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.     

Solubilization of nonhistone chromosomal proteins by carboxymethyldextran for chromatography

Chromosomal proteins extracted from calf thymus by 0.35 M NaCl were insoluble at the low salt concentration needed initially for ion-exchange chromatography. However, the addition of high-affinity carboxymethyl-dextran rendered the precipitated proteins soluble, permitting them to be fractionated by elution from DEAE-Sephacel. In this way, the separation of HMG-1 from HMG-2 was achieved, as well as a partial fractionation of the low mobility group proteins, under conditions that did not disturb the native conformation of the proteins.     

Negatively charged phosphopeptides of nucleolar nonhistone proteins from Novikoff hepatoma ascites cells.

To characterize the sites phosphorylated by endogenous kinases, phosphopeptides of isolated nucleolar nonhistone proteins were analyzed. Major phosphoprotein bands C23 and B23 were ³²P labeled $\text{in vitro}$ and electrophoretically isolated. Tryptic phosphopeptides were resolved by DEAE-Sephadex chromatography into fractions A, B and C for band C23 and α and β for band B23. Each of these fractions contained phosphoserine, had a distinct amino acid composition of 49–65% glx + asx and 4–11% lys, and had molecular weights of 7–11,000 determined on Sephadex G50. These data indicate that two nucleolar nonhistone proteins have similar phosphorylated regions of high negative charge density.     

Affinity chromatography of nonhistone chromosomal proteins from lymphocytes on DNA-agarose columns

A two step procedure is presented consisting of hydroxyapatite and DNA-agarose chromatography which allows the isolation of nonhistone chromosomal proteins with different affinities towards single stranded DNA. The application of this fractionation scheme to nonhistone chromosomal proteins from bovine lymphocytes is described.     

Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography

Phosphoproteome studies are hampered by the lack of methods which allow a comprehensive and fast analysis of intact phosphoproteins. Here we describe an immobilized metal-affinity chromatography (IMAC)-based technique for the enrichment of phosphorylated proteins, which allows recovery of up to 90% of phosphoproteins. This technique is compatible with 2-DE and can be applied to cultured cells and tissues.     

Carrier protein effects on DNA-cellulose chromatography of putative steroid receptors.

In the absence of carrier proteins, putative androgen receptors elute from DNA-cellulose in the range of 120 to 190 mM NaCl. However, in the presence of lysozyme, most of the receptor elutes in the range of 200 to 230 mM NaCl. This is the same range in which the lysozyme itself, a basic protein, elutes after being chromatographed in the same manner. Moreover, at low ionic strength, lysozyme also increases the sedimentation velocity of both androgen and estrogen receptors. In contrast, bovine serum albumin neither adheres to DNA-cellulose nor alters the sedimentation properties of these proteins. The lysozyme effects can account for some discrepancies reported in the literature. Thus, for qualitative elution studies, the use of lysozyme as a carrier protein is not advised, although its direct interaction with receptors might facilitate quantitative fractionation.     

Hydroxyapatite high-performance liquid chromatography: column performance for proteins

Hydroxyapatite columns for high-performance liquid chromatography that are reusable for a long time were developed; performance tests were carried out by using several types of protein. Using a high flow rate, a sharp chromatographic peak can be obtained for a homogeneous molecule. A very high level of chromatographic separation can be achieved by decreasing the slope of the gradient and increasing the total column length at the same time.     

Nuclear nonhistone proteins in mouse teratocarcinomas

Summary The nonhistone protein pattern of four murine teratocarcinomas with different capacities for differentiation were compared: a multidifferentiated teratocarcinoma OTT2289, a nondifferentiated teratocarcinoma OTT2158, a teratocarcinoma-derived rhabdomyosarcoma TDR114, and a teratocarcinoma-derived neuroblastoma TDN2151. Their nonhistone proteins (NHP) were separated by differential salt extraction and hydroxyapatite chromatography into three fractions, NHP-I, NHP-II and NHP-III. Comparison of the NHP fractions by twodimensional gel electrophoresis in combination with a sensitive silver staining method reveals that there are several tumour line specific proteins in each NHP fraction. We suggest that specific NHP, which can be used as biochemical markers for each of the four investigated tumour lines, may be involved in cell lineage specific control of gene expression.     

Large-scale ion-exchange column chromatography of proteins. Comparison of different formats

This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns.     

Organ discrimination through organ-specific nonhistone chromosomal proteins

Prefractionation of chromosomal proteins in 5 m urea with stepwise increase in NaCl molarity has been used to facilitate the examination of nonhistone chromosomal proteins isolated from various rabbit tissues. Electrophoretic analysis on polyacrylamide gels under denaturing conditions of the protein fractions derived from brain, liver, heart, and submandibular salivary gland chromatins displays reproducible compositional differences in nonhistone chromosomal proteins. The enzymatic removal of 48% of protein-bound phosphate with alkaline phosphatase does not significantly alter the electrophoretic mobility of these proteins. With the present technique, it is estimated that chromatin polypeptides (of average M_r 100,000) occurring in greater than 3 × 10⁴ copies per genome can be detected. At this level of sensitivity, a significant fraction of total nonhistone chromosomal proteins manifests organ specificity.     

Proteomic analysis of mammalian basic proteins by liquid-based two-dimensional column chromatography

To develop a standard method for separating highly basic proteins in mammalian cells, we established a 2-D LC separation system coupled with chromatofocusing/nonporous RP column chromatography (CF/NPRPC) in a ProteomeLab PF2D system. After standardizing conditions for 2-D LC, a 2-D liquid protein map of uninfected macrophage proteins with pH range 8.3-11.3 was constructed, and then compared with a macrophage protein map made after infection with Candida albicans. The results demonstrate that 2-D LC offers both high resolution and reproducibility for separation of highly basic, macrophage proteins. After protein identification using a nano 2-D LC-MS/MS Proteomics Solution System, quantitative determination of the changes in the differentially expressed proteins (e.g., galectin-3) in C. albicans-infected macrophages was also accomplished by measuring the peak area of the chromatogram in 2-D LC. The result from this measurement of galectin-3 expression shows a 3.41-fold decrease in the infected macrophage cells, which was further confirmed by that from the RT-PCR of mRNA of galectin-3. Thus, 2-D LC coupled with CF/NPRPC could be applicable to common analysis of highly basic proteins in a high-throughput manner.     

The removal of exogenous thiols from proteins by centrifugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.