Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Changes of Acetylcholinesterase Activity in Brain Areas and Liver of Sucrose- and Ethanol-Fed Rats

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.     

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.     

LOCAL CHANGES IN SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE (EC 1.14.2.1) AFTER BLOCKADE OF AXONAL TRANSPORT

Abstract— The distribution of DBH activity between soluble and sedimentable fractions of hypotonic homogenates was examined in rat sympathetic ganglia and nerves after interruption of axonal transport. Local application of colchicine to superior cervical ganglia caused an increase mainly in particulate DBH activity, which was presumably bound to membranes. Likewise, in sciatic nerves, particulate DBH activity accumulated on both sides of a ligature and disappeared from a region well below a ligature much faster than did soluble activity. On the other hand, 18 h after simultaneous application of two ligatures to the nerve, neither total DBH activity nor subcellular distribution of this activity changed in the isolated nerve region. More detailed analysis showed that ligation affected the distribution of DBH activity within a fraction that sedimented at 140,000 g after homogenization of nerves in isotonic sucrose. Just above a ligature, osmotically releasable DBH activity was a smaller proportion of the sedimentable activity than in control nerves. However, as compared to controls, osmotically releasable DBH activity was a larger proportion of the activity in the sedimentable fraction from a region well below a ligature. A model was developed which accounts for some of these results by postulating that DBH is associated with different compartments in sciatic nerve which have different rates of transport and different proportions of soluble and bound enzyme.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

Developmental changes in the distribution of rat brain pyroglutamate aminopeptidase,a possible determinant of endogenous cyclo(His-Pro) concentrations

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and Pyroglutamate aminopeptidase activity in adult and developing rat brains were studied. A comparison of the subcellular distribution of Pyroglutamate aminopeptidase activity in hypothalamic and cerebral cortical extracts from adult rats exhibited remarkable differences. In hypothalamus, the enzyme activity was mainly associated with the soluble fraction whereas in cortex it was predominantly associated with the particulate fractions. During postnatal development, the brain concentrations of cyclo(His-Pro) and Pyroglutamate aminopeptidase activities declined with age. These data suggest that Pyroglutamate aminopeptidase activity, but not TRH, plays an active role in determining the levels of endogenous cyclo(His-Pro) concentrations in brain.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

Ultrastructure of synaptosomes from one-day old and adult rat brain stem

Summary The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.This study was supported by a grant from the Paulo Foundation.     

DNA and DNA-polymerase activity in chicken brain regions during ontogeny

Abstract—

• 1 The DNA content of cerebral hemispheres, optic lobes, cerebellum and remainder was determined in chicken brains from the 11th day of embryonic life to 6 weeks after hatch. Each region showed a characteristic pattern of variation during development. The cerebellum showed the most rapid and the optic lobes the least rapid rate of DNA increase during the period studied. The concentration of DNA within these regions decreased continuously with age except for that of the cerebellum which passed through a maximum just before hatching.
• 2 The nature of the DNA-polymerase activity in soluble extracts from these brain regions seemed to be similar to the properties reported for this enzyme activity in other vertebrate tissues. Glycerol was stimulatory and denatured DNA was preferred to native DNA as primer. The requirements for magnesium ions and DNA were absolute. The requirement for deoxynucleoside triphosphates indicated this to be a replicative rather than a terminal addition enzyme. At nearly every age the level of enzyme activity was highest in extracts from the embryonic cerebellum.
• 3 The particulate fraction from brain homogenates decreased the DNA-polymerase activity observed in soluble brain extracts. Data are presented which indicate that this inhibition was the result of dephosphorylation of the deoxynucleoside triphosphate substrates by an ATPase in the brain particulate fraction whose activity increases during ontogeny.

     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide-hydrolysing activities in rat brain

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide hydrolysing activities (arylamidase activity) was studied from left and right rat brains, each including hemisphere, cerebellum and brain stem. Both soluble Leu- and Arg-beta-naphthylamide hydrolysing activities showed the highest levels in the synaptosomal fraction. However, the microsomal fraction presented the highest levels when membrane-bound activity was assayed. When we used Asp-beta-naphthylamide as substrate, there were no differences among fractions in the membrane-bound activity, and the highest soluble activity was present at the mitochondrial level. Two different patterns in the subcellular distribution of enzymatic activity were observed: One of them was the result of the use of Leu- or Arg-beta-naphthylamide as substrate and the other when Asp-beta-naphthylamide was employed. No differences between left and right brains in soluble or membrane-bound activities were found.     

Biotinidase in the porcine cerebrum

Biotinidase activities found in porcine brains (n = 3) were as follows: cerebrum, 4.4 +/- 0.2 pmol/min per milligram of protein; cerebellum, 7.6 +/- 0.3 pmol/min per milligram of protein; medulla, 2.9 +/- 0.3 pmol/min per milligram of protein. These values are relatively high compared with the activities in rat or guinea pig brains. Subcellular distribution of biotinidase was found mainly in the soluble cytoplasmic fraction (S3), i.e., in the supernatant of 0.32 M sucrose S2 solution after ultracentrifugation at 105,000g for 90 min. This is in contrast to the guinea pig livers, in which the subcellular distribution of biotinidase is mainly found in the microsomal fraction. After a seven-step purification (22,200-fold enrichment), porcine brain biotinidase is identified as a single polypeptide by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and its molecular weight is determined as 68,000 Da. The isoelectric point of the enzyme was 4.3. Sialidase treatment strongly suggests the presence of sialyl residues in this enzyme. Amino acid analysis indicates relatively high hydrophilicity and high content of glycine and serine. The enzyme activity is inhibited by organic mercurials, but not by diisopropylfluorophosphate. Abundant soluble biotinidase in brain cytoplasm may play an important role which has not been discovered yet.     

Polymorphism of Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

The molecular forms and membrane association of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and pseudocholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) were determined in the presence of protease inhibitors in dissected regions of developing human fetal brain, as compared with parallel areas from mature brain. All areas contained substantial cholinesterase activities, of which acetylcholinesterase accounted for almost all the activity. Two major forms of acetylcholinesterase activity, sedimenting at 10-11S and 4-5S, respectively, were detected on sucrose gradients and possessed similar catalytic properties, as judged by their individual Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M). The ratio between these forms varied by up to four- to fivefold, both between different areas and within particular areas at various developmental stages, but reached similar values (about 5:2) in all areas of mature brain. Acetylcholinesterase activity was ca. 35-50% low-salt-soluble and 45-65% detergent-soluble in various developmental stages and brain areas, with an increase during development of the detergent-soluble fraction of the light form. In contrast, pseudocholinesterase activity was mostly low-salt-soluble and sedimented as one component of 10-11S in all areas and developmental stages. Our findings suggest noncoordinate regulation of brain acetylcholinesterase and pseudocholinesterase, and indicate that the expression of acetylcholinesterase forms within embryonic brain areas depends both on cell type composition and on development.     

Subcellular fractionation of postmortem brain

Abstract— Procedures used to separate subcellular organelles from fresh brain were applied to brains which had been removed from guinea pigs (1) immediately after death; (2) after the dead animal was maintained at room temperature for 3 h, followed by 16–17 h at 4°C; or (3) after the dead animal was maintained for 19–20 h at room temperature. Subcellular fractionation of the brains in 0.32 M sucrose was followed by discontinuous density gradient centrifugation of the crude mitochondrial fraction. After overnight storage of brains at room temperature, there was a moderate shift in succinate dehydrogenase activity from sub-fraction C (mitochondria) to subfraction B (synaptosomes). There was little change in the distribution of galactolipid among particulate subfractions. There was little change in distributions of monoamine oxidase or acetylcholinesterase activities. Under the less extreme postmortem conditions, there were no shifts in the subcellular distributions of brain enzymes. Ultrastructural changes were much more profound and consisted of losses of identifiable mitochondria and synaptosomes and a progressive increase in very dense bodies. Our results suggest that in spite of the marked morphological changes, meaningful separation of subcellular organelles can be achieved with postmortem tissue.     

Influence of alloxan diabetes and insulin treatment on the activity of alanine aminotransferase in rat brain regions, liver and heart

Studies with brain alanine aminotransferase showed higher activity of the enzyme in the soluble fraction of cerebellum. Among the tissues, the liver soluble fraction was the richest source of the enzyme. Alloxan-induced diabetes caused both regional and time-dependent variations in the activity of brain alanine aminotransferase. Significant among these changes were the decrease in both soluble and particulate enzyme from cerebral hemispheres and an increase in the soluble enzyme activity from cerebellum at early stages of diabetes. Brain stem did not show any marked change in enzyme activity. Liver and heart enzyme, however, increased significantly after 1-2 weeks of diabetes. Insulin treatment to diabetic animals caused an 'over-shoot' in soluble alanine aminotransferase activity, particularly in cerebellum and liver.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.