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R Sterner

Publications and source records attributed to R Sterner.

At least 55 records · Page 3Linked to original sources
PubMed1983

Selective isolation of polypeptide chains bearing multiple types of postsynthetic modifications. Recovery of simultaneously acetylated and phosphorylated forms of histone H2A and high mobility group proteins 14 and 17.

In order to study coordinate or simultaneous modifications of chromosomal proteins by phosphorylation and acetylation, duck erythrocytes were incubated with [32P]orthophosphate and the thiol-containing acetate analogue, 2-mercaptoacetate. Enzymatic transfer of the analogue to the epsilon-amino groups of lysine residues permits the selective recovery of the newly thio-derivatized polypeptide chains by Hg-affinity chromatography, and this acetylated subpopulation can then be analyzed for [32P]phosphate uptake. The histones and high mobility group proteins were extracted from cell nuclei, purified, and finally analyzed for incorporation of [32P]phosphate and 2-mercaptoacetate. Several of the nuclear proteins, in particular histone H2A and the high mobility group proteins HMG-14 and HMG-17, were subjected to organomercurial-agarose chromatography. Significant amounts of these cysteine-free proteins were retained on the affinity column, and by this criterion were shown to have incorporated mercaptoacetate. The mercaptoacetylated proteins were further analyzed and found to contain the 32P label as well. These observations provide incontrovertible evidence that individual molecules of chromosomal proteins can carry postsynthetic modifications in the form of phosphorylation and acetylation at the same time, and also establish that both types of modification must have occurred during the short period in which the cells were exposed to the two precursors.

Acetylation↗
PubMed1983

The past, present, and future of clinical specular microscopy.

Since the introduction of specular microscopy into the field of clinical ophthalmology in 1975, many technological and methodological advances have been made. These have for the most part eliminated the previously discussed objections to the use of specular microscopy as a clinical tool. With the advent of new instrumentation, a number of specular microscopes are not available. The advantages and disadvantages of these clinical specular microscopes, current clinical practice with small-field and wide-field specular microscopy, and description of problems and possible future developments of specular microscopy are discussed.

Cornea↗
PubMed1982

In vitro mercaptoacetylation of chromosomal proteins. Selective recovery of newly modified protein molecules.

Duck erythrocytes were incubated with the thiol-containing acetate analog 2-mercaptoacetate. The high mobility group proteins and histones were then extracted from the cell nuclei and the resultant protein mixtures subsequently were fractionated to their individual components by established procedures of selective trichloracetic acid precipitation and ion exchange and gel exclusion chromatography. The protein mixtures and the proteins derived from them were then subjected to mercury-affinity chromatography on organomercurial agarose columns, and it was found that significant amounts of all of the high mobility group proteins and core histones were bound by the columns. Furthermore, chymotryptic digestion of histone H4 from mercaptoacetate-treated cells and subsequent mercury-affinity chromatography revealed that the only peptides which bound to the column were the NH2-terminal peptides, the ones containing the lysines known to be the normal sites of in vivo acetylation. Carboxymethylation of mercaptoacetate-treated H4 with iodo[3H) acetate acid yielded 3H-labeled H4 and subsequent chymotryptic digestion showed that the only radioactive peptides were again those containing the lysines known to be the normal sites of enzymatic acetylation. These results and other biochemical evidence strongly indicate that the proteins of the histone and high mobility group classes which are known to be subject to postsynthetic acetyl transfer reactions can undergo postsynthetic mercaptoacetylation in vivo and that the sites of such modification are the same as those for normal acetylation. The phenomenon of mercaptoacetylation of chromosomal proteins affords a means for the selective recovery of newly modified protein molecules and their associated chromatin.

Acylation↗
PubMed1981

Studies of acetylation and deacetylation in high mobility group proteins. Identification of the sites of acetylation in high mobility group proteins 14 and 17.

Duck erythrocytes were incubated with [3H]acetate both in the presence and absence of sodium butyrate. Subsequent perchloric acid extraction of the nuclei, followed by selective acetone precipitation, CM-Sephadex ion exchange chromatography, and gel filtration yielded radioactively labeled high mobility group (HMG) proteins HMG-14 and HMG-17 in pure form. Extensive enzymatic degradation of the proteins followed by amino acid analysis of the digests yielded a significant amount of material eluting in the position of epsilon-N-acetyllysine. Furthermore, automated Edman degradation of intact 3H-labeled HMG-14 and HMG-17 identified the specific sites of acetylation of these proteins. In both erythrocyte HMGs isolated from cells not exposed to butyrate, the lysine residue at position 2 was the only one found to be labeled. However, one additional site in HMG-14 and two additional sites in HMG-17 were found in the proteins from cells incubated in butyrate. Finally, studies of the enzymatic deacetylation of HMG-14 and HMG-17 confirmed that both nuclear proteins serve as deacetylase substrates and that butyrate inhibits their deacetylation, just as in the case of other HMG proteins and nucleosomal core histones.

Acetates↗
PubMed1978

Postsynthetic modification of high mobility group proteins. Evidence that high mobility group proteins are acetylated.

High mobility group proteins were isolated from calf thymus and duck erythrocyte nuclei and the possibility was investigated that these proteins undergo acetylation similar to that occurring in some histones. Dinitrophenylation of the proteins followed by acid hydrolysis and amino acid analysis indicated that 2 to 3% of the lysine residues present were unavailable for reaction with fluorodinitrobenzene. Extensive enzymatic degradation with trypsin and pronase and subsequent amino acid analysis showed a significant amount of material eluting at the position of epsilon-N-acetyllysine. Recovery and acid hydrolysis of this material generated a peak eluting in the lysine position. In vitro radioactive labeling of calf thymus nuclei with [3H]acetate yielded labeled high mobility group proteins. All of these findings are in accord with the conclusion that high mobility group proteins are acetylated and that acetylation occurs as a postsynthetic modification of these proteins.

Acetylation↗
PubMed1978

Covalent structural analysis of yeast inorganic pyrophosphatase.

The present paper describes the amino acid sequence analysis of the internal and COOH-terminal cyanogen bromide fragments of yeast inorganic pyrophosphatase (Sterner, R., Noyes, C., and Heinrikson, R.L. (1974) Biochemistry 13, 91-99). This information coupled with that derived from earlier structural studies of the enzyme (Sterner, R., AND Heinrikson, R.L. (1975) Arch. Biochem. Biophys. 165, 693-703) provides the complete covalent structure of the pyrophosphatase subunit. The majority of the sequence data was derived from automated Edman degradation of the intact cyanogen bromide fragments and the large tryptic peptides obtained from citraconylated derivates in which cleavages were restricted to arginyl residues. The structural determination was completed by analysis of tryptic and chymotryptic peptides from the decitraconylated fragments. The monomer peptide chain contains 285 amino acid residues and the molecular weight calculated from the sequence analysis is 32,042.

Amino Acid Sequence↗