ABSTRACT
Thiol compounds present in human malignant prostate cells (LNCaP) were investigated after reaction with a mercurial blocking reagent. After extracting the cellular glutathione and some other low molecular weight (LMW) thiols using trichloroacetic acid the resulting the protein precipitate was extracted with buffered 8 M urea containing 2-chloromercuri-4-nitrophenol in an equimolar amount to that of the thiol present. After removing the insoluble chromatin fraction the urea soluble labeled adducts formed were chromatographed on G15 Sephadex. Three yellow coloured (A410 nm) fractions were obtained; first, the excluded protein fraction containing 16.0 ± 4.1% of the applied label followed by an intermediate fraction containing 5.9 ± 1.2%. Finally a LMW fraction emerged which contained 77.2 ± 3.7% of the total label applied and this was further analyzed by column chromatography, first on an anion exchange column and then on a PhenylSepharose 6 column to give what appeared to be a single component. LC-MS analysis of this component gave a pattern of mercuri-clusters, formed on MS ionization showing possible parent ions at 704 or 588 m/z, the former indicating that a thiol fragment of molecular weight approximately 467 could be present. No fragments with a single sulfur adduct (a 369 m/z fragment) were observed The adduct was analyzed for cysteine and other amino acids, nucleic acid bases, ribose and deoxyribose sugars, selenium and phosphorus; all were negative leading to the conclusion that a new class of unknown LMW thiol is present concealed in the protein matrices of these cells.
Subject(s)
Chloromercurinitrophenols/chemistry , Lymph Nodes/chemistry , Prostatic Neoplasms/chemistry , Sulfhydryl Compounds/isolation & purification , Sulfhydryl Reagents/chemistry , Anion Exchange Resins/chemistry , Cell Line, Tumor , Chemical Fractionation , Chromatography, Liquid , Humans , Lymph Nodes/pathology , Lymphatic Metastasis , Male , Molecular Weight , Prostatic Neoplasms/pathology , Spectrometry, Mass, Electrospray IonizationABSTRACT
Although the unfolding and refolding of proteins have been extensively studied in the literature, relatively few attempts have been made to see how many residues of the total residues of a certain amino acid in an enzyme can be modified without seriously affecting its folding. Based on a statistical analysis of the quantitative relationship between the extent of modification of protein functional groups and the decrease in their biological activity, a method proposed by Tsou (Sci. Sin. 1962, 11, 1535-1558) is widely used to determine the number of residues essential for the catalytic activity of modified proteins. In the present paper, Tsou's method is applied to determine the number of cystein residues essential for the folding of creatine kinase. The thiol groups of the cysteine residues in fully unfolded creatine kinase were modified by 2-chloromercuri-4-nitrophenol (MNP). The relationship between the number of MNP-groups introduced and the recovery of activity after refolding was determined. Quantitative treatment of the data by Tsou's plot shows that among the cystein residue modified in each subunit of creatine kinase, only three are essential for its folding.
Subject(s)
Creatine Kinase/metabolism , Protein Folding , Sulfhydryl Compounds/metabolism , Animals , Chloromercurinitrophenols/chemistry , Muscles/enzymology , Protein Binding , Protein Denaturation , Rabbits , Sulfhydryl Reagents/chemistryABSTRACT
2-Chloromercurio-4-nitrophenol reacts reversibly with thiouridine, this reaction was applied to a chemical modification of E. coli transfer ribonucleic acid.
