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G R Reeck

Publications and source records attributed to G R Reeck.

At least 37 records · Page 2Linked to original sources
PubMed1986

Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds.

We have developed a large-scale method for recovering the corn inhibitor of trypsin and activated Hageman factor from a trypsin-agarose column predominantly in the single-chain form. To do so, inhibitor retained by the column was eluted with 1.0 M glycine buffer, pH 2.1. We have used reversed-phase high-performance liquid chromatography to further purify the inhibitor eluted from the trypsin-agarose column by separating the single-chain inhibitor from two-chain inhibitor (a small amount of which is present in the preparation after trypsin-agarose chromatography) and from still smaller amounts of another protein (apparently trypsin) that appears as a contaminant during trypsin-agarose chromatography.

Amino Acids↗
PubMed1985

Physical properties of chicken erythrocyte HMG-1, HMG-2 and HMG-E.

HMG-1, HMG-2 and HMG-E were purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions and subjected to several types of physical measurement. The principal conclusions drawn from the measurements were: none of the proteins has a strong tendency to self-associate, although HMG-1 does weakly self-associate; the frictional properties of HMG-1 and HMG-E (and probably HMG-2) indicate that the proteins deviate significantly from compact, moderately hydrated spheres; and each of the proteins contains approximately 40% helix and little if any beta-pleated sheet.

Animals↗
PubMed1984

Amino acid sequence and secondary structural analysis of the corn inhibitor of trypsin and activated Hageman Factor.

The amino acid sequence of a corn inhibitor for trypsin and activated Hageman Factor (Factor XIIa) was determined by automated Edman degradation from the intact inhibitor and two fragments generated by specific cleavage of the inhibitor. The 112-residue sequence is unique at each position except 91, where both Ala and Glu were found. The structural heterogeneity suggests the occurrence of two genes (possibly allelic) for the inhibitor. Based on analysis of fragments produced by the interaction of the inhibitor with trypsin-agarose, the reactive site peptide bond is identified as Arg 36-Leu 37. There is no strong similarity between the sequence of the corn inhibitor and the sequences published for other serine protease inhibitors. Thus, the corn inhibitor represents a new family of protease inhibitors. Circular dichroism measurements and a theoretical prediction of secondary structure indicate that the inhibitor has helix and beta sheet contents of approximately 40 and 20%, respectively.

Amino Acid Sequence↗
PubMed1983

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins.

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the protein's interaction with purified DNA).

Amino Acids↗
PubMed1983

Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: histones affect the migration of high mobility group nonhistone chromatin proteins.

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.

Animals↗
PubMed1982

Influence of nonhistone chromatin protein HMG-1 on the enzymatic digestion of purified DNA.

The effect of chicken erythrocyte High Mobility Group protein 1 (HMG-1) on the enzymatic hydrolysis of purified double-stranded and single-stranded bacteriophage lambda DNA was studied. HMG-1 was found to inhibit the digestion of single- and double-stranded DNA by S1 nuclease and DNase I, respectively. HMG-I increased the rate of hydrolysis of double-stranded DNA by micrococcal nuclease, particularly at low HMG-1/DNA ratios, and had little effect on the hydrolysis of single-stranded DNA by micrococcal nucleases, even at high HMG-1 DNA ratios. We also present a semi-quantitative estimate that HMG-1 and HMG-2 occur in chromatin from rapidly dividing, cultured rat hepatoma cells at about 8 times the level that they occur in adult rat liver chromatin.

Animals↗
PubMed1982

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA.

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.

Animals↗
PubMed1981

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.

Animals↗
PubMed1980

High mobility group chromosomal proteins isolated from muclei and cytosol of cultured hepatoma cells are similar.

Using sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA, we have purified a protein from the cytosol of an established line of cultured rat hepatoma cells that, by several criteria, is a high mobility group (HMG) protein. Analyses of DNA binding properties, electrophoretic mobilities, amino acid compositions, and immunochemical reactivities reveal that the cytosolic protein is the same protein as HMG-1 isolated from the purified chromatin of the same cell line. Thus, authentic HMG-1 appears to be at least partially responsible for the cytoplasmic fluorescence observed when mammalian cells are stained with fluorescece observed when mammalian cells are stained with fluorescent-labeled, affinity-purified antibodies against HMG-1 [Bustin, M., & Neihart, N.K. (1979) Cell 16, 181-189]. We suggest that HMG-1 cn shuttle between nucleus and cytoplasm, perhaps in response to the nucleus' need for helix destabilizing proteins.

Amino Acids↗
PubMed1980

Chromatographic change in histone H3-H4 preparations during short term storage and its reversal by bisulfite.

Chicken erythrocyte histone H3-H4 preparations stored at 4 degrees in 50 mM sodium acetate (pH 5.0) exhibit altered gel chromatographic properties that are detectable within 2 days. After 5-7 days, material with chromatographic properties of the H3-H4 tetramer is absent, but the H3 and H4 polypeptides are intact, as judged by gel electrophoresis. The change in chromatographic properties results from an oxidation reaction: The change is accelerated by bubbling oxygen through H3-H4 preparations, and it is reversed by exposure to dithiothreitol. Because chicken erythrocyte H3 contains a single cysteine residue the oxidation reaction is intermolecular disulfide bond formation between H3 molecules. High molecular weight aggregates formed by the H3 dimers and some histone H4 probably are the same aggregates observed but unexplained by other workers. The H3-H4 tetramer can be regenerated by exposure of aged preparations to bisulfite, presumably as a result of cleavage of disulfide bonds and formation of S-sulfonated H3.

Animals↗
PubMed1979

Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.

Amino Acids↗