摘要:

Abstract:Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5‐14C]citrate, [1‐14C‐acetyl]carnitine, [1‐14C]acetate, [2‐14C]pyruvate, or [U‐14C]glucose. The synthesis of acetyl‐labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2‐14C]pyruvate>[U‐14C]glucose>[1‐14C‐acetyl]carnitine>[1,5‐14C]citrate>[1‐14C]acetate. The utilization of [1,5‐14C]citrate and [1‐14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of and14C‐labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5‐14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcar‐nitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brain; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl‐CoA to the extramitochondrial

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00569.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:Slices of rat caudate nucleus were incubated in a solution of 123 mM‐NaCl, 5 mM‐KCl, 1.2 mM‐MgCl2, 1.2 mM‐NaH2PO4, 25 mM‐NaHCO3, 0.2 mM‐choline chloride, 0.058 mM‐paraoxon, 1 mM‐EGTA, and oxidizable substrates. (−)‐Hydroxycitrate, a specific inhibitor of ATP‐citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5‐14C]citrate by 82–86%, but that from [U‐14C]glucose by only 33%, from [2‐14C]pyruvate by 24% and from [1‐14C‐acetyl]carnitine by 8%; the production of14CO2from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM‐ and 5 mM‐citrate diminished it by 43% and 66%, respectively; the production of from [U‐14C]glucose and from [1‐14C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca2+and decreases the supply of mitochondrial acetyl‐CoA to the cytosol. The results with (−)‐hydroxycitrate indicate that the cleavage of citrate by ATP‐citrate lyase is not responsible for the supply of more than about one‐third of the acetyl‐CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)‐hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of ci

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00570.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the “PSD protein” (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [3H]fucose or [3H]N′‐acetylmannosamine. The incorporation of [3H]fucose into synaptic fractions decreased two‐ to threefold between 10 and 28 days whereas little change in the incorporation of [3H]N′‐acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with125I‐fucose‐binding protein or labeling of sialic acid with NaIO4NaB[3H4] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with125I‐fucose‐binding protein increased one‐ to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with125I‐wheat germ ag‐glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synap

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00571.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:A cholesterol‐esterifying enzyme which incorporates exogenous fatty acids into cholesterol esters in the presence of ATP and coenzyme A was demonstrated in 15‐day‐old rat brain. This enzyme was maximally active at pH 7.4 and distinct from the cholesterol‐esterifying enzyme reported earlier (Eto and Suzuki, 1971), which has a pH optimum at 5.2 and does not require cofactors. Properties of the two enzymes have been compared. Both the enzymes showed negligible esterification with acetate and were maximally active with oleic acid. The pH 5.2 enzyme esterified desmosterol, lanosterol and cholesterol at about the same rate, while the pH 7.4 enzyme was only 50% as active with lanosterol as it was with cholesterol and desmosterol. Phosphatidyl serine stimulated the pH 5.2 enzyme but not the pH 7.4 enzyme. Phosphatidyl choline and sodium taurocholate showed no effect on either of the enzymes. Both the enzymes were associated with particulate fractions, but the pH 7.4 enzyme was localized more in the microsomes. Purified myelin showed 2.6‐fold and 1.5‐fold higher specific activities of pH 5.2 and 7.4 enzymes respectively, when compared with homogenate. About 7–10% of total activity of both the enzymes was associated with purified myelin. Brain stem and spinal cord showed higher specific activity of pH 5.2 enzyme than cerebral cortex and cerebellum, while pH 7.4 enzyme specific activity was higher in cerebellum and brain stem than in cerebral cortex and spinal cord. Microsomal pH 7.4 activity showed progressive increase prior to the active period of myelination, reaching a maximum on the 15th day after birth and declined to 20% of the peak activity by 30 days. In contrast, pH 5.2 enzyme reached maximum activity about the 6th day after birth and remained at this level well into adulthood. In 15‐day‐old rat brain, pH 7.4 enzyme had five to six times higher specific activity than pH 5.2 enzyme, while in adults the activities were equal. The pH 7.4 enzyme showed a threefold higher specific activity than pH 5.2 enzyme in myelin from 15‐day‐old rats, but in adults

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00572.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:A rapid and simple technique using solvent extraction, ion‐pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3‐methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3‐methoxytyramine added to the samples is 45–50%. 3‐Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3‐methoxytyramine levels in rat striata, while catechol‐O‐methyltransferase inhibition with 3′,4′‐dihydroxy‐2 methylpropiophenone completely depleted 3‐methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D‐amphetamine were also examined. The 3‐methoxytyramine concentrations in the brains of animals killed by decapitation or by micr

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00573.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:A radioimmunoassay (RIA) using125I‐labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since125I‐β‐antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00574.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:Free sterol composition of the developing rabbit optic nerve was compared with that of the homologous cerebral white matter at corresponding stages of ontogeny. The sterols were detected and identified by means of combined gas‐chromatography and mass spectrometry. The following free sterols were found in both the optic nerve and cerebral white matter: cholesterol, desmosterol, lanosterol, two dimethylsterols, which are probably 4,4‐dimethyl‐5α‐cholest‐8,24‐diene‐3β‐ol, with a molecular weight of 412, and 4α,14α‐dirnethyl‐5α‐cholest‐7‐ene‐3β‐ol, with a molecular weight of 414 and probably cholestene, with a molecular weight of 368. The sterol spectrum of the developing optic nerve differed not only from that of the mature nerve but also from that of age‐matched white matter of the rabbit brain. The tri‐ and dimethyl‐sterols, detected for the first time in the rabbit optic nerve and cerebral white matter, are natural components of the developing nervous tissue but they were not found in the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00575.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:Brain microvessels were prepared from rat cerebral cortex. The purity was confirmed by phase‐contrast microscopy and by the measurement of an enzymatic marker, γ‐glutamyltranspeptidase. The microvessel preparation was subjected to radioreceptor assay using a125I‐labelled β‐adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80 μg per tube. It was saturated at 200 PM IHYP concentration. TheKD, value calculated by Scatchard analysis was 69.4 ± 9.9 PM. The maximum binding (Bmax) was 107 ± 4 fmol/mg protein. The binding reached equilibrium within 30 min and was dissociated by addition of (−)‐propranolol. The inhibitory effects of isomers of propranolol and iso‐proterenol on this binding showed that (−)‐isomers were two orders of magnitude more potent than the (+)‐isomers. Other neurotransmitters did not affect IHYP binding. The characteristics of the binding, saturability, high affinity, reversibility and stereospecificity, suggest that IHYP is bound to β‐adrenergic receptor sites lo

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00576.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:A simple and rapid purification method is presented for the two mouse cerebral isozymes of enolase (EC 4.2.1.11), E1and E3. The purity of the preparations was ascertained by electrophoresis under two different conditions. The biochemical and immunological properties of E1and E3were compared. The molecular weight of the cerebral enolases was analysed by column chromatography on Sephadex G 150 and by electrophoresis in the presence of SDS. Both E1and E3are homodimers with a subunit of molecular weight of 50,000. The procedure also yields a semi‐purified fraction of E2. Conditions ofin vitroformation of E2from pure or semi‐purified fractions of E1and E3show that it is likely to be a real hybrid, rather than an aggregate and that it is probably not an artefact formed during the purification. TheKmvalues (Km= 3–4·10−5M) for the substrate are not significantly different amongst the three forms. However, E1and E2but not E3are inhibited by excess substrate. Antisera against E1and E3have been obtained from rabbit and goat, respectively. Antibodies against each protein do not show any cross‐reactivity with each other. There is, however, a broad species cross‐reactivity, showing conservation of each enolase form during evolution. Both anti‐E1and anti‐E3sera react with the E2enolase fraction, in agreement with its hybrid structure. Anti‐E3serum does not react with extracts of other tested organs. Brain enolase 1 resembles liver enolase in its biochemical and immunological properties. A slight cross‐reactivity of anti‐E1serum with muscle extracts is observed. Heterogeneity of brain enolase 1 is observed by both biochemical and immunological methods; the nature of this hete

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00577.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract:Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L‐[35S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000g.Newly synthesised polypeptides in each fraction were resolved by SDS‐gel and two‐dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α1‐, α2and β‐tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two‐dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two‐dimensional gel electrophoresis and also by one‐dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikaryain vitroyielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesisedin situ.The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikaryain vitro, but to disproportionately weaker extents compared with th

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1981.tb00578.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY