Reversible Disulfide Bond Research Articles

Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.

GLUTATHIONE REDOX STATUS ALTERS THE DIMERIC-MONOMERIC EQUILIBRIUM OF HUMAN GLUTATHIONE REDUCTASE. + 1228

The major intracellular redox buffer in most eukaryotic cells is glutathione (GSH) and glutathione disulfide (GSSG). The NADPH-dependent conversion of GSSG to GSH is catalyzed by the flavoenzyme glutathione reductase (GR). When active, GR functions as a homodimer; the GR equilibrium greatly favors GSH formation at cellular NADPH/NADP* ratios. Steady-state levels of GSH and GSSG in cells are not constant, but may vary considerably in response to nutritional status, hormones, drugs and oxidative stress. Normal ratios of GSH/GSSG range from 100-400; however, under oxidative stress ratios may fall to as low as 1-10. Alterations in glutathione redox status due to a change in either the concentration of GSH, GSSG, or both, if coupled to the redox status of thiols and disulfides in specific proteins, could provide a unique regulatory mechanism affecting the biological activity of receptors, transporters, and transcription factors, as well as enzymes including GR. Studies have suggested that the inactivation/reactivation of GR involves a monomer-dimer equilibrium. Possibly, intramolecular disulfide bond formation between cysteine residues in the catalytic center of GR provide increased dimer stability. To determine whether alterations in the glutathione redox status might regulate the monomerdimer state of GR, equilibrium experiments were performed using purified recombinant human GR. Enzyme was incubated with varying ratios of GSH/GSSG at 37°C for 1 hr in phosphate buffer [0.1 M KPO4 + 5 mM EDTA, pH =7.4]. Samples were quenched with 200 mM N-ethylmaleimide, electrophoresed on a 8% SDS-polyacrylamide gel, transferred to immobolin PVDF membrane and analyzed by Western blot. Results show that normal physiological GSH/GSSG ratios favor the monometric form, while ratios below normal result in the enzyme being predominantly in its homodimeric state. This study suggests the possibility of an autoregulatory mechanism for the inactivation/reactivation of GR via reversible disulfide bond formation and supports the importance of reversible thiol/disulfide redox equilibria in biological processes.

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC 50 around 0.1 μM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

Thiolation and reversible immobilization of sweet potato δ-amylase on thiolsulfonate-agarose

Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide exchange reactions are unique because, simultaneously, they show a stable covalent bond and the possibility of eluting the protein by reduction when the enzymatic activity decays. The adsorbent can thus be reloaded. In this paper we report the use of the recently developed thiolreactive adsorbent thiolsulfonate-agarose, for the immobilization of sweet potato β-amylase. Since native, β-amylase thiol groups were not reactive towards the adsorbent, the enzyme was provided with ‘de novo’ thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP). When the SPDP/β-amylase molar ratio was changed between 3 and 100, up to sixteen exposed thiol groups per mol of enzyme were introduced. This was achieved without affecting the amylolytic activity. The immobilization yield for the intermediate thiolation level was 98%. However, only 19% of the applied enzyme activity was found in the gel suspension. Comparative studies were made on thiolsulfonate-agarose and on a commercial thiol-activated adsorbent (2-pyridyldisulfide-agarose). The immobilization of the thiolated enzyme through reversible disulfide bonds on both adsorbents showed similar results. A close analysis reveals that immobilization of proteins on thiolsulfonate-agarose is a very promising technique.

A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.