Isolation and analysis of a non-protein low molecular weight thiol-mercurial adduct from human prostate lymph node cells (LNCaP).

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.

Highlights

Thiols play pivotal roles in cellular metabolism and are important in the maintenance of the cellular redox balance and the control of oxidative stress
Many cellular thiol functions have been shown to be meditated via the cysteine groups found in enzyme or protein structures the main players in cellular metabolism are thought to be the mobile low molecular weight (LMW) thiols, often known as non-protein thiols (NPSH)
These results obtained using 2-mercuri-4-nitrophenol to label the cellular LMW thiols confirm the findings of previous experiments on lymph node androgen-sensitive human prostate adenocarcinoma cells (LNCaP) cells using the Ellman reagent

Summary

Introduction

Thiols play pivotal roles in cellular metabolism and are important in the maintenance of the cellular redox balance and the control of oxidative stress. In the case of cancer cells and tissues they have been shown to be important in radio-sensitization and protection, in resistance to chemotherapeutic drugs. Many cellular thiol functions have been shown to be meditated via the cysteine groups found in enzyme or protein structures the main players in cellular metabolism are thought to be the mobile low molecular weight (LMW) thiols, often known as non-protein thiols (NPSH). It has been reported that other reducing sulfur moieties, such as sulfides, sulfones (S◦), and persulfides, play important roles in thiol metabolism It has been reported that modified protein cysteine residues play an important role in redox stress signaling It has been reported that modified protein cysteine residues play an important role in redox stress signaling (e.g. [4])

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Conclusion