Azospirillum lipoferum is a soil bacterium known for its ability to colonize roots and to promote plant growth. Recently, a laccase-like polyphenol oxidase activity has been reported in a strain isolated from the rhizosphere of rice. Purification to apparent homogeneity of A. lipoferum laccase was achieved after cell disruption by sonication, protein solubilization by Triton X-100, acetone and protamine precipitation, hydroxyapatite column chromatography in the presence of urea (5 M) and NaCl (2 M), and finally by conventional hydroxyapatite column chromatography. After this final step, laccase was in an inactive form but could be reactivated by adding sodium dodecyl sulfate (SDS). Based on gel electrophoresis results, we suggest that native A. lipoferum laccase is composed of a catalytic polypeptide chain with low molecular mass (16.3 kDa) and one or two regulatory/structural chains with high molecular mass (81.5 kDa). The purified enzyme was found to be thermostable up to 70°C for 10 min, had an optimal pH of 6.0, and was inhibited by tropolone, a known inhibitor of metal-containing enzymes. Although the natural substrate of this enzyme is unknown, its biochemical characterization may facilitate further investigations on the ecological role of this laccase in the process of root colonization by A. lipoferum .

Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum

Carcinogenesis by aromatic amines

The changes in protein synthesis of salivary glands of Drosophila resulting from a brief exposure at 37 degrees have been analyzed on sodium dodecyl sulfate--acrylamide gels. In D. melanogaster and D. hydei this treatment induces nine and six new puffs, respectively, in the polytene chromosomes. After 20 min treatment seven new proteins are synthesized by the glands of D. melanogaster and six by those of D. hydei as detected by [35S]methionine labeling. Other agents, e.g., recovery from anaerobiosis, induce the same puffs and the same proteins. The extent of protein induction and the degree of puff induction are related to the severity of the temperature treatment. The new proteins are detected after 10 min treatmene at 37 degrees and their synthesis is inhibited by actinomycin D. Actinomycin D added 5 min after the start of temperature treatment has little effect on subsequent protein synthesis. The induced proteins are not tissue specific. Electrophoretic differences of two proteins exist between D. melanogaster and D. simulans, encouraging attempts to map the proteins' gene loci and to test directly whether or not the puffs code for them.

https://www.pnas.org/content/pnas/72/9/3604.full.pdf

Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila.

Publisher Summary This chapter describes a two-dimensional polyacrylamide gel electrophoretic fractionation technique. The development of a high-resolution, two-dimensional electrophoresis system provides a technique suitable for the analysis of the complex group of chromosomal proteins. The technique combines isoelectric focusing in the first dimension and sodium dodecyl sulfate (SDS) slab-gel electrophoresis in the second dimension. This chapter describes the two-dimensional electrophoresis procedure and some problems that are encountered with samples prepared from chromatin. The conditions for labeling cell protein to a high specific activity with radioactive methionine and the conditions for preparation of radioactive cell extracts are described. The limitation of the technique and difficulties in interpretation include (1) loss of basic proteins, (2) requirement for isotopically labeled proteins for high-sensitivity analysis, and (3) definition of a chromosomal protein.

Two-dimensional polyacrylamide gel electrophoretic fractionation.

1
A two-dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate electrophoresis has been used to analyse the components of 32P-labelled nonhistone protein fractions obtained by the chromatography of salt-urea dissociated chromatins on hydroxyapatite.

2
In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelectric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species.

3
Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific.

4
It was found that chromatography on QAE-Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion-exchange material is not entirely dependent on the overall charge of the polypeptide.

5
Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03258.x

The Heterogeneity of Mouse‐Chromatin Nonhistone Proteins as Evidenced by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis and Ion‐Exchange Chromatography

The non-histone proteins of chromatin, their isolation and composition in a number of tissues.

Publisher Summary This chapter provides methods for isolation and characterization of chromosomal nonhistone proteins. The methodology is based on the ability of hydroxyapatite to act as an ion exchange medium for macromolecules in the presence of high ionic strength and urea and consists of a single column procedure, yielding high recoveries of chromatin constituents. The methodology utilizes combination of salt and urea, which has been used as a solvent both for the dissociation and solubilization of chromatin and its specific reconstruction from previously isolated components. The complexity of nonhistone proteins is demonstrated by the presence of polypeptides of differing isoelectric points in sodium dodecyl sulfate–protein complexes that appear as individual components in the one–dimensional sodium dodecyl sulfate system. In addition, the two-dimensional separation procedure indicates that there is a considerable range of rapidly phosphorylated proteins in chromatin. The proteins comprising fractions H2 and H3 are markedly different because the latter possess more acidic isoelectric points. However, some overlap probably does occur between these two fractions because the proteins designated as P1 and P2 appear common to both, the more phosphorylated forms in the liver being present in fraction H3.

[12] Methods for isolation and characterization of chromosomal nonhistone proteins: Fractionation of chromatin on hydroxyapatite and characterization of the nonhistone proteins by ion exchange chromatography and polyacrylamide gel electrophoresis

R.J. Krauze

Papers

The non-histone proteins of chromatin, their isolation and composition in a number of tissues.

[12] Methods for isolation and characterization of chromosomal nonhistone proteins: Fractionation of chromatin on hydroxyapatite and characterization of the nonhistone proteins by ion exchange chromatography and polyacrylamide gel electrophoresis