DEAE system

Ethylene homopolymerization using Phillips catalyst PC600 calcined at 600°C followed by activation with DEAE cocatalyst during the slurry polymerization process was carried out with Al/Cr molar ratios of 7.5, 15.0, and 22.5 [84]. As shown in Fig. 14, a typical single-type polymerization kinetics corresponding to type b in Fig. 10b was observed, which was completely different from the kinetics with the same catalyst activated by TEA at the same conditions (as shown in Fig. 13). This t3 pe of polymerization kinetics could be ascribed to one type of active site (Site-B) formed in two ways. One was similar with the PC600 activated by TEA some chromate Cr(VI) species were reduced to Cr(II) species by ethylene monomer and coordinated with formaldehyde, then formaldehyde-coordinated Cr(ll) sites were transformed to DEAE-coordinated Cr(II) sites by substitution, as shown in Scheme 8. On the other hand, some chromate Cr(VI) species were reduced by DEAE, and then the Al-alkoxy product coordinated with the Cr(Il) sites. Site-B had relatively low activity and high stability. Based on the microstructure analysis, the relative amount of SCBs of polymers obtained from the DEAE systems was even more than that from TEA catalyst systems. This can be explained as follows. Firstly, the reduction ability of DEAE was weaker than that of TEA. More Cr(VI) species... 

DEAE system (Gold and Schweiger, 1971 Herrlich and Schweiger, 1974)... 

Various components have been added to either an S30 system or the DEAE-system, e.g. RNA polymerase, CAP factor, repressors, regulatory proteins. In each case one must observe whether the conditions optimal for enzyme synthesis are also optimal for the action of the added component. For... 

Fig. 9. Dependence of enzyme synthesis in vitro on ribosomes. Protein synthesis was performed in the DEAE system as described in Table 2. The amount of ribosomes was varied (O.D.jjo units per 50 pi incubation mixture). Upper graph crude ribosomes were used. Lower graph preincubated ribosomes were added. For a description of the enzyme assays see Herrlich and Schweiger (1974)...

In a crude extract or S30 system, no additional tRNA is required. The S30 or crude extract contains saturating amounts of tRNA. The DEAE-system is partially dependent on the addition of tRNA the protein fraction is almost free of tRNA, but tRNA is attached to the ribosomes. In fact, preincubated ribosomes still contain sufficient tRNA to allow 30-50% of the leucine incorporation which can be achieved upon addition of tRNA. However, some tRNA species are apparently limiting since meaningful protein synthesis is reduced to 5% (Gold and Schweiger, 1969a). Response to tRNA addition is improved by further purification of the ribosomes. 

In the DEAE system the protein fraction is obtained by chromatography of the supernatant Si 00 on DEAE-cellulose. There are differences in the saturating amounts of the protein fraction for synthesis of various enzymes. Thus, although maximal T7 lysozyme synthesis is reached with 60 [xg protein per standard reaction (0.050 ml), for maximal ligase formation slightly more is necessary (90 [xg) (Fig. 10) and maximal synthesis of T7 RNA polymerase needs more than 140 (xg protein fraction. (For maximal leucine incorporation as much as 200 [xg protein fraction is needed.) The reasons for these differences are unknown. 

Fig. 12. Dependence on template of RNA directed enzyme synthesis in vitro, A DEAE system of the composition described in Table 2 was started with varying amounts of RNA per incubation mixture. The isolation of in vivo RNA (in this case 10 minutes past T7+ infection) has been described (Herrlich and Schweiger, 1974), for the preparation of in vitro RNA (by E. coli RNA polymerase on T7 DNA) see Schweiger...

Fig. 15. Dependence of T3 DNA directed SAMase synthesis on Mg + and Mn + concentration. SAMase synthesis was performed in the DEAE system as described in Table 2. Synthesis was allowed to proceed at various concentrations of MgClg or MnClg. In the cases of manganese addition a basal concentration of 5 mM MgClj.was present in all samples because the system itself had been prepared with TMA buffer...

T3 and T7 Lysozyme. T3 and T7 lysozyme synthesis under the direction of the corresponding DNAs was performed in a DEAE system. The product consists of free enzyme and most probably ribosome-bound enzyme based... 

RNA dependent enzyme synthesis in the DEAE system is linear for 7-8 minutes. The half life of lysozyme messenger RNA appears to be approximately 7 minutes. A similar stability was measured for total RNA (unpublished data). This half life is longer than the one in vivo (Adesnik and Levin-THAL, 1970 ScHWEiGER et aL, 1972). The in vitro system may lack nucleases (DEAE cellulose chromatography of the Si 00 protein) or part of the natural mRNA degrading enzymes (Wetekam and Ehring, 1973)-... 

How does the rate of in vitro enzyme synthesis compare to in vivo synthesis Under optimal conditions, 0.2 [Jig / -galactosidase per ml is synthesized within 60 minutes of incubation (Chambers and Manley, 1973). Compared to maximal in vivo synthesis, this corresponds to an efficiency of 0.06%. In 08Odarg directed synthesis of N-a-acetyl-L-ornithinase, the in vitro rate was approximately 0.09% of the in vivo rate (Urm et al., 1973). T7 infected cells produce about 7X10 units of lysozyme per minute and per mg of ribosomes assuming that 3 X 10 cells contain 1 mg (1 500 O.D.geo) of ribosomes. The in vitro rate is 4 X 10 units per minute and per mg of ribosomes or 0.57% of the in vivo rate. All these data are of course approximations. Rates of enzyme synthesis in both the preincubated S30 system and the DEAE system are of the same order of magnitude. 

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