Influence of Buffers, Protein Concentration and Ionic Strength upon the Dissociation of <i>Pila</i> Haemocyanin

Cox, Jos A.; Elliott, Francis G.

doi:10.1111/j.1432-1033.1973.tb03134.x

Cited by 10 publications

(14 citation statements)

References 19 publications

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“…Inhibition of transcription by these agents [35,36] does not prove that RNA synthesis depends on initiations in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…The use of drugs which inhibit the attachment of polymerase to DNA as an approach to the problem of initiation has to be taken with scepticism, since all these agents (heparin [35], high salt, aurintricarboxylic acid [36], rifamycin AF/013 [37,38]) might also interfere with the binding of other chromosomal proteins to DNA and thereby indirectly interfere with the transcriptional process. Inhibition of transcription by these agents [35,36] does not prove that RNA synthesis depends on initiations in vitro.…”

Section: Discussionmentioning

confidence: 99%

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On the Activity of RNA Polymerase B in Lysates from Ehrlich Ascites Cells

Dreyer

Hausen

1978

European Journal of Biochemistry

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Transcription by endogenous RNA polymerase Bin lysates of Ehrlich ascites cells was investigated. The enzyme exhibits two salt optima at 0.025 M and at 0.3 M (NH,),SO, respectively. Preincubation of the cells with the nucleoside analogue 5,6-dichloro-1 -p-D-ribofuranosylbenzimidazole results in an inactivation of the polymerase molecules active under condition of low salt. This indicates two functional states of the enzyme in vivo.Initiations of RNA chains by polymerase B do not occur in vitro as judged by the incorporation of P-32P]GTP. Thus the two functional states seem to be both elongating polymerase molecules.Polymerase B does not occur in the lysates in a state ready to initiate on an exogenous template, in contrast to polymerase A and C which do occur in free form. Pretreatment with dichlororibofuranosylbenzimidazole in vivo does not result in an accumulation of free polymerase B.The ability of isolated nuclei to synthesize RNA suggests that they might be a suitable system in vitro for a detailed analysis of the processes involved in transcription. They offer certain advantages over alternative systems in vitro consisting of purified polymerase and isolated DNA or chromatin, since it is still unknown whether the purified enzymes retain all their functions during the purification procedure. RNA polymerases contained in whole nuclei presumably reflect the situation in vivo more accurately than the isolated enzymes. Secondly, chromatin is the natural template in isolated nuclei as well as in vivo, whereas naked DNA or even isolated chromatin may exhibit properties different from the situation in vivo.The activity of three classes of polymerases in isolated nuclei may be distinguished on the basis of their different sensitivity to a-amanitin. We have focussed our attention on the properties of RNA polymerase B, the enzyme responsible for the synthesis of messenger RNAs and their precursors.

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“…Inhibition of transcription by these agents [35,36] does not prove that RNA synthesis depends on initiations in vitro.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

On the Activity of RNA Polymerase B in Lysates from Ehrlich Ascites Cells

Dreyer

Hausen

1978

European Journal of Biochemistry

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“…These interactions may result in activation or inhibition of enzyme activity [36]. For example, heparin inhibits the activity of some enzymes such as lysosomal hydrolases [36], DNA polymerase [37], RNA polymerase [38], ribonuclease [39], ornithine decarboxylase [40] and casein kinase I1 [41, 421. Other enzymes are activated by heparin, such as lipoprotein lipase [43], tyrosine hydroxylase [44], rabbit skeletal muscle and rat liver phosphorylase kinase [45,461 and the p68 kinase. The nature of interaction of heparin with all these different enzymes is not very clear.…”

Section: The Signgicance Of' the P68 Kinase Activation By Heparinmentioning

confidence: 99%

The double‐stranded RNA‐dependent protein kinase is also activated by heparin

Hovanessian

Galabru

1987

European Journal of Biochemistry

114

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The double-stranded(ds)-RNA dependent protein kinase from human cells is a M , 68 000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin 111, heparin-binding protein. However, when antithrombin I11 is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-a~ido-[a-~~P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.A protein kinase activity dependent on double-stranded (ds)RNA is enhanced in mouse and human cells following treatment with interferon [l -41. The protein kinase from human cells is a Mr-68000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon [5]. The p68 kinase is characterized by two distinct protein kinase activities [6]. The first one is functional for its phosphorylation whereas the second one is responsible for the phosphorylation of exogenous substrates such as calf thymus histones and the CI subunit of protein synthesis initiation factor eIF2. When activated by dsRNA in the presence of MnZ+ and ATP, p68 kinase is autophosphorylated. This phosphorylated p68 kinase is then capable of catalyzing phosphorylation of eIF2. Phosphorylation of exogenous substrates (histone, eIF2) is not dependent on dsRNA [7]. It might occur as long as p68 kinase is phosphorylated, i.e. there is as strong correlation between the degree of phosphate content of p68 kinase and its kinase activity on exogenous substrates [7, 81. The protein kinase activities mediating autophosphorylation of p68 kinase and phosphorylation of exogenous substrates are independent of cyclic AMP or cyclic GMP and are markedly stimulated by Mn2 +. Previously we have suggested that the dsRNA-dependent protein kinase is composed of two subunits; a M , 48000 protein (p48), which phosphorylates a Mr-68000 protein (p68; [7]). However, these results now have been shown to be artifactual owing to partial proteolysis of p68 kinase during preparation of cell extracts. Under ex- Abbreviations....

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“…In all cases, the activity was inhibited by SO-80% in the presence of 1 pg cr-amanitin per 50 c(g of chromatin. Since heparin does not stimulate RNA synthesis in high salt ( fig.3C), it is concluded that a maximal elongation rate was attained, and that no initiation by released RNA polymerase was possible; an interference due to RNase activity is excluded in those conditions, since heparin, which is a powerful inhibitor of RNase [21], does not affect the results. It was important to estimate the length of the DNA pieces in the two fractions, since a difference in RNA chain elongation could be due to a difference in template size.…”

Section: Resultsmentioning

confidence: 91%

“…Another way to test the presence of endogenous RNA polymerase is to measure its activity in high salt in vitro ( fig.3); it is known [21] that this activity is essentially due to elongation of pre-existing RNA chains. By using this method, it is apparent that the first chromatin fraction contains a much greater concentration of RNA polymerase activity per pg of DNA, whether the assay is done before (fig.3A) or after shearing ( fig.3B), or after fractionation (fig.3C).…”

Section: Resultsmentioning

confidence: 99%