Changes in Glycoprotein Metabolism in the Cerebral Cortex Following First Exposure of Dark-reared Rats to Light

Abstract: The level of incorporation of [³H]fucose or [³H]lysine into subcel-lular fractions of the visual and motor cortices of 50-day-old dark-reared (D) and light-exposed (L) rats was determined. No differences were found between D and L rats in the incorporation of either precursor into subcellular fractions of the motor cortex, or in any fraction of the visual cortex except the synaptic-membrane fraction. After a 3-h light exposure the incorporation of [³H]fucose into the visual cortex synaptic-membrane fraction was elevated (L/D = 136%). Incorporation of [³H]lysine was elevated in the visual cortex synaptic-membrane fraction of L compared to D rats after a 1-h exposure (L/D = 118%). However, after a 3-h exposure the incorporation was depressed in this fraction (L/D = 79%). No differences could be found in the levels of activity of fucosyl transferase following first exposure to light but dark-rearing itself resulted in increased enzyme activity in the motor cortex compared to normal controls. First exposure of 20-day-old dark-reared rats to light led to an increase in the incorporation of [³H]fucose into soluble glycoproteins of both the visual and motor cortex and into particulate glycoproteins of the visual cortex only. These results are in contrast with those found with 50-day-old animals and suggest that the effects of light-exposure on [³H]fucose incorporation may be age-dependent.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

Incorporation of radioactive glucosamine into macromolecules at nerve endings

Mice were injected intracerebrally with [¹⁴C]glucosamine, and incorporation into macromolecules in various subcellular fractions of brain was studied at a number of times after administration of the precursor. The [¹⁴C]glucosamine was rapidly incorporated into macromolecules of all the subcellular fractions of brain including both the soluble and particulate fractions of isolated nerve endings. Incorporation into macromolecules in the soluble fraction of nerve endings was quite extensive 3 hr after administration of the precursor and the specific acitvity of this fraction fell thereafter. In contrast there was only slight incorporation of [¹⁴C] leucine into the soluble protein from isolated nerve endings in the first few hours after administration, whereas the other subcellular fractions were maximally labelled at that time. The data suggests that, unlike protein which is largely transported to nerve endings in the axoplasm, there is extensive incorporation of carbohydrate into macromolecules in nerve endings. Whereas the protein component of a glycoprotein or mucopolysaccharide may be transported to the nerve ending from the perikaryon, the structure and function of this protein may be modified at the nerve ending by further incorporation of glucosamine, sialic acid and possibly other carbohydrates. The carbohydrate-containing macromolecules could influence nerve ending function immediately after these final synthetic reactions since these reactions occur at the nerve ending and not in the perikaryon.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

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 with ¹²⁵I-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 with ¹²⁵I-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 with ¹²⁵I-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 synaptic contact has been formed.     

FUCOSE INCORPORATION INTO GLYCOPROTEINS OF MOUSE BRAIN

—Radioactive fucose was incorporated into glycoproteins of brain in vivo. After intracerebral administration of this precursor, radioactive glycoproteins were the sole detectable product. The glycoproteins formed appeared to have a slow turnover but this was due, at least in part, to re-utilization of fucose released from degraded glycoproteins. Incorporation of fucose into glycoproteins differed from that of glucosamine, since a much smaller proportion of the radioactive fucose was incorporated into soluble glycoproteins. Fucose was rapidly incorporated into glycoproteins of nerve endings, although there was relatively little incorporation into glycoproteins associated with the soluble component of the nerve-ending fraction. As found in previous studies with glucosamine, soluble glycoproteins of nerve endings turned over relatively rapidly. Pretreatment with acetoxycycloheximide markedly inhibited incorporation of fucose into glycoproteins of brain. In contrast to the results with glucosamine, comparable inhibition was observed for fucose in all subcellular fractions of brain including the particulate and soluble components derived from the nerve-ending fraction.     

Increased Incorporation of [3H]Fucose into Chick Brain Glycoproteins Following Training on a Passive Avoidance Task

Abstract: Incorporation of [¹H]fucose into the TCA-precipitable material from the particulate and soluble fractions of different regions of the brain in the 1-day-old chick was measured following training on a passive avoidance task. A significant increase in the level of incorporation of [³H]fucose into the particulate fraction of the anterior forebrain roof was observed in the trained birds as compared with untrained controls. The percentage increases in radioactivity in the particulate fraction of the anterior forebrain roof were 29% ( p < 0.01), 16% ( p < 0.01), and 26% ( p < 0.01) in the trained birds as compared with controls, 30 min, 3 h, and 24 h following learning, respectively, but had returned to control levels after 48 h. These results suggest either increased production of glycoproteins or increased fucosylation of preexisting proteins following training, and suggest one of the possible localized neurochemical changes associated with the learning of the passive avoidance response.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

Biochemical Demonstration of the Myelin-Associated Glycoprotein in the Peripheral Nervous System

Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [³H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [³H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [¹⁴C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

[2-3H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING

Abstract— When [2-³H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-³H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [³H]- and [¹⁴C]-labelled glycerol, the ratio of ³H to ¹⁴C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [³H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [¹⁴C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.     

PROTEIN SYNTHESIS IN THE BRAIN OF OCTOPUS VULGARIS, LAM

Abstract— The process of protein synthesis in the brain of Octopus vulgaris Lam has been examined after systemic administration of [³H]leucine and upon incubation of the tissue in sea water containing the radioactive precursor. After injection of [³H]leucine in the branchial heart, the radioactivity of the TCA-soluble fractions of the three main brain divisions reached a maximum in about 30 min and decreased thereafter, while incorporation into the protein fractions was complete in approx. 2 h. Per unit wet weight the radioactivity of brain proteins was higher than that of most other organs. In vitro the rate of incorporation of [³H]leucine in the protein fraction of the optic lobe remained low for more than 1 h, but increased several fold thereafter. Preincubation of the tissue in sea water abolished the lag period. Similar effects were observed in the vertical lobe as well as in the optic lobe of young and adult octopuses but not in the white body, a non-nervous organ. The process of protein synthesis in the optic lobe is markedly inhibited by puromycin, cycloheximide and chloramphenicol. Electrophoretic analysis on polyacrylamide gels indicated that the soluble proteins labelled in vitro and in vivo are similar.     

The Synthesis and Release of [3H-Tyrosine1]Methionine5-Enkephalin from Guinea Pig Brain Slices

Abstract: Stores of methionine-enkephalin were labelled on the N -terminal by incubation of whole brain slices with [³H]tyrosine (10 °Ci/ml). The ³H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD₂ and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [³H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N -terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [¹⁴C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N -terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 m M ) elicited a Ca²⁺-dependent release of the synthesised [³H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [³H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

SUBCELLULAR LOCALIZATION OF [14C]ADENINE DERIVATIVES NEWLY-FORMED IN CEREBRAL TISSUES AND THE EFFECTS OF ELECTRICAL EXCITATION

Abstract— Guinea pig neocortical tissues were incubated with [¹⁴C]adenine, dispersed in cold isotonic sucrose and subcellular fractions prepared by centrifugation. Some 98 per cent of the assimilated ¹⁴C was found as acid-soluble nucleotides in the incubated tissues. In primary fractions obtained by differential centrifugation, about 60 per cent of the [¹⁴C]-nucleotides were in supernatant fractions, in distinction to ATP of which the greatest molar quantity (61 per cent of that in the dispersion) was in the crude mitochondrial fraction. When the crude mitochondrial fraction was separated by density gradient centrifugation, most ¹⁴C was found in synaptosomal fractions and about 85 per cent of this ¹⁴C was adenine nucleotides.
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of ¹⁴C subsequently found in nucleotides (collectively) in the supernatant fraction, and increased their inosine and hypoxanthine. Stimulation increased the tissue's cyclic AMP but a preferential localization for this was not established. Results are tentatively interpreted in terms of liberation of an adenine derivative on excitation, and its action or reuptake at a tissue component different from that from which it was liberated. Fractionation of tissues which had been incubated with both [¹⁴C]-adenine and [³H]adenosine suggested that of the two compounds, more adenosine was taken up by synaptic regions in preference to other cellular regions of the tissue.