Susumu Kurioka

Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme

Abstract The hydrolytic rate of p-nitrophenylphosphorylcholine by phospholipase C is highly accelerated in a medium containing sorbital. Glycerol is also effective in increasing the hydrolytic rate but does not increase it as much as sorbitol. We propose a convenient spectrophotometric assay method for phospholipase C using p-nitrophenylphosphorylcholine together with sorbitol (60%, w/w). This method was found very useful for studying the metal and detergent requirement of phospholipase C. The results obtained using this method suggest that phospholipase C of Clostridium perfringens is a metalloenzyme possibly requiring Zn2+ and that no type of detergent has any activating effect on the enzyme itself.

Effects of Sodium and Bicarbonate Ions on γ-Aminobutyric Acid Receptor Binding in Synaptic Membranes of Rat Brain

Abstract: Crude synaptic membranes treated with Triton X‐100 (TX) bound γ‐aminobutyric acid (GABA) to two classes of receptor site in Na+‐free 10 mM‐Tris‐sulfate buffer (pH 7.4), but to only a single class of receptor site in 10 MM Tris‐sulfate buffer (pH 7.4) containing 150 mM‐NaCl. The high‐affinity receptor site in TX membranes was specifically masked in the presence of Na+. However, TX membranes incubated in Krebs‐Ringer bicarbonate solution (pH 7.4) bound GABA to two classes of receptor site despite the presence of Na+. It was found that addition of bicarbonate ions to the Na+‐containing 10 mM‐Trissulfate buffer (pH 7.4) could restore the high‐affinity class of GABA receptors, rendering both classes detectable. This finding suggests that both Na+ and HCO‐3 may have a regulatory function on GABA binding to the receptor.

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD+‐dependent 4‐aminobutyraldehyde dehydrogenase activity was purified about 360‐fold from rat brain extract. AMP‐Sepharose chromatography was effective in separating the enzyme from other NAD+‐dependent aldehyde dehydrogenases included in the extract. The Kms for the substrates NAD+ and 4‐aminobutyraldehyde were 4.8 × 10−4 and 8.3 × 10−5M, respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4‐aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4‐aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

Abstract: [3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X‐100 [membranes (TX)] involved two classes of GABA binding sites (KD= 38.7 and 78.0 nM) in the absence of Na+, but the high‐affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD= 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high‐affinity class of GABA receptor.

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.

Influence of Sodium‐Independent γ‐Aminobutyric Acid Binding on the Assay of Sodium‐Dependent γ‐Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [3H]GABA was bound to crude synaptic membrane fractions of rat. by sodium‐independent process in a medium that contained 100 μM [3H]GABA used for assaying GABA uptake site. This [3H]‐GABA binding was different from receptor binding of GABA. It was confirmed that this sodium‐independent [2H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium‐independent GABA binding had a negligible influence on sodium‐dependent GABA binding.

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.

Dispersion of fibrin using cationic detergent

By treating fibrin with cationic detergent in a medium containing 5M urea, a fibrincationic detergent complex was formed. The complex was soluble in distilled water, and was observed to be in a dispersed state by electron microscopy. The complex was precipitated and resolubilized in the presence of salt. This behavior of the complex was due to adsorption of the salt anions onto the complex. When an aqueous solution of the complex was incubated with fresh serum, the complex soon aggregated and then was converted into a firm clot which was not soluble in distilled water.