Metabolic stability of nonhistone chromosomal proteins in two different classes of developing rat brain nuclei.

The metabolic behaviour of NHCP was studied in rat brain nuclei from fully differetiated cells and in nuclei from still differentiating cells. Eight-day-old rats were injected intracisternally with [14C]thymidine and [3H]tryptophan and the 3H/14C ratio of the chromatin was followed for a period of 33 days. It was found that in the fraction of differentiated cells this ratio remained unchanged, showing the presence of metabolically stable NHCP. At the same time in the fraction containing nondifferentiated cells a substantial part of the NHCP was turned over, which was indicated by a sharp decrease in their 3H/14C ratio with time.     

DNA-binding nonhistone proteins: DNA site reassociation.

The DNA-binding nonhistone proteins (NHP) have been demonstrated to fractionate the rat genome into protein-bound and unbound DNA sequences. Twenty percent of highly sheared rat DNA [approximately 350 base pair (bp)] can be retained on membrane filters as protein complexes. When extracted from the filter and retitrated with the NHP, a 4- to 5-fold enrichment of binding sites is present in the bound DNA with few, if any, sites detected in the unbound DNA. Rat DNA restricted by EcoRI endonuclease can be fractionated by its DNA-binding NHP retention characteristics. Reassociation kinetics of the bound restricted sequences indicate that 45.6% is a subset of total single-copy sequence of the rat genome an 26.9% is repetitive sequences. Cross hybridization studies indicate the repetitive sequences of the bound DNA are not enriched as much as the slow component of the rat genome. Thus a 4-fold enrichment of a subset of the rat genome has been observed via NHP-DNA interactions.     

Fractionation of chylomicrons by heparin-sepharose chromatography. Characterization of two heparin-binding proteins

Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed.     

Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Degradation of phosphorylated chromosomal nonhistone proteins

In experiments with in vivo 32P-labelled nonhistone proteins of rat liver nuclei it was shown that these components are more sensitive against degradation than the mass of the nonhistone proteins. In the presence of 0.1 mM phenylmethylsulfonylfluoride and 1 mM sodium molybdate, however, they are protected against degradation.     

Purification from cultured hepatoma cells of two nonhistone chromatin proteins with preferential affinity for single-stranded DNA: apparent analogy with calf thymus HMG proteins.

Sequential chromatography on double-stranded DNA and single-stranded DNA columns selects two proteins with marked preference for single-stranded DNA from the complex set of proteins that is released by NaCl from chromatin of cultured hepatoma cells. By a number of criteria, these two proteins appear to be analogous to the calf thymus chromatin proteins HMG-1 and HMG-2.     

DNA sequence selection by tightly-bound nonhistone chromosomal proteins.

Extraction of chicken reticulocyte chromatin with 2.0 M NaCl removed 96% of chromosomal protein and yields two DNA components after dialysis and high-speed centrifugation. The bulk of chromosomal DNA (ca. 99%) is rendered free of protein, and is thus soluble in 10 mM Tris-HCl, pH 8.0. The other component (ca. 1%) displays a high protein/DNA ratio, and is insoluble in 10mM Tris-HCl, pH 8.0. These DNAs can be separated on the basis of their solubilities. Analysis of the reassociation kinetics with total chicken DNA of these DNAs reveals marked differences. Whereas total DNA and the soluble component (DNA-S) have rapidly reassociating components, the insoluble component (DNA-P) is devoid of these components, and is therefore composed completely of unique sequence DNA. Cot 1/2 values indicate that DNA-S is substantially depleted of some DNA-P sequences. We conclude that this segregation, as determined by tightly-bound nonhistone chromosomal proteins, selects a subset of total genomic DNA sequences, and suggests sequence-specific interaction between the tightly-bound nonhistones and DNA.     

RNA associated with nonhistone chromosomal proteins of dog liver

The nonhistone chromosomal proteins were separated on Sephadex G-200 into 3 fractions of which two were associated with 3S RNA. The RNA eluted with fraction I (guanine + cytosine content 54%) is tightly bound to the proteins from which it can be separated only after digestion with pronase. The RNA associated with fraction III (guanine + cytosine content 64%) can be separated from the proteins directly by chromatography on DEAE-Sephadex A 25. No dihydropyrimidines have been detected in any of the two RNAs.     

beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions.

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).     

Fractionation of nonhistone proeins on a column of daunomycin-CH-Sepharose 4B

The molecular weight of a partially purified alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) from the halotolerant yeast Debaryomyces hansenii was estimated to 110,000 by gel filtration. The isoelectric point determined by electrofocusing was at approximately pH 4.4. The enzyme had a broad specificity against phosphomonoesters and also attacked some acid anhydrides. Arsenate, molybdate, and orthophosphate acted as competitive inhibitors. Various metal-binding agents inhibited enzyme activity. A zinc addition almost completely reversed the EDTA inhibition. Magnesium stimulated enzyme activity and was required for maintenance of activity at high concentrations of Na+. Increasing glycerol concentration increased the value of the Michaelis constant (Km) and decreased the maximum velocity (V). Solutions equimolar in KCl and NaCl stimulated enzyme activity by increasing V, whereas the Km was almost unaffected by salt concentration. Enzyme extracted from cells cultured at low salinity was indistinguishable from that of cells grown in the presence of 2.7 M NaCl with respect to several criteria.     

Fractionation of proteins with two-sided electro-ultrafiltration

Downstream processing is a major challenge in bioprocess industry due to the high complexity of bio-suspensions itself, the low concentration of the product and the stress sensitivity of the valuable target molecules. A multitude of unit operations have to be joined together to achieve an acceptable purity and concentration of the product. Since each of the unit operations leads to a certain product loss, one important aim in downstream-research is the combination of different separation principles into one unit operation. In the current work a dead-end membrane process is combined with an electrophoresis operation. In the past this concept has proven successfully for the concentration of biopolymers. The present work shows that using different ultrafiltration membranes in a two-sided electro-filter apparatus with flushed electrodes brought significant enhancement of the protein fractionation process. Due to electrophoretic effects, the filtration velocity could be kept on a very high level for a long time, furthermore, the selectivity of a binary separation process carried out exemplarily for bovine serum albumin (BSA) and lysozyme (LZ) could be greatly increased; in the current case up to a value of more than 800. Thus the new two-sided electro-ultrafiltration technique achieves both high product purity and short separation times.     

Synthesis and application of two reagents for the introduction of sulfhydryl groups into proteins

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate-oxidized N termini of proteins, provided that the N-terminal residue is serine or threonine. 3-(Phenyldithio)propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.