated by random in vitro mutagenesis using hydroxylamine. In this research, two yeast mutants, yITB254 and yITB264, were characterized based on sensitivity toward dithiothreitol (DTT), ability to secrete killer toxin, reductase activity, and protease sensitivity. yITB254 and wild type yeast appeared to have similar DTT sensitivity. They also secreted killer toxin at the same level. The abilities of crude enzyme extracts isolated from yITB254 and yITB264 to reduce insulin were 172% and 123% higher than that of the wild type. PDI isolated from yITB254 and wild type yeast showed different degradation patterns after treatment with proteinase-K at concentration of 100, 200, and 300 (myu)g/mL. These results indicate that conformation of these two PDI was different. To determine mutation that gave rise to the observed phenotypes, PDII gene of yITB254 and yITB264 has been sequenced. PDII from yITB254 had silent mutation at Ser41 (TCT - TCC) and G1u44 (GAA - GAG), and also contained mutation at Asp436 (GAC) - Asn (AAC). These mutations do not change isomerase s activities of PDI, but PDI might bind peptide more easily and the conformation of PDI might have been altered. Nucleotide sequence analysis of PDII from yITB264 showed no alteration. The phenotype of y1TB264 could be due to mutation on other gene related with oxidation-reduction system.