Keith R. Shelton

Evidence that glutathione participates in the induction of a stress protein

A step in the induction of a 30- to 35-kD stress protein may be the reaction of chemical inducers with glutathione. Effective inducers are sulfhydryl reagents. Further, a comparison of three reagents, 1-chloro-2,4-dinitrobenzene, diethylmaleate, and N-ethylmaleimide, indicates that the first two, which have considerable selectivity for glutathione, are strong inducers of the stress protein but the third, which is much more reactive with protein sulfhydryls, is either a poor or ineffective inducer. A decrease in cellular glutathione does not appear to be inductive, however. An increase in modified glutathione remains as a possible signal for the induction of this stress protein.

Major oligomeric structural proteins of the HeLa nucleus

Abstract The three predominant polypeptides of the insoluble proteinaceous fraction from the HeLa cell nucleus polymerize in vitro upon oxidation of intrinsic sulfhydryl groups. The ease and specificity of this reaction indicate that these polypeptides exist as ordered oligomers in vivo. The comparable insoluble fraction from the rat liver nucleus also contains three predominant polypeptides of the same molecular weights, 65,000 71,000, and 75,000. The insoluble protein of the avian erythrocyte nuclear envelope consists principally of the 71,000- and the 75,000-dalton polypeptides. Indeed, in the avian erythrocyte nucleus these are the predominant polypeptides of the entire nucleus (Shelton, K., Cobbs, C., Povlishock, J. and Burkat, R., 1976, Arch. Biochem. Biophys. 174 , 177). Further, these avian polypeptides each form homogeneous covalently linked oligomers upon sulfhydryl oxidation (Cochran, D., Cobbs, C. and Shelton, K., 1977, J. Cell Biol. 75 , 151a). The insolubility, oligomeric disposition, and relative prominence of these polypeptides in a wide variety of cells indicate a fundamental structural role in the nucleus. Morphological features which may reflect this structural or skeletal role could be the nuclear envelope, the fibrous lamina, or perhaps an intrachromatinic matrix. The metabolism of the oligomeric polypeptides has been investigated in HeLa cells. Turnover of the HeLa insoluble nuclear protein is similar to that of the histones which are known to be stable proteins. The insoluble protein, including the oligomeric polypeptides, is synthesized in G1, S, and G2 phases of the cell cycle. This metabolic behavior indicates that the oligomeric polypeptides are reutilized in successive cell cycles and that synthesis accompanies nuclear and cellular expansion rather than deoxyribonucleohistone synthesis. This suggests that neither degradation nor selective synthesis of oligomeric polypeptides at a particular phase of the cell cycle are responsible for the breakdown and reformation of the interphase cell morphological features that occur during mitosis.

Dodecyl sulfate-polyacrylamide gel electrophoresis of N′-dimethylaminonaphthalene-5-sulfonyl-proteins: A covalently attached fluorescent label

Abstract Five reduced and alkylated proteins have been labeled with l-dimethylamino-naphthalene-5-sulfonyl (DNS) chloride in sodium dodecyl sulfate (SDS) solution. The fluorescent DNS-proteins have been subjected to disc gel electrophoresis. Sensitivity of the method is such that a few micrograms of labeled protein can be easily revealed on conventional gels by UV light. By reduction of the gel diameter to 1 mm, a minimum of 0.05 μg of protein can be detected. The DNS group has at most a minor effect on the migration of the protein in the SDS-disc gel electrophoresis system.

Analysis of the nuclear envelope polypeptides by isoelectric focusing and electrophoresis

Abstract The more insoluble polypeptides of the avian erythrocyte nuclear envelope have been characterized by a two-dimensional electrophoretic procedure. Most of the polypeptides occur in two classes with isoelectric points of approximately 6.4 and 5.7 respectively. The more acidic class contains two polypeptides, P71 and one which contributes to an electrophoretic band previously identified as P55. The more basic class includes P75, P68, P61 and two or more polypeptides from the P55 band. There are four to six isoelectric point variants of each polypeptide in the more basic class, and the relative stain intensities for the variants are similar for the different polypeptides. These similarities in ionic properties suggest a chemical relationship between the polypeptides. These results are discussed in relation to the in vitro conversion of P75 to polypeptides of the same molecular weight as P68, P61 and P55.

Nuclear envelope proteins: Identification of lamin B subtypes

Abstract The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.

On the variation of the major nuclear envelope (lamina) polypeptides.

Abstract Three polypeptides which predominate in nuclear envelope and nuclear pore complex-lamina fractions are usually identified by their migration rates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unfortunately, their migration relative to proteins frequently used as molecular weight standards can vary significantly under different electrophoresis conditions. However, the three maintain the same order of migration. They have been separated on the basis of both isoelectric point and apparent molecular weight and characterized by their tryptic peptides. Their identity as unique proteins has been established. One appears to be a cleavage product of another while the third has regions of unique sequence. This cleavage model is supported by an in vitro cleavage reaction.

The acid-dependent conversion of major nuclear oligomeric polypeptides into a smaller species.

Abstract Oligomeric polypeptides predominate among the nonhistone nuclear proteins of the avian erythrocyte. Two of these are converted to smaller species by several minutes in boiling aqueous buffer at pH values of 4.0–4.5 and below. Polypeptides corresponding to the species generated by this treatment are also isolated from the cell in the absence of acid treatment. This result indicates that several nuclear structural proteins could derive from a common polypeptide chain.