Ehrenfried Mehl

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate.

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.

Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A

1. 1.A method was developed for isolating a pure enzyme with cerebroside-sulfatase activity from the pig kidney. It was found that arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was confined to the same isolated enzyme preparation. As disc electrophoresis revealed, this enzyme is strikingly different from the arylsulfatase A from ox live which was isolated in Roys laboratory by an essentially different procedure. Nevertheless, Roys enzyme also exhibited, like ours, a slight degree of cerebroside sulfatase activity which in both preparations was strongly enhanced by addition of the same heat-stable ‘complementary fraction’ obtained from pig kidney. Analytical disc electrophoresis under various conditions indicated purity of the two arylsulfatases A. The quotient of the sulfatase activities with aryl and cerebroside sulfate as substrate remained constant after further disc-electrophoresis separation. 2. 2.As sulfatase activity with 35S-labelled synthetic galactose 6-sulfate and cerebroside 6-sulfate could not be detected, the enzyme is highly specific for its naturally occurring substrate, cerebroside 3-sulfate (Km = 0.105 mM) (see ref. 5). 3. 3.Galactose 3-sulfate, galactose 6-sulfate and cerebroside 6-sulfate were found to inhibit the activity with cerebroside 3-sulfate as substrate as well as with arylsulfate (2-hydroxy-5-nitrophenyl sulfate). 4. 4.Cerebroside 3-sulfate was found to inhibit sulfatase activity with 2-hydroxy-5-nitrophenyl sulfate as substrate and vice versa; a result which indicates a common enzyme for both substrates. 5. 5.Arylsulfatase B is only slightly active towards cerebroside sulfate, provided the heat-stable complementary fraction is added. Thus it is very evident that cerebroside 3-sulfate is a naturally occurring substrate of arylsulfatase A.

GABA and glycine in synaptic vesicles: storage and transport characteristics

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.

The protein components of the erythrocyte membrane and their molecular weights.

Abstract Membrane proteins from pig erythrocytes were solubilized by a phenol formic acid water solvent. Such treatment yielded monomers which were subjected to electrophoresis across a polyacrylamide gel gradient. The heterogeneity of the protein components was demonstrated by the appearance of 14–16 zones. The major components comprising about 30 and 13% of the total protein had molecular weights of 48000 and 27000, respectively. With the exception of one zone, the other components, which each contributed about 5% or less to the total protein mass, ranged in molecular weights from 26000 to 65000. One zone accounting for about 8% of the protein may have had a molecular weight as high as 180000, but it could not be proved to be a monomeric protein. The variation of results reported by others is explained by difficulties in completely dissolving the membrane proteins to monomers rather than by species differences.

Na+K+-dependent conformation change of proteins of excitable membranes

Abstract The K + , Na + and Ca 2+ form of excitable membranes of rat brain were investigated by infrared, ORD and CD spectroscopy. It is shown that with the K + form the conformatio of relatively large parts of the membrane proteins occurs as an antiparallel β structure. No β structure is found with the Na + and Ca 2+ form. In the presence of these ions the proteins are largely helical. This suggests that during the action potential, membrane proteins change their conformation depending on the cations shifted.

Identification of water-insoluble membrane proteins by immunoelectrophoresis in a solubilizing urea-triton solvent

Abstract A procedure was developed which renders possible the identification of water-insoluble membrane proteins by immunoelectrophoresis in a solubilizing solvent. Purified erythrocyte membranes from the pig were extracted with butanol, and the protein components were solubilized in a Tris-HCl buffer containing urea and Triton X-100 at reduced concentrations throughout the procedure. After removal of 12% insoluble protein by centrifugation, 50% of the remaining proteins appeared to be monomers and 50% polymers, i.e. tetramers. Immunoelectrophoresis in the above buffer mixtures resulted in six precipitation lines. The specificity of the reaction was established. It seems that the multiple-site interactions of antigen and antibody have sufficient energy to allow some reduction by addition of the solubilizing agents, urea and Triton X-100, without losing specificity.

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.

Heterogeneity of LSD-displacing factors and multiple types of high affinity LSD-binding sites

Abstract Heterogeneity of high affinity LSD-binding sites was confirmed by displacement studies with 2-bromo (+)—LSD and with apamin, a peptide neurotoxin. In line with the concept of multiple binding sites, a number of fractions of putative endogenous ligands could be separated from rat brain extract. The LSD-displacing β-fraction was not detectable in tissues lacking high affinity LSD-binding sites. High affinity dopamine- and serotonin-binding was differentially affected by the β-fraction.