Roger N. Rosenberg

A radioisotopic, chromatographic microassay for acetyl-coenzyme a synthetase and some preliminary studies on the rat brain enzyme

Abstract 1. 1. An isotopic, chromatographic assay for acetyl-CoA synthetase (acetate:CoA ligase (AMP), EC 6.2.1.1) has been developed which is both sensitive and rapid to perform. 2. 2. The rat brain synthetase activity behaves the same as the synthetase activity from other sources when exposed to increasing concentrations of Na+, K+, or NH4+. 3. 3. The subcellular location of the rat brain synthetase is primarily mitochondrial, but 13% of the activity was found in the cytoplasm. 4. 4. Kinetic studies on the rat brain synthetase preparation indicated higher substrate affinities than previously reported and marked substrate inhibition which occurred at moderate substrate concentrations. 5. 5. In view of the differences in substrate affinities and substrate inhibition of the rat brain acetyl-CoA synthetase compared to the synthetases from other sources, it is suggested that the results of comparative studies be interpreted cautiously.

Kinetics of acetylcholine synthesis and hydrolysis in myasthenia gravis

Kinetic studies for choline-O-acetyltransferase (CAT) (E.C. 2.3.1.6) and acetylcholinesterase (E.C. 3.1.1.7) were performed on serum, skin fibroblasts in culture, and biopsied sartorius muscle from normal and myasthenic subjects. There was a significant decrease of CAT activity per milligram of protein in myasthenic muscle compared to normal muscle, and there was no difference in acetylcholinesterase activity per milligram of protein in the same muscle homogenates. Substrate concentration curves for acetyl coenzyme A and CAT also showed a significant reduction in the maximum rate of product formation (Vmax) per milligram of protein between myasthenic and normal muscle. It is postulated that binding of substrate to CAT is being inhibited by an inhibitor present in muscle.

Neuronal and glial enzyme studies in cell culture

SummaryNewborn BALB/c mouse brain was cultured as disaggregated cells after serial trypsin dissociations. The ontogeny of the cultures was followed by assays of cell number, deoxyribonucleic acid, and protein content and by the activities of three enzymes considered to be markers of neuronal differentiation. Aliquots of the freshly dissociated cells were assayed for choline acetylase, acetylcholinesterase, and glutamic acid decarboxylase activities and compared with intact brain. The percentages of recovery of activities, expressed as14C product formed per mg of protein per 10 min, at pH 6.8 and 37°C, were 37% for choline acetylase, 54% for acetylcholinesterase, and 24% for glutamic acid decarboxylase. The remainder of the freshly dissociated cells were placed into culture; enzyme assays were performed as the cells multiplied and then when the cultures became static. Choline acetylase activity increased as the cells rapidly divided, and glutamic acid decarboxylase activity increased only after the cultures became confluent. Under the culture conditions, acetylcholinesterase was not induced, despite active synthesis of acetylcholine.Neuroblastoma clone N18, C1300 cell line, was grown in cell culture, and the activity of acetylcholinesterase was measured as the cells multiplied and came to confluency. The specific activity of mouse neuroblastoma acetylcholinesterase increased 25-fold when the rate of cell division was restricted. The rate of cell division could be regulated by adjusting the serum concentration. By removing fetal calf serum during the growth period, cell division ceased, and acetylcholinesterase activity was significantly and rapidly induced. Choline-O-acetyltransferase specific activity was measured in rapidly dividing and in static cultures. Its specific activity was highest in nondividing cultures, compared to cultures containing actively dividing cells (6-fold), and the specific activity of thymidylate synthetase was increased 2.5-fold in actively dividing cultures, compared to static cultures.Glioblastoma cells obtained from the rat astrocytoma, clone C6, were grown in culture, and glucose metabolism was measured in control cultures, and in cultures containing norepinephrine (0.017 mg per ml). Norepinephrine produced a 50% inhibition in the incorporation ofd-[14C]glucose. Cells incubated for 2 hr in the presence ofd-[14C]glucose, washed and then incubated in control medium or in medium containing norepinephrine, resulted in the release of greater than 50% of radioactive metabolites in the norepinephrine treated plates. Norepinephrine caused a 50% increase in14CO2 production in glioblastoma cells incubated withd-[1-14C]glucose. Norepinephrine, under similar conditions, did not affect the metabolism of glucose in clone C46, C1300 mouse neuroblastoma cells.