A. M. Kidd

Metabolically Active Synaptosomes Can Be Prepared from Frozen Rat and Human Brain

Abstract: Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue‐derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1‐5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter‐related studies.

Purification and characterization of a neuropeptide-degrading aminopeptidase from human brain

Abstract: The major aminopeptidase from human post‐mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion‐exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2‐mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal‐ion chelating agents, sulphhydryl blocking agents, bestatin, and puromycin. A series of amino acyl‐7‐amido‐4‐methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 μM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5‐enkephalin (Km 180 μM), cholecystokinin octapeptide, and Arg8‐vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5‐enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.

Agonal status affects the metabolic activity of nerve endings isolated from postmortem human brain

Isolated nerve endings (synaptosomes) that show high rates of metabolic activity have been prepared up to 24 h postmortem from the brains of patients who have died suddenly. In contrast, similar preparations from brains of patients dying after a prolonged terminal illness showed little or no respiration. These data suggest that the agonal state of the patient is of major importance when investigating specific defects in neurotransmitter function in cerebral disorders and effects of neuroactive drugs on human tissue.

Use of post-mortem human synaptosomes for studies of metabolism and transmitter amino acid release

Synaptosomes have been prepared from human brain obtained at autopsies carried out up to 24 h postmortem (p.m.). They showed generally good retention of morphology, as well as accumulation of tissue potassium and linear rates of oxygen uptake. In response to veratrine depolarization they showed increased respiration rate, decreased tissue potassium content and the specific release of transmitter amino acids. Regression analysis indicated that metabolically and functionally active preparations may be obtained up to ca. 25 h p.m. Preparations obtained from patients dying with brain injury were inactive.

Release of Aspartate and Glutamate Caused by Chloride Reduction in Synaptosomal Incubation Media

Abstract: Release of the amino acid neurotransmitter candidates aspartate and glutamate from synaptosomes can be stimulated by reduction of [Cl−] in the incubation medium by replacement with the impermeant anions pro‐pionate and isethionate. Replacement by the permeant anion Br− is without effect on amino acid release. This release offers a biochemical method of studying the Cl−ion channel in CNS membrane. Key Words: Synaptosomes — Chloride — Transmitters — Release — Amino acid—Permeability. Hardy J. A. et al. Release of aspartate and glutamate caused by chloride reduction in synaptosomal incubation media.

Amino acid transport by synaptosomes isolated from post mortem human brain.

Synaptosomes isolated from post mortem human brain release the transmitter amino acids upon depolarisation. Active preparations can be isolated for up to 24 hours post mortem. The amount of depolarisationinduced release of the transmitter amino acids is correlated with the rate of oxygen uptake. Only when this correlation is taken into consideration would it be possible to detect disease-related changes in presynaptic aminoacid transport systems.

Ion-exchange, gel-filtration and reversed-phase high-performance liquid chromatography in the isolation of neurotensin-degrading enzymes from rat brain

Brain and pituitary peptidases involved in the metabolism of neurotensin were identified by ion-exchange (TSK-545 DEAE) and gel-filtration (TSK-G4000 SW) high-performance liquid chromatography (HPLC). Reversed-phase HPLC on radial compression cartridges was used in a rapid assay of neurotensin-hydrolysing activity in column fractions and at the same time the bond specificity of the activity was determined. Semipreparative isolation of the major enzyme which cleaves neurotensin at Arg8-Arg9 was carried out and some of its characteristics were determined.