Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.