Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein

In order to assess the selectivity of the distribution patterns of individual nonhistone chromosomal proteins (NHC proteins), immunofluorescent staining experiments were performed on Drosophila polytene chromosomes. Antisera have been prepared against three individual NHC proteins which were isolated by sequential preparative slab gel isoelectric focusing and SDS polyacrylamide gel electrophoresis. In two cases, immunofluorescent staining of the chromosomes indicated a specific limited distribution pattern; apparently the antigen in each case is present at a reproducible and distinct subset of chromomeres. This type of pattern has also been obtained with antisera prepared against molecular weight subfractions of NHC proteins (Silver and Elgin, 1977). Each selective fluorescence distribution pattern obtained so far is reproducible and unique to the antiserum under study. In a third case, an antiserum caused prominant staining at dense chromomeres and the chromocenter in a pattern mimicking DNA (and presumably histone) distribution. Indirect radioimmunostaining of SDS and isoelectric focusing gels on which total NHC proteins had been separated confirmed that this antiserum reacted specifically with a protein(s) of molecular weight 21,000 D and pI 5.2. The data in conjunction with absorption experiments indicates that the chromosomal staining is due to an interaction of antibodies with NHC protein(s) and not with histones. This finding suggests that at least one major acidic NHC protein plays a very general role (comparable to that of the histones) in maintaining chromatin structure.     

Fractionation of chromosomal proteins of pea seedlings using procedures which avoid denaturation

Chromatin was prepared from the buds and cotyledons of Alaskapea seedlings. The dissociated chromosomal components in thepresence of 2 M NaCl and 5 M urea were completely fractionatedinto DNA and proteins with a Bio-Gel A50 column. The proteinswere recovered by (NH₄)₂SO₄ and further fractionated into histonesand non-histone proteins using a Bio-Rex 70(Na⁺) column. Thedifference in the ratios of histones to non-histone proteinsbefore and after chromatography with the Bio-Rex 70 was lessthan 10%. The histones and non-histone proteins thus preparedshowed typical protein absorption spectra. Polyacrylamide gelelectrophoresis of histones showed that the histone compositionsin buds and cotyledon were similar, but the amount of HI histoneswas a little less in cotyledons than in buds. Unlike histones,non-histone proteins fractionated by SDS-polyacrylamide gelelectrophoresis indicated distinct differences between the twotissues. Buds had more heterogeneous non-histone proteins, atleast 13 polypeptides, than cotyledons did. On the other hand,non-histone proteins of cotyledons showed less heterogeneityand lacked proteins of high molecular weight which were foundin buds. (Received May 6, 1976; )     

Isolation of all proteins from the 50S subparticles of ribosomes of Escherichia coli

The paper proposes a method of preparative isolation of all proteins from the 50S subparticle of E. coli ribosomes. The method is based on (1) preliminary fractionation into protein groups and ribonucleoprotein particles by a consecutive treatment of the 50S particles with increasing LiCl concentrations, and (2) chromatographic separation of protein groups on DE- and CM-cellulose and gel-filtration of separate fractions. The method allows to obtain any protein required for studies in preparative amounts avoiding many chromatographic stages. A detailed scheme of isolation of all proteins is given together with quantitative data of yields of individual proteins calculated per 6 g of the 50S subparticles.     

Identification of a glucocorticoid-sensitive histone protein from mouse fibroblast nuclei.

The acid-soluble proteins from mouse fibroblast nuclei show a typical elution profile for histone proteins on Bio-Rex 70 columns. Glucocorticoid treatment of cells growing $\text{in}\text{vitro}$ decreases labeled amino acid incorporation into one specific histone region, corresponding to a peak of a lysinerich histone. The single histone affected is present in only small amounts in the cell; it normally represents about 6% of the H₁ protein, and less than 1% of the total histones. As measured by total protein nitrogen of the affected peak, glucocorticoid inhibition of this protein is first apparent between one and two hours after the start of steroid treatment.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

Free-flow electrophoresis of proteins in phenol-containing solvents at various pH values

1. Several proteins were found to migrate when subjected to free-flow electrophoresis in buffered phenol-ethanediol-water (3:2:3, w/v/v) solvent mixtures. Mobility of these proteins changed with changing pH (apparent) values of this medium. A pH value of zero mobility for each individual protein could be estimated. 2. Founded on these observations, a high-voltage electrophoresis method in free-flowing buffer films was worked out. The method as presented here was particularly suitable for the separation of proteins on a preparative scale. Application of this and other protein fractionation techniques in dissociating media for the investigation of structural and other insoluble proteins was discussed.     

Purification and characterization of wheat germ DNA topoisomerase I (nicking-closing enzyme)

Wheat germ contains an enzyme capable of removing supercoils from circular DNA. We have purified this enzyme using Polymin P fractionation, ammonium sulfate precipitation, and chromatography on Bio-Rex 70 and phenyl-Sepharose. Renaturation after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels shows that topoisomerase activity is associated with a polypeptide with a Mr = about 111,000. The enzyme is similar to other eukaryotic type I DNA topoisomerases (nicking-closing enzymes) by the following criteria: it is capable of increasing or decreasing the topological linking number of covalently closed DNA substrate; it is capable of restoring an equilibrium distribution of linking numbers to DNA substrate with a single unique linking number; and it does not require magnesium ion or ATP for activity.     

Z protein: Isolation and characterization of multiple forms in rat liver cytosol

The Z protein fraction of rat liver cytosol contains one or more proteins which have been associated with organic anion transport, fatty acid metabolism, and aminoazodye binding. To study the possible identity of these proteins and investigate their function, Z was purified using ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Three protein fractions were obtained (pI 5.2, 6.0, 7.3) which reacted specifically with anti-Z IgG. These three fractions were homogenous as determined by several electrophoretic systems. Monospecific antibody prepared against two of the proteins cross-reacted specifically with all three. Each fraction bound BSP with different affinity; acidic Z bound the least BSP. The molecular weight of each fraction was 12,500 as determined by SDS-gel electrophoresis. Amino acid analyses of the three Z protein bands were virtually identical. Heterogeneity in Z probably results from interaction of the protein with ampholytes or exogenous ligands.     

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the ³²P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60^src kinase.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

Isolation of complement protein D from urine of patients with Fanconi's syndrome

Complement protein D is the least abundant of all complement proteins and, thus, one of the most difficult to purify. We report a new method for obtaining pure D from urine of patients with Fanconi's syndrome. The method is simple and allows the purification of milligram amounts of D within a few days. It involves three chromatographic steps using Bio-Rex 70, hydroxylapatite HPLC, and reverse-phase HPLC. Protein D purified by this method is suitable for both functional and structural studies.     

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.     

Comparison of nonhistone chromosomal proteins from neuronal and glial chromatin by isoelectric focussing and microdisc electrophoresis.

The present study describes optimal conditions for preparative fractionation of nonhistone chromosomal proteins from neuronal and glial nuclei.We have detected about 1200 nonhistone chromosomal proteins. Microgels of glial origin contained 20% more protein bands than those of neuronal origin. However, neuronal bands prevailed clearly in the acidic respectively in the high-molecular range. Amino acid analysis confirmed the observed heterogeneity.     

Conservation of nonhistone chromosomal proteins through dipteran evolution

We have investigated the possibility that some high molecular weight nonhistone chromosomal (NHC) proteins may have been conserved through the evolution of two distantly related diptera-Drosophila melanogaster and Sciara coprophila. Antisera produced against three NHC protein subfractions were analyzed for cross-reactivity with Sciara polytene chromosomes. The indirect immunofluorescent staining technique used couples an assay for immunologic cross-reactivity with an assay for the in situ distribution of the proteins under study. The results indicate that the - and NHC protein antigens have been conserved since the divergence of Drosophila and Sciara, while the Drosophila NHC protein antigen is not present on Sciara chromosomes. In one case, with anti-- serum, we have identified a highly conserved, very high molecular weight NHC protein (or class of proteins) which appears to interact strongly with all chromatin in a manner which is not DNA sequence-specific. In the second case, with anti- serum, we have identified an NHC protein which may have evolved an additional function(s) in Sciara relative to its function(s) in Drosophila.     

A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.     

Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Evaluation of immobilized boronates for studies of adenine and pyridine nucleotide metabolism

Three different immobilized boronates were compared with respect to their possible utility in studies of adenine and pyridine nucleotide metabolism. These included boronate derivatives of polyacrylamide, Sepharose, and the cation-exchange resin, Bio-Rex 70. The relative binding affinities, binding capacities, and elution properties were compared. Under the conditions utilized, the Sepharose and Bio-Rex 70 derivatives selectively retained nucleotides containing two or more sets of 1,2 cis-diol groups. Since the bulk of cellular nucleic acids contain but a single set of 1,2 cis-diol groups or less, these boronate derivatives are very useful for the isolation of the acid-soluble nucleotides, NAD and diadenosine 5',5"'-tetraphosphate, and for the acid-insoluble polymer, poly(ADP-ribose). The Bio-Rex 70 derivative had a particularly useful combination of binding selectivity, capacity, and elution characteristics. Specific applications of this resin for studies of NAD and poly(ADP-ribose) metabolism are presented.