bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of Succinyl‐CoA:3‐Oxo‐Acid CoA‐Transferase from Rat Brain
Authors: Russell Joseph J. , Patel Mulchand S.
Published: October 2006

Abstract: Rat brain succinyl‐CoA:3‐oxo‐acid CoA‐transferase (3‐Oxo‐acid CoA‐transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3‐150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl‐CoA) are consistent with a “ping pong” mechanism. Substrate inhibition appears above approximately 1 mM acetoacetate. Apparent Km, values were 70 μM for acetoacetate and 156 μM for succinyl‐CoA (the forward reaction), and 59 μM for acetoacetyl‐CoA and 25 mM for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.
Kinetic Studies of Mouse Brain Transketolase
Authors: Blass John P. , Piacentini Silvia , Boldizsar Eva , Baker Andrea
Published: October 2006

Abstract: The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme‐coupled spectrophotometric assay. The apparent Km for ribose‐5‐phosphate was 330 μM, for d‐xylulose‐5‐phosphate was 120 μM, and for thiamine pyrophosphate was 7 μM. However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate‐ dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.
Antibodies Specific for α‐N‐Acetyl‐β‐Endorphins: Radioimmunoassays and Detection of Acetylated β‐Endorphins in Pituitary Extracts
Authors: Weber Eckard , Evans Christopher J. , Chang Jaw‐Kang , Barchas Jack D.
Published: October 2006

Abstract: Antibodies specific for α‐N‐acetyl‐β‐endorphins have been prepared by injecting into rabbits either α‐N‐acetyl‐β‐endorphin(1‐31) or [α‐N‐acetyl, ε‐acetyl‐Lys9]‐β‐endorphin(1‐9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α‐N‐acetyl‐β‐endorphins. The radioimmunoassays were used to measure α‐N‐acetylated β‐endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β‐endorphin and α‐N‐acetyl‐β‐endorphin immunoreactivity, a relative ratio of β‐endorphin acetylation was obtained. The relative acetylation of β‐endorphin was highest in rat posterior‐intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β‐endorphin. Human pituitary extracts did not contain acetylated β‐endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β‐endorphin were resolved in extracts of rat posterior‐intermediate pituitaries. Two of these peptides corresponded to α‐N‐acetyl‐β‐endorphin(1‐31) and ‐(1‐27). One acetylated β‐endorphin fragment had the same size as α‐N‐acetyl‐β‐endorphin(1‐27) but was eluted earlier from the cation exchange column. This peptide had full cross‐reactivity with antibodies directed against the middle and amino‐terminal parts of β‐endorphin. Compared with α‐N‐acetyl‐β‐endorphin(1‐27), it had much less cross‐reactivity with antibodies directed against the COOH‐terminal part of β‐endorphin, suggesting that it was a COOH‐terminally modified derivative of β‐endorphin(1‐27). The remaining N‐acetylated β‐endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y‐endorphin, i.e., β‐endorphin(1‐17), but smaller than β‐endorphin(1‐27). They had full cross‐reactivity in an amino‐terminally directed β‐endorphin radioimmunoassay and a greatly diminished cross‐reactivity with antibodies to the middle region of β‐endorphin.
The Regional Concentrations of S‐Adenosyl‐l‐Methionine, S‐Adenosyl‐l‐Homocysteine, and Adenosine in Rat Brain
Authors: Gharib A. , Sarda N. , Chabannes B. , Cronenberger L. , Pacheco H.
Published: October 2006

Abstract: The concentrations of S‐adenosyl‐l‐methionine (SAM), S‐adenosyl‐l‐homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.
Immunochemical Identification of 2′, 3′‐Cyclic Nucleotide 3′‐Phosphodiesterase in Central and Peripheral Nervous System Myelin, the Wolfgram Protein Fraction, and Bovine Oligodendrocytes
Authors: Sprinkle T. J. , Sheedlo H. J. , Buxton T. B. , Rissing J. P.
Published: October 2006

Abstract: Myelin proteins and the total Wolfgram protein fraction were isolated from the CNS of several mammalian species and characterized with rabbit anti‐bovine 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) antisera after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose membranes. The corresponding CNP proteins cross‐reacted across all species examined, suggesting that the CNP amino acid sequence was fairly well conserved in all six species. The same corresponding proteins were also identified immunochemically in the crude total Wolfgram protein fraction in the region of the W1 myelin protein, thus further supporting and extending two different previous reports indicating a relationship between CNP and the W1 protein. In addition to these CNS enzyme sources, peripheral nervous system CNP (rabbit and rat sciatic nerve) was also recognized by these same rabbit anti‐bovine (CNS) CNP antisera. CNP was also detected in freshly isolated delipidated bovine oligodendrocyte membranes. These results suggest that rabbit anti‐bovine CNP antisera may be of use in localization and structural studies of this enzyme in several different species and will permit clear identification of CNP in oligodendrocytes and their isolated membrane fractions.
Chemical Analysis of Organotypic Cultures of Mouse Spinal Cord in Normal, Demyelinative, and Nondemyelinative Conditions
Authors: Roth German A. , Yu Robert K. , Bornstein Murray B.
Published: October 2006

Abstract: Several biochemical parameters were analyzed in cultured embryonic mouse spinal cord during various stages of normal myelinogenesis or demyelination. In cultures demyelinated by exposure to anti‐whole CNS tissue serum plus complement, the activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37) was decreased 70%, whereas in cultures that did not show morphological changes with complement‐inactivated anti‐CNS serum or anti‐myelin basic protein serum, the activity was 30% lower than in control cultures. The lipid composition of these cultures was quantitated by means of high‐performance thin‐layer chromatography densitometry technique. Cultures with normal nutrient medium alone or with the addition of 5% normal rabbit serum plus 10% guinea pig serum had 30% of the total lipid content of that present in newborn mouse spinal cord of the corresponding age. There were, however, relatively more lysophospholipids, cholesterol esters, triglycerides, and free fatty acids and less phosphatidylethanolamine and galactolipids in cultures as compared with normal spinal cord. Explants demyelinated by exposure to anti‐CNS serum plus complement demonstrated principally a 70% decrease in the content of galactolipids with respect to normal cultures. When complement was inactivated, total lipids increased 42% (with increases of 40–70% in individual lipids). Inclusion of anti‐myelin basic protein serum plus complement in the medium produced no significant changes in the lipid composition of the cultures.
Sulfatides in Prenatal Metachromatic Leukodystrophy
Authors: Baier W. , Harzer K.
Published: October 2006

Abstract: In one 21‐week‐old fetus with prenatally diagnosed metachromatic leukodystrophy, galactolipid contents were determined in the forebrain cortex, cerebellum, brainstem, spinal cord, and kidney and compared to an appropriate control. Spinal cord and kidney showed the highest sulfatide accumulation as a consequence of deficient cerebroside sulfatase activity. No increase, but a measurable amount of sulfatide, was detected in the forebrain. The prenatal neural sulfatides contained a high proportion of the hydroxy fatty acid component. The galactosyl ceramides were not reduced in the early stage of the demyelinating disease.
Phospholipid Methylation Increases [3H]Diazepam and [3H]GABA Binding in Membrane Preparations of Rat Cerebellum
Authors: Di Perri B. , Calderini G. , Battistella A. , Raciti R. , Toffano G.
Published: October 2006

Abstract: The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ([3H]γ‐aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S‐Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0°C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and β‐adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro‐α‐ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]‐diazepam binding.
Modulation of [3H]Muscimol Binding in Rat Cerebellar and Cerebral Cortical Membranes by Picrotoxin, Pentobarbitone, and Etomidate
Authors: Quast Ulrich , Brenner Otto
Published: October 2006

Abstract: Modulation of [3H]muscimol binding by picrotoxin, pentobarbitone, and etomidate was investigated in rat cerebellar and cerebral cortical membranes. In cerebellum, at 37°C in the presence of chloride ions (150 mM), picrotoxin and picrotoxinin decreased specific [3H]‐muscimol binding to 43 ± 3% of control, with an EC50 of 1.2 ± 0.1 μM. [3H]Muscimol saturation experiments in the presence and absence of picrotoxin indicated that the picrotoxin effect was primarily due to a loss of high‐affinity muscimol sites with KD∼ 10 nM. Pentobarbitone enhanced specific [3H]muscimol binding to 259 ± 3% of control, with EC50= 292 ± 37 μM, and etomidate increased binding to 298 ± 18%, with EC50= 7.1 ± 1.0 μM. The influence of temperature and chloride ion concentration on these effects was investigated by comparing experiments at 37 and 0°C in the presence or absence of chloride at constant ionic strength. The results indicate that studies at 0°C underestimate the coupling between GABA receptors and barbiturate sites and that they greatly overestimate the importance of chloride ions in this phenomenon. In cerebral cortical membranes (37°C, 150 mM Cl−), the effect of picrotoxin was similar to that observed in cerebellum, whereas the effects of pentobarbitone and etomidate were greater, but occured at higher concentrations.
Metabolic Consequences of Inhibition of Cerebral Adenosylmethionine Decarboxylase by Methylglyoxal bis(Guanylhydrazone)
Authors: Hietala Oili A. , Pulkka Anitta E. , Lapinjoki Seppo P. , Laitinen Päivi H. , Pajunen Antti E. I.
Published: October 2006

Abstract: Effects of intracerebroventricularly injected methylglyoxal bis(guanylhydrazone) on polyamine metabolism in mouse brain were recorded during 180 h after a single dose of 3.4 μmol/kg body weight. Cerebral concentrations of 31 other amino compounds were also asayed during the experiment. The drug caused a significant inhibition of adenosylmethionine decarboxylase that lasted for 50 h, with the maximal decrease, about 70%, occurring between 5 and 10 h after injection. Significant decreases of brain spermidine and spermine concentrations were observed in three phases. Two transient decreases occurred at 10 and at 35 h, and a longer‐lasting one between 60 and 100 h. Ornithine decarboxylase was stimulated within 5 h after the injection, reaching a maximal level about 30‐fold above normal at 60 h, and returned to control level at 140 h. This stimulation was accompanied by significant accumulation of the reaction product, putrescine, in the brain. It was maximally > 10‐fold above normal at 160 h, and was still significantly above control at the end of the observation period. The time course of changes in the parameters of polyamine metabolism was regarded as support of a previously presented hypothesis that limiting putrescine concentration may play a role in the regulation of cerebral polyamine metabolism. In addition, the present results emphasize the possibility that changes in the activities of catabolic reactions may also contribute to the regulation of cerebral polyamine concentrations. Of the 31 amino compounds analyzed, only the concentrations of ornithine, urea, glutamine, and glutamate showed significant changes from normal. Ornithine concentration first was significantly increased at 25 h, whereafter it decreased and was somewhat below normal for most of the period between 60 and 180 h. The urea concentration showed a tendency to increase throughout the experiment, being significantly elevated at the end. These changes were regarded as suggesting that the increased need for ornithine in putrescine synthesis is satisfied mainly by increased arginine uptake and degradation. The magnitude of urea accumulation suggested that metabolism of ornithine to glutamate was also accelerated. An unexpected shift toward glutamine in the glutamine/glutamate relationship was observed during the first 100 h. However, the total concentration of these two compounds was quite constant throughout the experiment. Inhibition of ornithine decarboxylase by intraventricular injection of 2‐difluoromethylornithine was tried during the study, but sufficient doses could not be used without induction of acute side effects.
Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum
Authors: Kuno Takayoshi , Saijoh Kiyofumi , Tanaka Chikako
Published: October 2006

Abstract: D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3–[(3‐cholamidopropyl)‐dimethylammonio]‐1 ‐propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]‐spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of do pamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL‐4B column chromajography as a large molecule, with a Stokes radius of ∼90 Å. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.
Anterograde Axonal Transport in Rats During Intoxication with Acrylamide
Authors: Sidenius P. , Jakobsen J.
Published: October 2006

Abstract: Anterograde axonal transport was examined in sensory nerves of rats intoxicated with a low dose (group I) or a high dose (group II) of acrylamide. After injection of either [35S]methionine and [3H]fucose or [3H]proline into the dorsal root ganglia of the 5th lumbar roots, distribution of protein label was measured in 3‐mm segments of the sciatic nerve at intervals of 2 h, 4 h, 10 days, and 26 days. No difference in ganglion incorporation was present at 4 h, and the fast transport velocity of methionine label also remained normal [14.7 ± 1.3 mm/h (mean ± SD) in controls versus 14.6 ± 0.3 mm/h and 15.4 ± 1.2 mm/h in acrylamide group I and II, respectively]. Neither was there any decrease in transport velocity of proline label of slow component b (4.18 ± 0.29 mm/day in controls versus 4.29 ± 0.17 mm/d and 4.22 ± 0.29 mm/day in acrylamide group I and II, respectively). In slow component a, however, a significant reduction in the fractional amount of proline label was found (20.8 ± 4.0% in controls versus 17.6 ± 14.9% and 9.7 ± 5.9% in acrylamide group I and II, respectively). Again no decrease in transport velocity was observed (1.03 ± 0.02 mm/day in controls versus 1.06 ± 0.08 mm/day and 1.07 ± 0.03 mm/day in acrylamide group I and II, respectively), and closer inspection of the activity along the nerve did not reveal any alteration in skewness or ‘peakedness’ of the distribution curve. The reduction in amount of protein carried in the slow axonal transport component in rats with severe acrylamide neuropathy (group II) could be associated with fibre breakdown at a late stage of the neuropathic process. The most important consequence of the study is, however, that in contrast to previous suggestions, during acrylamide intoxication no changes are present in protein incorporation or in anterograde axonal transport which can explain the initial pathological or functional abnormalities of the distal axons.
Transport of Histidine into Synaptosomes of the Rat Central Nervous System
Authors: Chudomelka Patricia J. , Murrin L. Charles
Published: October 2006

Abstract: Histidine transport into synaptosomes was studied in order to characterize this aspect of histamine synthesis in neurons. Histidine transport was found to be independent of sodium, calcium, and magnesium ions and dependent upon potassium and chloride ions. Histidine transport was also found to be energy dependent, and subcellular fractionation studies suggested it was highly localized to nerve terminals. Kinetic analysis of histidine transport in several brain regions indicated the presence of two uptake sites, a high‐affinity site with a Km of approximately 35 μM and a low‐affinity site with a Km in the millimolar range. Density of the high‐affinity site, as reflected by Vmax, correlates well with density of proposed histaminergic innervation. Rate of histidine transport was not altered by prior depolarization of the synaptosomes, indicating that histidine transport probably does not play a regulatory role in histamine synthesis.
Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine
Authors: Porta Raffaele , Schatz Robert A. , Tatter Stephen B. , Sellinger Otto Z.
Published: October 2006

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S‐adenosyl‐homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4‐14C]putrescine, the specific radioactivity (sra) of the newly formed [14C]spermidine remained unchanged. Conversely, after i.vt. l‐[3,4‐14C]methionine, the other polyamine precursor, significantly higher sra values for [14C]spermidine and [14C]spermine were recorded in the brains of the MSO‐treated animals. [14C]S‐ adenosylmethionine in the brain of the MSO‐treated animals was also more highly labeled following [1‐14C]‐methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4‐14C]‐methionine. The results of these experiments show both significantly lower sra values for [14C]spermidine and [14C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.
Release of Endogenous Amino Acids from Superior Colliculus of the Rabbit: In Vitro Studies After Retinal Ablation
Authors: Sandberg Mats , Corazzi Lanfranco
Published: October 2006

Tissue slices from the superior colliculi (SC) of the rabbit were superfused and investigated 1 week after unilateral eye removal. Amino acid levels were determined both in the tissue slices and in the medium after chemical depolarisation (56 mM K). The amino acid determinations were done fluorimetrically by precolumn derivation and HPLC separation. Colliculi contralateral to the enucleation exhibited a 16% reduction in glutamate compared with the ipsilateral colliculi. The Ca‐dependent release of glutamate or other amino acids tested was not appreciably affected by enucleation. However, both the total and the Ca‐independent release of glutamate was lower from contralateral SC slices compared with the ipsilateral slices. The results do not favour glutamate as the major optic nerve transmitter in the rabbit, but do not rule out glutamate as a transmitter in a minor population of retinal fibres.
Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period
Authors: Sarlieve L. L. , Bouchon R. , Koehl C. , Neskovic N. M.
Published: October 2006

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T4) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP‐galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T4 was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T4 also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T4 and GH may have both independent and complementary actions on the myelin‐associated enzymes during the early postnatal period of brain development.
Multiple Forms of Glutamate Decarboxylase in Porcine Brain
Authors: Spink David C. , Wu Susan J. , Martin David L.
Published: October 2006

Three forms of glutamate decarboxylase from hog brain (termed α‐, ß‐, and γ‐GAD) were separated by hydrophobic interaction chromatography on phenyl‐Sepharose, by isoelectric focusing, and by polyacryl‐amide gel electrophoresis. When rechromatographed on phenyl‐Sepharose, each form migrated as a single entity, indicating that the forms are not readily interconvertible. The three forms are not different‐sized aggregates of one form, since all three have the same approximate molecular weight (100,000) as determined by Sephadex G‐200 chromatography. The pIs of the three forms separated by phenyl‐Sepharose were determined by isoelectric focusing. The values obtained (5.3, 5.5, and 5.8 for α‐, ß‐, and γ‐GAD, respectively) were comparable to the pIs of the three peaks of activity observed upon focusing of enzyme that had been subjected to phenyl‐Sepharose chromatography. These results indicate that phenyl‐Sepharose chromatography and isoelectric focusing separate the same three components. When synaptosomal extracts were analyzed by phenyl‐Sepharose chromatography without intervening purification steps, all three forms were present, but the proportion of ß‐GAD was somewhat higher and that of γ‐GAD somewhat lower than in the usual preparations.
The Identity of Hexokinase Activities from Mitochondrial and Cytoplasmic Fractions of Rat Brain Homogenates
Authors: Needels Dwight L. , Wilson John E.
Published: October 2006

Cytoplasmic hexokinase (ATP: d‐hexose 6‐phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran‐Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially‐bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.
The Presence of Phospholipase D In Rat Central Nervous System Axolemma
Authors: DeVries George H. , Chalifour Robert J. , Kanfer Julian N.
Published: October 2006

An axolemma‐enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma‐enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.
Effect of Colchicine on 45Ca and Choline Uptake, and Acetylcholine Release in Rat Brain Synaptosomes
Authors: O'Leary M. E. , Suszkiw J. B.
Published: October 2006

The effects of 1 and 10 mM colchicine on the K+‐evoked release of preformed and newly synthesized acetylcholine and on the K+‐depolarization‐stimulated uptake of 45Ca were compared in rat brain synaptosomes. Preincubation of synaptosomes with 1 mM colchicine had little effect on transmitter release and on uptake of 45Ca; 10 mM colchicine inhibited both the release of transmitter and uptake of 45Ca by 58%. Since 1 mM colchicine has been shown to disaggregate intrasynaptosomal micro‐tubules almost completely, but to be without effect on release of either preformed or newly synthesized acetylcholine in our experiments, it is concluded that colchicine modifies transmitter release by reducing Ca2+ influx, rather than by its postulated intracellular action on microtubule‐mediated transmitter mobilization. In addition, 1 and 10 mM cholchicine significantly inhibited the high‐affinity choline uptake in synaptosomes. This hemicholinium‐like action of colchicine may contribute to the reduction of transmitter release.
Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α‐N‐Acetyl Derivative
Authors: Boarder Michael R. , Weber Eckard , Evans Christopher J. , Erdelyi Elizabeth , Barchas Jack
Published: October 2006

A sensitive assay, which cross‐reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ˜15 fmol of β‐endorphin per incubation tube, i.e., ˜ 100‐fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β‐endorphin) to 1.6 nM (Met‐enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu‐ or Met‐enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.
Tyrosine Hydroxylase Inactivation Following cAMP‐Dependent Phosphorylation Activation
Authors: Vrana Kent E. , Roskoski Robert
Published: October 2006

Tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP‐dependent protein kinase (largely by decreasing the Km of the enzyme for its pterin co‐substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho‐diesterase, cAMP‐dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent Km for pterin co‐substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent Km, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP‐dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent Vmax with no change in the low apparent Km for pterin co‐substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low‐molecular‐weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos‐phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.
Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor
Authors: DeVries George H. , Anderson M. G. , Johnson David
Published: October 2006

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose‐salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20‐40% (wt/wt) linear sucrose gradient in a Beckman Ti‐14 Zonal Rotor. After centrifu‐gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′‐cyclic nucleotide 3′‐phos‐phodiesterase (EC 3.1.4.37), myelin‐associated glycopro‐tein (MAG)]; microsomes [CDP‐choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho‐line hydrolase, EC 3.1.1.7), 5′‐nucleotidase (5′‐ribonu‐cleotide phosphohydrolase, EC 3.1.3.5), Na+,K+‐adeno‐sine triphosphatase (EC 3.6.1.3), [3H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin‐related markers were most enriched in the 20‐26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma‐related markers were most enriched in the 28‐32% region of the gradient, whereas the microsomal and mitochondrial‐related markers were enriched in the 35‐40% region of the sucrose density gradient. Mixing experiments utilizing 125I‐labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un‐ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20‐25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.
Comparative Hydrolysis of Sphingomyelin and 2‐N‐(Hexadecanoyl)‐Amino‐4‐Nitrophenyl‐Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH
Authors: Levade Thierry , Salvayre Robert , Douste‐Blazy Louis
Published: October 2006

Hydrolysis of sphingomyelin and 2‐N‐(hexade‐canoyl)‐amino‐4‐nitrophenyl‐phosphorylcholine (HDA‐PC), a synthetic analogue of sphingomyelin, by acid and Mg‐dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl2 nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA‐PC to sphingomyelin is ˜2). In contrast, Mg‐dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl2, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA‐PC to sphingomyelin is ˜0‐0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg‐dependent neutral sphingomyelinase.
Presence of Somatostatin, Enkephalins, and Substance P‐Like Peptides in Cultured Neurons from Embryonic Chick Cerebral Hemispheres
Authors: Louis J. C. , Rougeot C. , Bepoldin O. , Vulliez B. , Mandel P. , Dray F.
Published: October 2006

Abstract: The presence of peptides in pure cultures of neurons from 8‐day‐old chick embryo cerebral hemispheres has been investigated by means of specific radioimmunoassays and chromatographic purification. Somatostatin, Met‐enkephalin, Leu‐enkephalin, and substance P immunoreactive substances have been detected in 8‐day‐old cultures grown in serum‐free culture medium. The peptides were present in the cellular extracts, as well as in the culture medium extracts. β‐Endorphin, thyroliberin, luteinizing hormone‐releasing hormone, and ACTH could not be detected. The largest amount was accounted by somatostatin (48 ± 2 ng/mg protein). Some 60% of the somatostatin‐immunoreactive material was found in the culture medium. Met‐enkephalin, Leuenkephalin, and substance P were present at lower concentrations: 1.61 ± 0.27, 0.24 ± 0.02, and 0.14 ± 0.005 ng/mg protein, respectively. The identities of somatostatin‐ and enkephalin‐immunoreactive materials were confirmed by high pressure liquid chromatography. The findings suggest that cultured neurons that express dopaminergic and GABAergic properties contain peptides similar, if not identical, to somatostatin, Met‐enkephalin, Leu‐enkephalin, and substance P.
Studies on Subcellular Fractions Which Are Involved in Myelin Assembly: Labeling of Myelin Proteins by a Double Radioisotope Approach Indicates Developmental Relationships
Authors: Pereyra Pedro M. , Braun Peter E. , Greenfield S. , Hogan E. L.
Published: October 2006

Abstract: The question of developmental relationships amongst myelin‐related membranes in subfractions of myelinating mouse brain (15 days) was investigated by a time‐staggered double isotope protocol using [3H]leucine and [14C]leucine. Preliminary results are interpreted and discussed in the context of a mathematical conceptualization of pulse‐labeling kinetic analyses of myelin proteins in subcellular membrane compartments. Differences in ratio of the two leucine labels among proteins of myelin‐containing subfractions are interpreted as confirming metabolic differences relating to various stages of development rather than precursor‐product relationships. The incorporation into myelin of 14K, 17K, and 18.5K basic proteins (MBPs) occurs with relatively short delay times, following their synthesis (less than 5 min), and seems to occur simultaneously into all compartments. The 21.5K MBP and the proteolipid protein, on the other hand, require 10–14 min and 14–20 min, respectively. A scheme is presented to illustrate the probable assignment of subfractions to various myelin “compartments” during myelination, and to serve as a working hypothesis for studies on precursor‐product relationships.
The Influence of Specific Phospholipids on the Interaction of Hexokinase with the Outer Mitochondrial Membrane
Authors: Moller F. , Wilson John E.
Published: October 2006

Abstract: Repeated washing of a brain mitochondrial fraction results in a progressive decrease in the proportion of mitochondrially bound hexokinase that can be solubilized during a subsequent incubation with glucose‐6‐phosphate (glucose‐6‐P). Phospholipids removed during the washing procedure can be added back to washed mitochondria, resulting in enhancement of the solubilization by glucose‐6‐P. Column and thin‐layer chromatographic methods have been used to isolate and identify active phospholipids. Additional studies were performed with purified lipids obtained commercially. Both lysophospholipids and acidic phospholipids were active in enhancing solubilization of hexokinase by glucose‐6‐P. Phospho‐inositides, particularly diphosphoinositide, were quite effective, raising the possibility that the actively metabolized phosphoinositides may be involved in regulation of hexokinase binding in vivo.
Presence of a Membrane‐Bound Acetylcholinesterase Form in a Preparation of Nerve Endings from Torpedo marmorata Electric Organ
Authors: Li Zhi‐Yi , Bon Cassian
Published: October 2006

Abstract: We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings from Torpedo, to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabainresistant and ‐sensitive ATPase, lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1–5 μm diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic in Torpedo electric organs. We characterized this form as a membrane‐bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission in Torpedo electric organs and perhaps also in other cholinergic synapses.
Stress‐Induced Alterations in Metabolism of Γ‐Aminobutyric Acid in Rat Brain
Authors: Yoneda Yukio , Kanmori Kouji , Ida Shuji , Kuriyama Kinya
Published: October 2006

Abstract: The effect of a stressful manipulation on the metabolism of γ‐aminobutyric acid (GABA) in the rat brain was studied. Application of an immobilized stress to animals induced a significant increase in the striatal and hypothalamic GABA contents without affecting those in other central structures examined. It was also found that the increase in striatal GABA level preceded that in the hypothalamus. This increase in steady‐state levels of GABA in the striatum and hypothalamus disappeared at 12 h after the termination of the application of stress for 3 h, which exhibited a maximal stimulatory action on the GABA contents in both central areas. The activity of l‐glutamic acid decarboxylase was found to be significantly elevated in the striatum and hypothalamus following the stress application with a concomitant decrease in the content of L‐glutamic acid, which is converted to GABA by the catalytic action of the latter enzyme. The in vivo turnover of GABA in the brain was estimated by taking advantages of the postmortem accumulation of GABA following decapitation and of the selective inhibitory action of a low dose of aminooxyacetic acid on the GABA degrading systems, respectively. Analysis using these two different methods revealed that the cerebral turnover of GABA in vivo was not significantly altered under stressful situations despite of the increase in its steady‐state level. These results suggest that central GABA system may respond to the input of painful stimuli resulting from the application of a severe physical and psychological stressor, in addition to the well‐known functional alterations in catecholamine neurons. The functional significance of these alterations in the central GABA neurons is also discussed.
Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3′: 5′‐Cyclic Monophosphate in Cultured Murine Brain Cells
Authors: van Calker Dietrich , Löffler Fridolin , Hamprecht Bernd
Published: October 2006

Abstract: Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half‐maximally stimulating concentrations, EC50, in parentheses) ACTH‐(1–24) (10 nM) > α‐, β‐MSH (30 nM) > ACTH (≥ 100 nM) > γ‐MSH, ACTH−(1‐10), −(4‐10), −(4‐11) (≥0.5 μM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an α‐adrenergic receptor), adenosine (acting via an A1 receptor) and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and α‐and β‐MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals and (2) ACTH and α‐MSH occur in brain.
Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor
Authors: Bigbee John W. , Bigner Darell D. , Pegram Charles , Eng Lawrence F.
Published: October 2006

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K‐dalton) GFA peptides. SDS (0.15%)‐soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two‐dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS‐soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.
Protein Carboxyl Methylation Increases in Parallel With Differentiation of Neuroblastoma Cells
Authors: Kloog Yoel , Axelrod Julius , Spector Ilan
Published: October 2006

Abstract: Cells of mouse neuroblastoma clone N1E‐115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane‐bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6–7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane‐bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property in neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation and a major group that may be related to ionic permeabilitys mechanisms of the excitable membrane.