PubMed · 5112939
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P J Weiss. Laboratory quality assurance program.. https://pubmed.ncbi.nlm.nih.gov/5112939/
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Interleukin-2 (IL-2) is a cytokine essential for the growth and proliferation of T-cells. The purpose of this research was to study the effects of altering the "kink" caused by Pro in an alpha-helix of the protein. The Pro-47 residue was chosen because it was originally, but mistakenly, traced to the kink of an alpha-helix [1]. Pro-65 is now recognized to be situated in the middle of helix B [2]. To study the significance of this Pro for the bioactivity and overall conformation of IL-2 it was mutated to Gly and Ala. We successfully obtained 17 different mutants at position 47 and two mutants at position 65. Certain amino acid substitutions representing different categories of amino acids, namely, acidic, neutral and helix stabilizing, were chosen for more thorough investigation. The results showed that Asn-47 and Asp-47 decreased the bioactivity of these mutants by 50- and 700-fold respectively, while the Kd to its high affinity receptors was increased 180- and 90-fold respectively, compared to IL-2. The intermediate binding affinity of Asn-47 and Asp-47 was decreased 8- and 37-fold, respectively. On the other hand, Gly-47, Gly-65 and Ala-65 showed less dramatic decreases in bioactivity and high affinity binding. The intermediate binding affinity of these mutants decreased from 5- to 3-fold and low affinity binding decreased approximately 4-fold suggesting some structural and conformational changes. From these observations, we conclude that Asn-47 or Asp-47 disrupt the hydrophobic packing of the core and thus changed the overall conformation of the protein, thereby giving rise to partial agonists. Although Pro-65 lies within the helix, it may be near the surface of the protein but may not be the actual binding site and thus any conservative mutation can be better tolerated.
A rapid bioluminometric technique for real-time detection of known single-base changes is presented. The concept relies on the measurement of the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymerase-catalyzed primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyrén (1987) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-base detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' end are designed, one precisely complementary to the nonmutated DNA sequence and the other precisely complementary to the mutated DNA sequence. The primers are hybridized with the 3'-termini over the base of interest and the primer extension rates are, after incubation with DNA polymerase and deoxynucleotides, measured with the ELIDA. We show that the relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini. The possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.
Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-8 can have different biological specific activities, even though all were produced using E. coli. It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays. In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.