Viktor Holoubek

Dependence of the composition of the protein moiety of nuclear ribonucleoprotein particles on the extent of particle purification as studied by electrophoresis including a two-dimensional procedure

Extraction with 0.1 M NaCl in 0.01 M Tris-HCl buffer, pH 8.0 releases from liver nuclei 30-40-S ribonucleoprotein particles containing newly synthesized RNA. Separation of the protein moiety of these particles by acid-urea gel electrophoresis depends on the concentration of beta-mercaptoethanol in the buffer used for the solubilization of the particles. At low concentration or with short time of solubilization, only a polypeptide chain with apparent molecular weight 38 000 penetrates into the gel and can be detected by electrophoresis. By introduction of two-dimensional polyacrylamine gel electrophoresis, we succeeded to separate the protein moiety of these particles into a core group of 4 major and 6 minor polypeptides with molecular weights ranging from 38 000 to 50 000 and a second group of 19 polypeptides ranging in molecular weight from 50 000 to 120 000. The composition of the protein moiety of these particles is dependent on the extent of purification. Polypeptides with molecular weight below 50 000 represent 55% of the total protein of particles purified only by centrifugation through a 15-30% sucrose gradient. If the particles were first purified by gel filtration through Bio-Gel A-50m followed by centrifugation in sucrose gradient, the low molecular weight proteins represent 80% of all the proteins of the particles. The purification removed selectively the minor high molecular weight polypeptides without resulting in any extensive release of the four major polypeptides with molecular weight below 50 000 which form a stable core particle. By repeated purification it is possible to strip the particles of the high molecular weight polypeptides even further. An increase in the NaCl concentration of the extraction buffer to 0.35 M will extract additional 30-40-S particles associated with a newly synthesized RNA from the cell nucleus. These particles contain the same polypeptides as particles extracted at lower salt concentration. Extraction with 0.1 M and 0.35 M NaCl at pH 8.0 removed from the nucleus approximately 55% of all RNA labeled in 30 min after intraperitoneal injection of [3H] orotic acid to the rats.

Similarity of the 0. 35 M NaCl soluble nuclear proteins and the nonhistone chromosomal proteins

Abstract Nuclear proteins which are extractible with 0. 35 M NaCl and the nonhistone chromosomal proteins which are not soluble at this salt concentration separate on analytical polyacrylamide gel electrophoresis into the same 11 main fractions. Only one fraction (less than 7% of the total proteins) is specific for the nonhistone chromosomal proteins and is not found among the proteins soluble in 0. 35 M NaCl.

Binding of 3'-methyl-4-dimethylaminoazobenzene to nuclear ribonucleoprotein particles.

Abstract The binding of 3 H-3′-methyl-4-dimethylaminoazobenzene to various nuclear fractions from liver of rats administered with the dye was studied. The highest specific radioactivity was found in a nonhistone nuclear protein fraction which was extracted with 0.1 M Tris-HCl buffer,pH 7.6, and contained a large amount of proteins identical to those of nuclear ribonucleoprotein particles. The ribonucleoprotein particles isolated by the combination of extractions at different pH and sucrose gradient centrifugation also showed the highest specific radioactivity.

RNA associated with nonhistone chromosomal proteins of dog liver

Abstract 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.

Fractionation of nuclear proteins by extraction with solutions of different ionic strength

Abstract 1. 1. Rat liver nuclear proteins were fractionated according to their solubility in solutions of different ionic strength. The extracted fractions separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis into 17–22 main polypeptide bands (with an apparent molecular weight from 98,000 to 14,600) six of which were histones. 2. 2. Only a few of the proteins were selectively extracted in one given fraction. Most of the bands were found in the electrophorograms of all fractions but in different quantitites. 3. 3. The proteins of the informofers were in part solubilized by extraction with 0.1 M Tris-HCl buffer pH 7.6 and completely solubilized by extraction with 0.35 NaCl. 4. 4. Only eight bands were found to be exclusively present in the electrophorograms of proteins removable from the nucleus by extraction with 0.14 M NaCl-0.01 M sodium citrate. Other proteins extracted at this salt concentration had identical mobility with the bands found in electrophorograms of chromosomal proteins.

Protein composition of liver nuclear ribonucleoprotein particles of rats fed carcinogenic aminoazo dyes

Abstract The feeding of carcinogenic 3′-methyl-4-dimethylaminoazobenzene to rats alters the protein composition of liver nuclear 30S ribonucleoprotein particles which are proposed to be involved in the processing and transport of the newly synthesized RNA. After 10 weeks of feeding of the carcinogenic aminoazo dye, one of the major proteins is missing from these particles but not from the particles isolated from liver of animals fed with noncarcinogenic 4-aminoazobenzene. In all the groups of rats studied, the RNA associated with the isolated particles was of high specific activity.

Separation of proteins of nuclear ribonucleoprotein particles by high-performance liquid chromatography

Abstract High-performance liquid chromatography (h.p.l.c.) is used to fractionate the proteins of the 30–40 S nuclear ribonucleoprotein particles. The major core proteins of the particles are eluted from a SynChropak AX-300 anion-exchange column before the more acidic higher-molecular-weight minor particle proteins. Each of the three major core proteins which can be separated from the other particle proteins by preparative polyacrylamide gel electrophoresis are eluted from the SynChropak AX-300 column as one peak. Isoelectric focusing separates each of these three apparently homogeneous peaks into a series of charge isomers ranging in isoelectric pH (pI) from 5.5 to 9.0. The core proteins of the ribonucleoprotein particles have a strong affinity to each other and form aggregates. The elution of each of the charge isomers of the three major proteins in one peak and their elution from an anion exchanger before the elution of the more acidic higher-molecular-weight minor proteins of the nuclear ribonucleoprotein particles is explained by the formation of these aggregates. The separation of the total proteins of the nuclear ribonucleoprotein particles by h.p.l.c. is similar to the separation which can be obtained by preparative electrophoresis but the l.c. technique is simpler, substantially quicker, and adaptable to large-scale preparation.

Electrophoretic spectra of nuclear proteins from embryos ofXenopus laevis

SummaryThe changes in saline-soluble, 0.35 M NaCl-soluble and the residual fraction of nuclear proteins during early development ofXenopus were studied by analytical electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The fractions were obtained by consecutive extraction of nuclei from the blastula, neurula and tail-bud stage of development. No qualitative and only limited quantitative differences were found when the proteins of any of the three fractions isolated from the neurula stage were compared with the proteins of the corresponding fraction isolated from the tail-bud stage. But the electrophoretic pattern of each of the three fractions of the nuclear proteins from the blastula stage differs significantly from the electrophoretic pattern of the same fraction isolated from the neurula or tail-bud stage. Compared with the blastula stage, in the two later stages the relative amounts of chromosomal proteins with apparent molecular weights below 30,000 are decreased. Proteins which migrate in electrophoresis in the positions of the very lysine-rich histones and of the proteins of the nuclear ribonucleo-protein particles are indicated among the chromosomal proteins of the blastula stage, and are visible as strong bands in the electrophorogram of 0.35 M NaCl-soluble proteins extracted from neurula or tail-bud stage nuclei.

Isolation and characterization of the predominant protein in nuclear ribonucleoprotein particles from rat liver

The predominant protein of the nuclear ribonucleoprotein particles of rat liver was isolated by polyacrylamide gel electrophoresis. The polypeptide represented 35% to 40% of the total mass of the protein moiety. Its molecular weight was estimated to be 38 000 and its NH2-terminal residue was found to be threonine. The amino acid composition is unique in having a high content of glycyl residues (20%) and NG-dimethylarginine (14% of total arginyl residues).

Early effects of carcinogenic aminoazo dyes on the protein patterns and metabolism in rat liver

Abstract 1. 1. The feeding of the carcinogenic 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) resulted in a pronounced stimulation of the incorporation of 14C amino acids into nearly all fractions of nuclear proteins of rat liver cells with one exception. The incorporation was suppressed in the fraction which contains the proteins of the nuclear ribonucleoprotein particles which are involved in the processing of newly synthesized nuclear RNA. 2. 2. Only limited quantitative changes were observed in the electrophoretic patterns of liver nuclear proteins of animals fed 3′-Me-DAB. 3. 3. In the cytoplasm the feeding of 3′-Me-DAB induced high increase in the amount of glutathione-S-transferase and of a specific microsomal hemoprotein. A smaller increase in the amounts of both proteins was observed also in animals fed non-carcinogenic 4-aminoazobenzene. 4. 4. The induced microsomal hemoprotein differed in mol. wt from the microsomal hemoproteins induced by phenobarbital and by 3-methylcholanthrene. 5. 5. Increased glutathione-S-transferase was identified to be identical with the minor cytoplasmic binder of 3′-Me-DAB.