The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate.

A method for determining the optical purity of amino acids using HPLC and precolumn derivatization is described. (+)-1-(1-Naphthyl)ethyl isocyanate reacts with racemic amino acids, in high yield, to form naphthylethyl carbamoyl derivatives. The resulting diastereoisomeric pairs were separated on reversed-phase C18 columns and detected fluorometrically.

Damage to the high-affinity ?-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP).

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K(+)-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [(14)C]?-aminobutyric acid uptake.

Rates of protein synthesis in brain and other organs.

We previously found a decrease in protein synthesis in brain during development, which was much greater as measured in brain slices than in brain in vivo. In the present work such changes in brain were compared to those in other organs. With measurement of incorporation of flooding doses of [14C]valine into proteins of organs, the highest synthesis rate in the adult animal in vivo was found in liver (2.

Effects of caffeine on monoaminergic systems in mouse brain.

Effects of caffeine on monoamine systems in the mouse brain were studied in three lines of experiments. First, concentrations of 10(-7) M to 10(-2) M of caffeine were tested for their potency in inhibiting the carriers involved in the neuronal uptake of dopamine, norepinephrine, and serotonin. The IC50 of caffeine in inhibiting the first two carriers was approximately 10(-2) M, and that in inhibiting the serotonin was 2 x 10(-3) M.

Regional and cellular differences in rat brain protein synthesis in vivo and in slices during development.

We compared the rate of protein synthesis in immature and adult rat brain in vivo to that in brain slices. After the incorporation of a flooding dose of [14C]valine, in vivo and in brain slices, the label in proteins was measured in CNS regions and in neuron- and glia-enriched fractions. In regions in vivo in the adult, incorporation rates in corpus callosum were lower than in other regions, which were similar; in the young, cerebellum showed the highest rates and hypothalamus and cord the lowest.

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations.

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range.

Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase.

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly.NH(2)), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly.NH(2)) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes.

Breakdown of brain tubulin by cerebral cathepsin D.

The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.

Membrane-bound tubulin: Resistance to cathepsin D and susceptibility to thrombin.

In recent studies we found that cytoplasmic tubulin from brain was rapidly split by brain cathepsin D. Two pools could be established; the major portion was split at 18%/h, a minor portion at 2%/h, under our experimental circumstances. In the present work these experiments were extended to membrane-bound tubulin from brain.

Separate metabolic pathways for Leu-enkephalin and Met-enkephalin-Arg(6)-Phe(7) degradation by rat striatal synaptosomal membranes.

Metabolism of Leu-enkephalin and Met-enkephalin-Arg(6)-Phe(7) was studied using synaptosomal plasma membranes prepared from rat corpus striatum and whole brain. Cleavage of the pentapeptide was mediated largely by an aminopeptidase leading to the release of Tyr and Gly-Gly-Phe-Leu. Bestatin, an aminopeptidase inhibitor, prevented the release of Tyr and the tetrapeptide, but not secondary cleavage at the Gly Phe site leading to the release of Tyr-Gly-Gly and Phe-Leu.

Metabolism of potent enkephalin analogs (FK 33-824, D-Ala2, pentafluorophenylalanine-4-enkephalinamide and a dimer of D-Ala2-enkephalin) and D-amino acid substituted derivatives of human beta-endorphin.

New analogs of the opiate peptides containing novel substitutions were compared in terms of their metabolic stability in the presence of an ultrafiltrate of mouse brain. The enkephalin analog FK 33-824 was more stable at short incubation periods (30 min) than Met-enkephalin but less stable than D-Ala2-enkephalinamide at longer periods (180 min) as shown by the complete release of N-terminal Tyr. In contrast, D-Ala2-enkephalinamide substituted in position 4 with pentafluorophenylalanine was completely stable at the time periods tested.