Kazuaki Ohsawa

Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis

A rapid and highly sensitive silver stain and color stain were developed for visualizing proteins. The procedure is simple and the bands were clear. This silver stain detects 100 pg quantities of proteins. In order to stain quickly, sensitively, and sharply a protein matrix in a gel, the repeated shrinkage and swelling gel was developed with a hyper- and hypotonic solution to remove the sodium dodecyl sulfate (SDS) from SDS-protein complex and to generate influx of staining solution into the gel. We have found that the silver staining method with the repeated exposure to hyper- and hypotonic solution and a narrow well produced 10 fg order of proteins.

Surface potential and surface charge density of the cerebral-cortex synaptic vesicle and stability of vesicle suspension

Using a microelectrophoresis instrument employing the Lazer-Zee system, the electrophoretic mobility of synaptic vesicles isolated from Guinea-pig brain cortex was measured under conditions. The mobility was found to depend on both pH and ionic concentration of the solution. The surface of the synaptic vesicle was shown to be negatively charged under physiological conditions. The isoelectric point was observed at pH 4.0 in 0.01 M NaCl solution. Effects of divalent cations were examined and reversal of surface charge was observed in 0.1 M CaCl2 solution. Interaction of vesicles was also considered on the basis of the DLVO theory of colloid stability by using calculated values of surface charge density and surface potential of the synaptic vesicle.

ATP content in isolated mammalian nerve cells assayed by a modified luciferin-luciferase method.

ATP content in a nerve cell isolated from dorsal root ganglia of adult guinea-pigs by collagenase was measured by a newly developed technique modified from the conventional luciferin-luciferase methods. A small volume (4 microliters) of the nerve cell suspension, which contained 10-300 nerve cells (3-100 X 10(-4) microliters of cellular volume) under view of an inverted, phase-contrast microscope, was heat-treated for about 1 s by flame of an alcohol lamp. This heat-treated cell suspension was then reacted with a luciferin-luciferase solution. Light flux from the bioluminescence thus elicited gave an ATP content in single nerve cell, 27 pg (mean) +/- 10 pg (S.D.). ATP concentration in a nerve cell was calculated as 1.7 mM (mean) +/- 0.6 mM. The ATP content in a nerve cell was reduced when the nerve cells were exposed to KCN (5 microM) or dinitrophenol (20 microM), respectively.

Purification of intestinal brush border membrane vesicles by the use of controlled-pore glass-beads column

Abstract A simple rapid method for obtaining highly purified and efficiently transporting intestinal brush border membrane vesicles was developed. The new method consists of two major steps: Ca-treatment of the homogenate of the scraped epithelium (rabbit ileum) and the subsequent chromatography of the supernatant of the Ca-treated homogenate through a controlled-pore glass beads column. The fraction showing the peak value of the optical density at 280 nm and two subsequent fractions were identified as those containing purified brush border membrane vesicles as judged from the activities of the marker enzymes (sucrase and alkaline phosphatase) and the rate of D-glucose uptake. Whole procedures could be completed in about 90 min. Specific activities of sucrase and alkaline phosphatase increased to 35.5 and 34 times, respectively, while Na, K-ATPase activity decreased to one tenth as compared with the initial homogenate. Overshoot uptake of D-glucose in the presence of a NaSCN gradient showed peak at 1–1.5 min after the start of incubation, when it reached 10–40 nmoles/mg protein. Electron microscopic examination revealed that the highly purified vesicles had fairly homogeneous size, the average diameter being about 80 nm.

Purification of bursh border membrane vesicles from rat renal cortex by size-exclusion chromatography

Abstract Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of d -glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is a suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.

Fluid-potentiometer and acetylcholine bioassay with clam entire heart

Abstract Acetylcholine-chloride (Ach) was assayed on isolated entire heart of clam for more than 15 hours with newly designed fluid-potentiometer, a simple balance mechanism with one fulcrum. Beating preparations were accurately depressed by Ach of picogram order and cardiac arrest appeared with a large quantity of Ach. The heart was excited by 5-hydroxytriptamine (5-HT) and teh distortion was found in the falling phase. With these facilities the dose-response curve of Ach was measured by frequencies of beat and this bioassay was applied to the measurement of trace amounts of Ach in ultra-purified synaptic vesicles from the brain (1).

Froth-floating measurements of detergent

This study shows correlation between concentration and the froth produced by five detergents: Triton X-100 (TX100), octylglucopylanoside (OGP), Nonidet P-40 (NOP), Na deoxycholate (DOC), and cholate. Concentrations of TX100 in the order of 10−10% were measured by the height of a floating froth.