On the spontaneous adherence of myelin basic protein to T lymphocytes.

We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.     

"In vivo" (35S)methionine interaction with rat liver tRNA

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin++ lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.