Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro.

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.     

A low molecular weight calf pineal peptide with an inhibiting effect on the growth of L1210 and HL60 cells.

The peptide isolated by us from calf pineal gland causes a reduction of RNA synthesis in vitro in L1210 and HL60 tumoral cells. This peptide also causes inhibition of cell proliferation; the cell viability is not modified. The effects are dose-dependent and reversible.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase 'C' with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to beta- and gamma-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K+ at 2-20 mmol/l, for Mn2+ at 0.8 mmol/l, for Mg2+ at 4.0 mmol/l, the pH optimum is 8.0. Its Km is 1.5 mumol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

High molecular weight glycosylated protease in calf brain. Purification and partial characterization

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K⁺ at 2–20 mmol/l, for Mn²⁺ at 0.8 mmol/l, for Mg²⁺ at 4.0 mmol/l, the pH optimum is 8.0. Its K_m is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

Evidence that DNA polymerase-alpha of calf thymus contains a subunit of molecular weight 155000.

Small samples of the 8-S species of enzymes (A1 and A2) occurring in the DNA polymerase-alpha fraction of calf thymus, have been extensively purified using non-denaturing (normal) polyacrylamide gel electrophoresis. When peak fractions of activity on normal gels were subjected to dodecylsulphate-polyacrylamide gel electrophoresis, a polypeptide at 155000 correlated with polymerase activity. Samples of the 7.3-S (C) enzyme prepared from A2 by treatment with 2.4 M urea or isolated directly without exposure to urea, also showed the presence of a 155000-Mr polypeptide. It is concluded that the 7.3-S (C) enzyme, of previously estimated molecular weight 155000-170000, is a single polypeptide and that the 8-S enzymes A1 and A2 contain an additional subunit of 50000-70000 molecular weight.     

Purification and immunochemical properties of human low molecular weight kininogen.

A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.     

Purification and characterization of rat low molecular weight kininogen

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.     

The ataxia telangiectasia clastogenic factor is a low molecular weight peptide

Summary The clastogenic factor in the plasma of ataxia telangiectasia (AT) patients and in conditioned medium from AT skin fibroblast cultures is a peptide with a molecular weight in the range of 500 to 1000. No clastogenic activity could be demonstrated in extracts of cultured AT fibroblasts.     

Purification of low molecular weight copper proteins from copper loaded liver.

Purification of low molecular weight copper binding proteins from the livers of copper loaded male rats was achieved by sequential ultracentrifugation (186,000g, 2h), ultrafiltration (Amicon PM 30), gel filtration (Sephadex G-75) and anion exchange chromatography (DEAE - Biogel A) of soluble tissue extracts. The three major copper-associated polypeptides obtained which had molecular weights of about 7000, 9,000, and 12,000 daltons contained approximately 2.5g atoms of copper per mole. Amino acid analyses indicated a similarity between these proteins and the copper protein ‘L-6D’ isolated earlier from livers of Wilson's disease patients and distinguished them from metallothioneins which have been isolated from animals administered other trace metal ions.     

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions.

1. A soluble protein with a molecular weight of 11-12-10(3) has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH4OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP-Pi exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 mumol-min-1-mg-1 of the factor protein. However, the NH4OH-EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20-40 mug factor per mg particle). 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. The factor is inhibited by mercurials and N-ethylmaleimide. The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. The NH4OH-EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.     

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 μg/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.