5''-Nucleotidase in Rat Brain Myelin

Rat brain myelin showed substantial activity of 5'-nucleotidase. The specific activity in myelin was enriched two- to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5'-nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5--9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5'-AMP, 5'-UMP, and 5'-CMP were the preferred substrates, and 5'-GMP was hydrolyzed at approximately one-half the rate of the other mononucleotides. The very low rates of cleavage of beta-glycerophosphate and 2'-AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5'-nucleotidase activity in myelin. The 5'-nucleotidase was inhibited by concanavalin A and was protected by alpha-methyl-D-mannoside against inhibited by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5'-nucleotidase in the rat brain.     

Regional Localization and Subcellular Compartmentalization of Thyrotropin-Releasing Hormone in Adult Human Brain

In the current study, we sought to define the subcellular compartmentalization of thyrotropin-releasing hormone (TRH) in adult human brain tissues. Upon evaluating tissues (3-24 h post mortem) from 62 humans, ranging in age from 5 to 75 years, we found that TRH was widely distributed throughout the brain. The highest TRH concentration (ng/mg protein) was in the stalk-median eminence region of the hypothalamus (19.3 +/- 3.3, mean +/- SE); the TRH concentration in the hypothalamus, exclusive of the stalk-median eminence, was much lower (1.7 +/- 0.2). Substantial quantities of TRH also were detected in the medulla oblongata (0.26 +/- 0.08), mammillary bodies (0.33 +/- 0.25), and optic chiasm (0.14 +/- 0.07). Lower levels of TRH were found in the amygdala (0.060 +/- 0.015) and the corpus striatum (0.033 +/- 0.010). TRH was near or below the limits of detection in tissues of the cerebral and cerebellar cortices, the olfactory bulbs, the pons, and the hippocampus. When homogenates of medial basal hypothalamic tissue (prepared in 0.32 M sucrose-10 microM CaCl2) were fractionated by means of differential centrifugation, most of the TRH was recovered in subcellular particles which were pelleted at 10,000 X g and which contained the highest amounts of occluded LDH activity. When the nuclei-free supernatant fluid (900 X g S) was fractionated on discontinuous sucrose density gradients or continuous sucrose density gradients, most of the TRH was recovered in subcellular fractions containing synaptosomes. The subcellular distribution of TRH appeared to be stable for up to 24 h post mortem in rat and human brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic Distribution of the Cholinergic-Specific Antigen Chol-1 and 5''-Nucleotidase in Rat Brain, as Determined by Complement-Mediated Release of Neurotransmitters

Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, gamma-aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4-dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement-mediated lysis. Three antisera were used: anti-5'-nucleotidase, anti-Chol-1, and anti-rat cerebrum. Anti-5'-nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti-Chol-1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Anti-rat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5'-nucleotidase was not present on any of the terminals studied, and that Chol-1 is only present on cholinergic terminals.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.     

Soluble 5'-Nucleotidase Activities in Rat Brain

5'-Nucleotidase activity was assayed in 105,000-g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5'-nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.     

The Subcellular Distribution of Peptide Histidine Isoleucine Amide-27-Like Peptides in Rat Brain and Their Release from Rat Cerebral Cortical Slices In Vitro

The subcellular distribution of peptide histidine isoleucine amide (PHI)-27-like peptides (PLP) was investigated in rat cerebral cortex and whole rat brain in comparison with the distribution of vasoactive intestinal peptide (VIP). The highest content of PLP was found in the crude mitochondrial fraction (P2) and was also detected in the microsomal pellet. PLP was recovered in synaptosomes when further fractionation of P2 was performed. This distribution of PLP closely follows that of VIP and is suggestive of possible storage in vesicles at the nerve terminal. Basal release of PLP from rat cerebral cortical slices was below the detection limit of the PHI radioimmunoassay. However, depolarization by 55 mM potassium induced measurable PLP release. This release was calcium-dependent. These findings support the hypothesis that PLP could play a role in neurotransmission.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical Studies on 5''-Nucleotidase of Schwann Cells in Degenerated Nerve

This report describes the partial characterization of 5'-nucleotidase (5'-AMPase) in Schwann-cell plasmalemmae (PM) prepared from degenerated cat sciatic nerve. 5'-AMPase was enriched 3.7-fold in the PM fraction over that of the crude homogenate preparation. The plant lectin concanavalin-A (Con-A) reduced Schwann cell PM 5'-AMPase activity in a concentration-dependent manner (30-600 micrograms/ml). Plasma membrane 5'-AMPase activity was maximally inhibited to 20% of control values by Con-A (400-600 micrograms/ml), and activity returned to control levels by pretreatment with the hapten sugar alpha-methyl-D-mannoside (50 mM). Equimolar concentrations of UDP and ADP (100 microM) reduced the rate of hydrolysis of labeled AMP to labeled adenosine in PM to 45% and 35% of control, respectively. This is the first study to characterize a Schwann-cell PM enzyme and demonstrates that 5'-AMPase may be used as a Schwann-cell PM marker enzyme.     

Biogenic Aldehyde Metabolism in Rat Brain: Subcellular Distribution of Aldose Reductase and Valproate-Sensitive Aldehyde Reductase

Reductase activity towards two aldose substrates has been examined in subcellular fractions prepared from rat brain. The reduction of glucuronate, which is sensitive to inhibition by the anticonvulsant drug sodium valproate, corresponds to the major high-Km aldehyde reductase in brain. Xylose reduction that is insensitive to valproate inhibition has characteristics consistent with the activity of aldose reductase (EC 1.1.1.21). Both enzymes are predominantly localized in the cytosolic fraction. The significance of the location of these two reductases is discussed in relation to the compartmentation of catecholamine metabolism in brain.     

Analytical Subcellular Fractionation of Rat Cortex: Resolution of Serotonergic Nerve Endings and Receptors

Abstract: An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two-step procedure involving a differential centrifuga-tion using the five-fraction scheme and an isopycnic cen-trifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S₂ receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mito-chondrial (M and L) fractions. After equilibration in density gradients, serotonin S₂ receptors revealed two peaks, which were similarly affected after treatment with ami-triptyline and/or yohimbine. There is no evidence to suggest that serotonin S₂ receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S₂ receptors and 5′-nucleoti-dase are very similar, but differ from that of UDP-galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5-HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5′-nucleotidase, and receptor binding), is rapid and clearly differentiates pre-and postsynaptic constituents.     

Evidence for Neuronal Localization of Histamine-N-Methyltransferase in Rat Brain

Abstract: There is evidence that histamine may be a neurotransmitter in mammalian brain. Histamine in neurons of the central nervous system is easily released and rapidly turned over. The cellular localization of histamine- N -methyltransferase, the proposed histamine-inactivating enzyme, was investigated by measuring its activity in rat striatum after applying neurochemical or electrolytic lesions. The results indicate a major neuronal localization of the enzyme in this area.     

Newly Synthesized Cholecystokinin in Subcellular Fractions of the Rat Brain

Abstract: The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [³⁵S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, P₂, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, K_av , of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tet-rapeptide amide, CCK-4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.     

PO Protein and 2'',3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity in the Peripheral Nerve and Subcellular Fractions of the Trembler Mouse

To investigate the biochemical abnormalities of the Trembler mouse, the level of the PO protein (as % of total protein) and the activity of CNP was compared in the sciatic nerve and subcellular fractions of normal and mutant littermates. There was a significant decrease in both of these myelin markers in total nerve homgenates of the neurological mutant compared with the control animals. Immunoassay of the PO protein and polyacrylamide gel analysis of proteins indicated an accumulation of a protein with an apparent molecular weight of 67K in mutant nerve extracts. The mutant nerve also had relatively decreased levels of a protein of molecular weight about 41K that cross-reacted with antibody to PO protein. The Trembler mouse exhibited a larger percentage recovery of PO protein and CNP activity in subcellular fractions denser than the myelin sheath. Together these results are consistent with the theories that these denser components represent immature forms of myelin and that the Trembler mutant is characterized by hypomyelination.     

Regional and Subcellular Localization in Rat Brain of the Enzymes That Can Synthesize γ-Hydroxybutyric Acid

Abstract: Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.     

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and Immunocytochemical Evidence for a Deficiency of Normal Interfascicular Oligodendroglia in the CNS of the Dysmyelinating Mutant (md) Rat

Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant.     

Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Subcellular Distribution of Mammalian Tachykinins in Rat Basal Ganglia

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.     

Metabolism of Extracellular Adenine Nucleotides by Cultured Rat Brain Astrocytes

Intact astrocytes cultured from newborn rat cerebral cortex rapidly converted extracellular ATP to ADP. The ATPase responsible was apparently not saturated, even at 750 microM ATP. In contrast, the conversion of ADP to AMP was slow, and the reaction was limiting for the subsequent dephosphorylation process. Adenosine formation was the only fate for AMP. The reaction was catalyzed by 5'-nucleotidase with an apparent Km of 55 microM for AMP and appeared to be inhibited by high concentrations of ATP and ADP. Astrocytes were able to take up adenosine with an apparent Km value of 45 microM. Uptake was inhibited by dipyridamole but not by anti-5'-nucleotidase IgG. The results support the proposal that astrocytes play a role in modulating synaptic events involving ATP and adenosine.