Quantitative determination of lectins in mistletoe preparations.

Assay of lectins in mistletoe preparations was based on an improved and validated version of ELLA (enzyme-linked lectin assay) to meet the requirements given in the guidelines for drug tests. The monoclonal antibody used has more than 90% cross reactivity with the three known mistletoe lectins, so that total lectin content is determined with much greater accuracy. With the detection and quantitative analysis limit below 5 ng/ml and a linear measuring range of 5-50 ng/ml, dosages in therapeutic range can be assayed. Tests to establish the accuracy of the analytical method showed that up to 26% of lectin activity is suppressed by other constituents of the extract, so that the recovery must be taken into account. The recovery increases following ultrafiltration to remove low-molecular constituents. Analysis for precision gave a variation coefficient of < or = 7.7% and a confidence interval < or = 5.7% (p = 0.05) for total lectin concentrations of approx. 250 ng/ml. This level of precision, which is good for an immunologic assay, makes it possible to standardize mistletoe preparations.

Influence of dimethylsulfoxide on transcription by bacteriophage T3-induced RNA polymerase.

Dimethylsulfoxide (DMSO) up to 25% (v/v) does not cause irreversible alterations of T3 DNA at 42.5 degrees C as assayed by transcription with T3-specific RNA polymerase. The optimal temperature for the formation of polyanion-resistant ternary complexes of the enzyme, T3 DNA, and nascent RNA chains is lowered by 12.5 degrees C in the presence of 20% (v/v) DMSO. The same solvent concentration, however, decreased the temperature optimal for T3 RNA chain elongation by only 2.5 degrees C, indicating that DMSO preferably affects the initiation of T3 RNA synthesis. DMSO accelerates the loss of T3-specific RNA polymerase activity at 24.5 degrees C. Nevertheless, the speed with which the binary complexes between the phage RNA polymerase and DNA are inactivated by heat (42.5 degrees C) is not altered in presence of 20% (v/v) DMSO. The binding of T3-induced RNA polymerase to T3 DNA in polyanion-resistant ternary complexes is influenced by DMSO which makes the enzyme accessible to the inhibitory action of polyvinyl sulfate. Elongation of T3 RNA chains is slowed down by 20% (v/v) DMSO.

Effect of temperature on the transcription by bacteriophage T3-induced RNA polymerase.

Bacteriophage T3-induced RNA polymerase is rapidly inactivated at 42 degrees C. Addition of T3 DNA delays this process for 30 s and reduces the rate with which the enzyme activity is lost indicating that a labile binary complex between T3 DNA and polymerase must have been formed. The ternary complex between T3-specific RNA polymerase, T3 DNA, and nascent RNA chains obtained when the enzyme is incubated with T3 DNA, GTP, ATP, and UTP is stable to heat (42 degrees C) and only slowly inactivated by polyvinyl sulfate. The optimal temperature for the formation of polyanionresistant ternary complexes is 30 degrees C while the elongation of T3 RNA chains proceeds fastest at 38 degrees C.

[Effect of temperature on the transcription of T3 DNA b y T3-specific RNA polymerase in cell-free extracts of Escherichia coli CRT266]. / Einfluss der Temperatur auf die Transkription der T3-DNA durch T3-spezifische RNA-Polymerase in zellfreien Extrakten von Escherichia coli CRT266.

Cell free extracts were prepared from E. coli CRT266 9 min after infection with T3 phages. RNA synthesis in these extracts is almost entirely due to T3 RNA polymerase. The inactivation of T3 RNA polymerase in these extracts proceeds rapidly at 42 degrees C. 90% of the activity is lost within 10 min at this temperature. Under conditions where the formation of a stable initiation complex with T3 DNA is possible, i.e., in the presence of GPT, APT, and UTP the T3 RNA polymerase becomes protected against heat inactivation losing only )0% of its activity during an exposure to 42 degrees C for 10 min. Studies on the time course of RNA synthesis have shown that reinitiation is still possible at 37 degrees C and 42 degrees C. At 44 degrees C, however, RNA synthesis stops abruptly after 3 min indicating that reinitiation does no longer take place. The elongation of already initiated T3 RNA chains is rather resistant to heat. At 44 degrees C the same elongation rates are observed as at 37 degrees C and 42 degrees C, respectively.