Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.     

The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) spectra of thyroid cell membranes and their total lipid extracts, in the presence of 1-anilino-8-naphthalenesulfonate (ANS), have been studied. The addition of ANS causes a shifting of the head group PMR signal, a splitting of the signal into two components and an increase in total spectral intensity. The data suggest that ANS interacts with phospholipid in the membrane as it does in total lipid vesicles. Evidence is also presented for the removal of lipids from the membrane, by ANS, and the subsequent formation of micelles. The membrane results are compatred with our earlier work on the interaction of ANS with egg phosphatidycholine (P.C.) vesicles and the results are used in explaining the inhibition of iodide transport in isolated thyroid slices.     

Synthesis and application of a new phosphorylating agent: S-(N-monomethoxytritylaminoethyl)-O-(o-chlorophenyl)phosphorothi oate

A new phosphorylating agent, S-(N-monomethoxytritylaminoethyl)-O-(O-chlorophenyl) phosphorothioate, was prepared and reacted with a 5'-hydroxyl group of an oligonucleotide using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) as a condensing agent. After base labile protecting groups were removed, the partially deprotected oligonucleotide was separated on a reversed phase column and converted to the oligonucleotide with an aminoethyl or a phosphoryl group at the 5'-end by treatment with 80% acetic acid or iodine-water, respectively. The syntheses of ppT, pppT, A5'pp5'T and A5'ppp5'T were also performed by treatment of 5'-O-(N-monomethoxytritylaminoethylthiophosphoryl) thymidine with tri-n-octylammonium salt of phosphoric acid, pyrophosphoric acid, pA and ppA, respectively.