Comparative enzymatic development in chick embryonic brain areas

The activities of glutamic acid decarboxylase (GAD), choline acetylase, dopa decarboxylase, and tyrosine hydroxylase were measured by radioactive assays and of acetylcholinesterase by a colorimetric procedure on homogenates of the tectum, forebrain, and cerebellum of the chick from the third embryonic day to 3 weeks post-hatch. GAD showed a rapid development beginning about day 9 and peaking at or before hatching: there were generally similar levels in all 3 areas during development although in the oldest chicks the tectum had significantly higher GAD levels than the forebrain, the cerebellar levels being intermediate. The other enzymes all showed a somewhat later development with sharp increases beginning on or after day 11 and peak levels being reached only after hatching. The different brain regions also showed much greater disparity in levels of these other enzymes than found for GAD. The tectum contained the greatest concentrations of choline acetylase and acetylcholinesterase, and the forebrain had the most tyrosine hydroxylase and dopa decarboxylase. The data may be useful for correlation with morphological developmental studies.     

DEVELOPMENTAL CHANGES OF CHOLINESTERASES AND MONOAMINE OXIDASE IN CHICK EMBRYO SPINAL AND SYMPATHETIC GANGLIA

Abstract— Total cholinesterase, acetylcholinesterase, (AChE) and monoamine oxidase (MAO) activity and protein content were determined throughout the embryonic life of the chick in spinal and sympathetic ganglia. The greatest part of total cholinesterase activity was due to AChE.
AChE and MAO activity increased in both spinal and sympathetic ganglia very similarly from the 6th to the 12th day of incubation; from this day on a significant divergence occurred, mainly owing to a steady fall in spinal ganglion AChE, which decreased to approximately one tenth of the maximum value. The ratio of MAO activity in sympathetic and spinal ganglia increased from the 8th day onwards and approached 5·0 at hatching. The ratio between sympathetic and spinal ganglia, for AChE, choline acetylase (ChAc) and MAO activity, suggests a relationship between the maturation of the synapse in the sympathetic ganglia and the maximal activity of these enzymes.     

Brain levels and turnover rates of presumptive neurotransmitters as influenced by administration and withdrawal of ethanol in mice

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [³H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment     

Early second trimester maternal plasma choline and betaine are related to measures of early cognitive development in term infants

Background

The importance of maternal dietary choline for fetal neural development and later cognitive function has been well-documented in experimental studies. Although choline is an essential dietary nutrient for humans, evidence that low maternal choline in pregnancy impacts neurodevelopment in human infants is lacking. We determined potential associations between maternal plasma free choline and its metabolites betaine and dimethylglycine in pregnancy and infant neurodevelopment at 18 months of age.

Methodology

This was a prospective study of healthy pregnant women and their full-term, single birth infants. Maternal blood was collected at 16 and 36 weeks of gestation and infant neurodevelopment was assessed at 18 months of age for 154 mother-infant pairs. Maternal plasma choline, betaine, dimethylglycine, methionine, homocysteine, cysteine, total B12, holotranscobalamin and folate were quantified. Infant neurodevelopment was evaluated using the Bayley Scales of Infant Development–III. Multivariate regression, adjusting for covariates that impact development, was used to determine the associations between maternal plasma choline, betaine and dimethylglycine and infant neurodevelopment.

Results

The maternal plasma free choline at 16 and 36 weeks gestation was median (interquartile range) 6.70 (5.78–8.03) and 9.40 (8.10–11.3) µmol/L, respectively. Estimated choline intakes were (mean ±SD) 383±98.6 mg/day, and lower than the recommended 450 mg/day. Betaine intakes were 142±70.2 mg/day. Significant positive associations were found between infant cognitive test scores and maternal plasma free choline (B = 6.054, SE = 2.283, p = 0.009) and betaine (B = 7.350, SE = 1.933, p = 0.0002) at 16 weeks of gestation. Maternal folate, total B12, or holotranscobalamin were not related to infant development.

Conclusion

We show that choline status in the first half of pregnancy is associated with cognitive development among healthy term gestation infants. More work is needed on the potential limitation of choline or betaine in the diets of pregnant women.     

Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-β-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm² centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm² in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm², P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy.     

Effect of static magnetic field on some enzymes activities in rats

The magnetic field of 0.008 T and 0.15 T inductions influence lasting 7 weeks (7 days a week), 1 h daily determines the increase of the activity of cytoplasmatic enzymes (glutamic pyruvic transaminase, glutamic oxalacetic transaminase, lactic dehydrogenase), the decrease of cholinesterase activity and the growth of alkaline phosphatase activity in the plasma of the examined animals. The observed changes were reversible. 2 months after the exposure had been stopped, the tested parameters were back to normal.     

Neuronal and glial enzyme studies in cell culture

Summary Newborn 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. Portions of this work were supported by a research grant (6-444946-58605) from the American Cancer Society.     

Choline permeability in cardiac muscle cells of the cat

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm²·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm²·sec in 137 mM choline and at 1.3–3.5 pmoles/cm²·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.     

The effect of enzyme inhibitors on the resting potential and on the ion distribution of the sartorius muscle of the frog

Isolated frog sartorii were exposed for 30 minutes to HETP—an irreversible anti-cholinesterase, and were then soaked in Ringer's at 15°C. for 16 hours. At the end of the period of soaking the mean resting potential of the muscle fibers was only 29 mv. The decrease in the resting potential of the HETP-treated muscles was accompanied by a loss of potassium and a gain in sodium by the muscles. The effect of anticholinesterases on sodium extrusion was studied by incubating the muscles in a Ringer's containing half of the normal amount of sodium. The muscles respond by extruding sodium against a concentration gradient into the external medium. Sodium extrusion was blocked by prior exposure of the muscle to HETP, and reversibly blocked by exposure to physostigmine. The inhibition of sodium extrusion by physostigmine was correlated with the inhibition of the intracellular cholinesterase. Sodium extrusion was also blocked by high concentrations of 2-methyl-1,4-napthaquinone 8-sulfonic acid and by α-ketoglutarate, which are known to inhibit choline acetylase in vitro. But sodium extrusion was not affected by a third inhibitor of choline acetylase, phenobarbital. Sodium extrusion was unaffected by KCN and partially blocked by IAA. The IAA block was eliminated by the addition of pyruvate. It is concluded that either glycolysis or oxidative metabolism can furnish the energy needed for sodium extrusion.     

Acetylcholine Translocating Protein: Mediatophore at Rat Neuromuscular Synapses

The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium-dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.     

AXONAL TRANSPORT OF CATECHOLAMINE SYNTHESIZING AND METABOLIZING ENZYMES

The rates of accumulation of the catecholamine synthesizing and metabolizing enzymes proximal to a ligation on the sciatic nerve of the rat were studied. Dopamine-β hydroxylase (EC 1.14.2.1) and tyrosine hydroxylase (EC 1.14.3a) accumulated at a similar rapid rate, and catechol-O-methyl-transferase (EC 2.1.1.6), choline acetyltransferase (EC 2.3.1.6) and monoamine oxidase (EC 1.4.3.4) accumulated at the same slow rate, whereas DOPA decarboxylase (EC 4.1.1.26) accumulated at an intermediate rate. Based on clearance of the rapidly accumulating enzymes, absolute flow rates were estimated to be: 106-167 mm/24 h for tyrosine hydroxylase; 138-185 mm/24 h for dopamine-β-hydroxylase; and 36-86 mm/24 h for DOPA decarboxylase. In contrast, the mean rate of transport of the slowly accumulating enzymes (monomine oxidase, catechol-O-methyltransferase and choline acetyltransferase) was approximately 3 mm/24 h. Colchicine and vinblastine completely blocked the axonal transport of both the rapidly and slowly transported enzymes. Studies of the subcellular distribution of each enzyme failed to confirm the suggestion that particulate enzymes are transported rapidly and soluble enzymes slowly. Our results suggest that the transport and inactivation of dopamine-β-hydroxylase, DOPA decarboxylase, and tyrosine hydroxylase are under different controls than monoamine oxidase and catechol-O-methyltransferase.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

CHOLINE ACETYLTRANSFERASE AND CHOLINESTERASE IN SKELETAL MUSCLE REGENERATION

—The activities of choline acetyltransferase and cholinesterase have been measured in homogenates of rat anterior tibial muscles following intramuscular injection of the myotoxic agent Marcaine (plus hyaluronidase). Marcaine causes rapid degeneration of skeletal muscle followed by complete regeneration. By the first day after administration of Marcaine the activities of both enzymes decreased markedly. The activities then increased concomitantly with regeneration of drug-treated muscles. Choline acetyltransferase attained the control level by the 19th day following Marcaine treatment. Cholinesterase activity became greater than the control value by the seventh day and decreased to control activity by the 19th day following injection of Marcaine. The results are discussed in terms of the relationship between nerve and muscle during Marcaine induced degeneration and regeneration.     

EFFECTS OF SYMPATHECTOMY ON AXOPLASMIC TRANSPORT OF SELECTED ENZYMES INCLUDING MAO AND OTHER MITOCHONDRIAL ENZYMES

Abstract— Axoplasmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of various enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC 1.4.1.3) activity was unaffected by Sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged.
Despite impressive inhibition of MAO accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by Sympathectomy. The existence in nerve of isozymes of MAO was demonstrated using the inhibitors clorgyline and deprenyl. Transported MAO activity was almost entirely type A; intrinsic activity was 2/3 type A and 1/3 type B.
The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.     

Transported Enzymes in Sciatic Nerve and Sensory Ganglia of Rats Exposed to Maternal Antibodies Against Nerve Growth Factor

Abstract: The accumulations by axoplasmic transport of selected enzyme activities proximal and distal to a ligature placed on the sciatic nerve were monitored in rats exposed in utero to maternal antibodies to nerve growth factor (NGF) and in control rats. Littermates of the animals exposed to anti-NGF were shown elsewhere to have had a 70% reduction in the number of sensory neurons in dorsal root ganglia and a 90% reduction in number of neurons in superior cervical (sympathetic) ganglion. The accumulation of F^--sensitive acid phosphatase activity was depressed 75% both proximal and distal to the tie. Accumulation of F^--resistant acid phosphatase activity was depressed nearly 50% proximal to the tie. Distal accumulation of this activity did not occur in either group of rats. Accumulation of acetylcholinesterase activity was not affected. Proximal accumulation of glutamic dehydrogenase activity was depressed 30%. Distal accumulation of the activities of β-glucuronidase and hexokinase was depressed 50%. In the lumbar dorsal root ganglia, dry weight was reduced 40%, and the activities of peroxide-sensitive, F^--resistant acid phosphatase and of the mitochondrial enzymes hexokinase, glutamic dehydrogenase, glutamic-oxalacetic transaminase, and NAD-dependent isocitric dehydrogenase were all reduced a little more, 45–50% per ganglion. However, the activities of the lysosomal enzymes, F^--sensitive acid phosphatase and β-glucuronidase, of the peroxide-resistant, F^--resistant acid phosphatase, and of the mitochondrial enzyme glutaminase were all reduced about 60% per ganglion. The results of these measurements were interpreted to suggest that much, and perhaps all, of the F^--sensitive acid phosphatase activity in motion in peripheral nerve in rat is confined to sensory axons.     

Direct comparison of the rapid axonal transport of norepinephrine and dopamine-β-hydroxylase activity

Stop-flow techniques were used to examine the rapid axonal transport of norepinephrine in rabbit sciatic nerves. When the midpoint of a nerve incubated in vitro was cooled to 2°C while the remainder was kept at 37°C, norepinephrine accumulated proximal to the cooled region at a rate corresponding to an average transport velocity between 5 and 6 mm/hr in a distal direction. Since only about half of the norepinephrine appeared to be free to move, the mean velocity of the moving fraction was probably twice as great. No norepinephrine accumulated distal to a broad cooled region under conditions in which there would have been a significant accumulation of dopamine-β-hydroxylase activity. Therefore, unlike dopamine-β-hydroxylase, norepinephrine may not be subject to rapid retrograde transport. When nerves that had been locally cooled for 1.5 hr were rewarmed uniformly to 37°C, a wave of norepinephrine moved exclusively in a distal direction. The peak of this wave moved at a velocity of 12.2 ± 0.5 mm/hr or 293 ± 12 mm/day; the front of the wave moved at about 18 mm/hr. or 430 mm/day; and the tail probably moved faster than 6 mm/hr. This spectrum of velocities was virtually identical to the one displayed by the wave of dopamine-β-hydroxylase activity that was generated under the same conditions. Our results are consistent with the conclusion that all axonal structures containing norepinephrine also contain dopamine-β-hydroxylase, but they are not consistent with the converse.     

Enzyme Activities in Relation to pH and Lactate in Postmortem Brain in Alzheimer-Type and Other Dementias

Phosphate-activated glutaminase, glutamic acid decarboxylase, pyruvate dehydrogenase, succinic dehydrogenase, pH, and lactate were measured in frontal cortex and caudate nucleus of postmortem brains from cases of Alzheimer-type dementia (ATD), Down's syndrome, Huntington's disease, and one case of Pick's disease, as well as from sudden death and agonal controls. Lactate levels were higher and pH, phosphate-activated glutaminase, and glutamic acid decarboxylase levels were lower in the agonal controls than in the sudden death controls. Phosphate-activated glutaminase and glutamic acid decarboxylase were correlated with tissue pH and lactate, and also were reduced by in vitro acidification, suggesting that the low activities of these enzymes in agonal controls were related to decreased pH consequent upon lactate accumulation. Compared with control tissues at the same pH, phosphate-activated glutaminase and glutamic acid decarboxylase were unaltered in ATD and Down's frontal cortex and reduced in Huntington's caudate nucleus, and glutamic acid decarboxylase was reduced in Huntington's frontal cortex. These data suggest that GABAergic neurons are not affected in ATD and confirm the GABAergic defect in Huntington's disease. Pyruvate dehydrogenase and succinic dehydrogenase activities were the same in agonal controls and sudden death controls and were unaffected by acid pH and lactate in vitro, and pyruvate dehydrogenase was not correlated with pH or lactate. Reduced pyruvate dehydrogenase in frontal cortex of individual ATD, Down's, and Pick's cases, and in the caudate nucleus of Huntington's and Down's cases, was accompanied by gliosis/neuron loss. We conclude that decreased pyruvate dehydrogenase reflects neuronal loss.     

EFFECTS OF COLCHICINE ON AXONAL TRANSPORT IN PERIPHERAL NERVES

—Colchicine injected intracisternally markedly inhibited the rapid migration (300-400 mm/day) of labelled proteins in the hypoglossal and vagus nerve of the rabbit. The transport of acetylcholinesterase (EC 3.1.1.7) and choline acetyltransferase (EC 2.3.1.6) previously shown to move with the slow (5-26 mm/day) phase of axoplasmic transport in these nerves, was only partially blocked. In view of this differential effect on axonal flow, we suggest that the neurotubules, on which colchicine acts preferentially, are primarily involved in the rapid (300-400 mm/day) axoplasmic flow. After local injection of colchicine into the nerves both the rapidly migrating labelled proteins and the enzymes (AChE and ChAc) accumulated above the site of injection to the same degree as they accumulate above a nerve ligation. Since this blockage of enzyme transport occurred after concentrations of colchicine much higher than those used for intracisternal injections these findings after local injection may represent more severe effects on axonal transport systems.     

Quantitation of Cholinergic Synaptosomes from Guinea Pig Brain

An antiserum raised to nerve terminal sacs derived from the electric organ and Torpedo marmorata was used to lyse guinea pig brain synaptosomes in the presence of complement. From the release of the cytoplasmic enzymes choline acetyltransferase, lactate dehydrogenase, tyrosine hydroxylase and glutamate decarboxylase it appears that the antiserum binds specifically to cholinergic terminals. The amount of lactate dehydrogenase released was used to estimate the proportion of cholinergic nerve terminals in different synaptosome preparations.     

FREE-FLOW ELECTROPHORETIC SEPARATION AND ELECTRICAL SURFACE PROPERTIES OF SUBCELLULAR PARTICLES FROM GUINEA PIG BRAIN

Continuous free-flow electrophoretic separation has been used to obtain relatively pure preparations of synaptosomes and synaptic vesicles from crude fractions of guinea pig brain homogenates. Measurements of the contents of protein, neuraminic acid, and bound acetylcholine; the activities of succinic dehydrogenase, adenosine triphosphatase, choline acetylase, and 5'-nucleotidase; and the uptake of ¹⁴C-labeled choline arid acetylcholine in the presence and absence of hemicholinium, all confirm the electron microscope evidence that the electrophoretic preparations are at least as pure as those obtained by ultracentrifugal methods. The electrophoretic mobility measurements have been used to calculate zeta potentials and surface charge densities for these particles.