STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Aspects of the protein and the lipid composition of myelinoid marchi-positive bodies from mammalian spinal cord

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.     

Study of a floating fraction obtained during preparation of myelin from degenerating goldfish optic tract

A floating fraction that layers on top of 0.25 sucrose has been obtained during the preparation of myelin from intact and 9 day degenerating goldfish optic tracts. The proportion of total tract protein isolated in floating fraction rises from 6.6% to 11.0% during degeneration. This increase is paralleled by a morphologically observed splitting of myelin lamellae. Floating fraction contains all of the major myelin proteins but shows a 40% increase in the proportion of basic protein and a 2–3 fold decrease in the proportion of IP proteins (intermediate molecular weight glycoproteins) and a 36 Kd (X) protein. The lipid to protein ratio is slightly higher in floating fraction than myelin. Lipid composition is characterized by 1/2–1/3 the myelin levels of galactolipids and twofold increased levels of triglycerides and cholesterol esters. Electron microscopy of floating fraction shows a mixture of myelin fragments with few lamellae and single membrane fragments. Taken together the results indicate that floating fraction in the degenerating goldfish optic tract is at least partially derived from the breakdown of myelin.     

STUDIES ON THE MECHANISM OF DEMYELINATION: TRIETHYL TIN-INDUCED DEMYELINATION

The metabolism of myelin undergoing breakdown as a result of edema induced by chronic administration of triethyl tin (TET) dissolved in the drinking water (10 mg/l.) was examined. The spinal cord showed more edema and loss of myelin than the brain. Uptake in vitro of [1-¹⁴C]acetate into myelin lipids of slices of brain or spinal cord from TET-treated rats was depressed until 4–5 weeks after the beginning of the regime, then rose to above normal levels. The uptake of [l-¹⁴C]leucine into myelin protein rose within several weeks of TET treatment to levels averaging over 300 per cent of normal and remained high even after the TET was removed. The high levels of [l-¹⁴C]leucine incorporation were inhibited by cycloheximide and were not explained by an increase in the size of the free amino acid pool. The three classes of myelin proteins, basic, proteolipid protein, and Wolfgram protein shared in the increased incorporation. Spinal cord myelin showed the greatest metabolic response, brain stem myelin less, and myelin from the forebrain was minimally affected by the TET treatment. Myelin prelabelled by intracisternal injection of [l-¹⁴C]acetate and [l-¹⁴C]leucine before the onset of TET administration showed faster turnover in myelin proteins in relation to the myelin lipids than the control in the most severely affected animals, but not in others less affected. A ‘floating fraction’ was observed floating on 10.5% (w/v) sucrose during the myelin purification. This fraction showed metabolic characteristics typical of myelin, and myelin-labelling studies at various stages of the animal's development showed it to be derived from recently synthesized myelin. The floating fraction from the brain contained less cerebroside and more lecithin than myelin, while the spinal cord floating fraction composition was much like that of myelin. The floating fractions contained less protein typical of myelin (basic and proteolipid protein) and more highmolecular-weight protein which may have been derived from contaminating microsomes. The floating fraction was presumed to be partially deproteinated myelin. The use of TET-treatment as model for demyelination as a result of edema and proceeding in the absence of macrophages is discussed.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.     

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

Poster Sessions BP03: Myelin,Demyelination and Diseases of Myelin. Myelin and myelination in PLP‐null mice

The myelin proteolipid protein (PLP) is the major structural protein of CNS myelin, accounting for approximately half of total myelin protein. We studied synthesis and accumulation of myelin components for two months postnatally in PLP‐null mice and age‐matched controls. Accumulation of myelin, as assayed by levels of whole brain cerebroside and myelin basic protein, was normal in the knockout mice. The rate of cerebroside synthesis in the knockout mice was also normal. Myelin was isolated at several ages during development, using a standard subcellular fractionation protocol. The yield of ‘purified myelin’ isolated from a large particle (crude mitochondrial) fraction was reduced in PLP‐null mice, but increased amounts of ‘myelin’ were obtained in the small particle (crude microsomal) fraction. This ‘myelin’ in the crude microsomal fraction was identified as such by flotation on 0.85 m sucrose and the myelin‐characteristic 2 : 1 molar ratio of cholesterol to cerebroside. This suggests myelin from PLP‐null mice is physically more fragile than normal myelin, and that during tissue dispersion, much more PLP‐null myelin is fragmented into small vesicles than is the case for normal myelin. Three hours after intracranial injection of tritiated acetate into PLP‐null mice, cerebroside in myelin isolated from the large particle fraction was at a similar specific radioactivity to that isolated from the small particle (crude microsomal) fraction, suggesting that the most recently deposited PLP‐null myelin is not preferentially unstable. The increased fragility evident during tissue dispersion is indicative of an underlying structural abnormality in PLP‐null myelin. Whether this inherent structural instability affects myelin metabolism is under investigation. Acknowledgements: Supported by USPHS & NMSS grants.     

Activation of calpain-1 in myelin and microglia in the white matter of the aged rhesus monkey

Ultrastructural disruption of myelin sheaths and a loss of myelin with age are well-documented phenomena in both the human and rhesus monkey. Age-dependent activation of calpain-1 (EC 3.4.22.52) has been suggested as a plausible mechanism for increased proteolysis in the white matter of the rhesus monkey. The present study documents activation of calpain-1 throughout brain white matter in aged animals, evidenced by immunodetection of the activated enzyme as well as a calpain-derived spectrin fragment in both tissue section and Triton X-100-soluble homogenate of subcortical white matter from the frontal, temporal, and parietal lobes. Separation of myelin fractions from brain stem tissue into intact and floating myelin confirmed previous reports of an age-related increase in activated calpain-1 in the floating fraction. Measurements of calpain-1 activity using a fluorescent substrate revealed an age-related increase in calpain-1 proteolytic activity in the floating myelin fraction consistent with immunodetection of the activated enzyme in this fraction. Double-immunofluorescence demonstrated co-localization of activated calpain-1 with human leukocyte antigen-DR (HLA-DR), a marker for activated microglia, suggesting that these cells represent the major source of the increase in activated calpain-1 in the aging brain. These data solidify the role of calpain-1 in myelin protein metabolism and further implicate activated microglia in the pathology of the aging brain.     

Entry of Newly Synthesized Gangliosides into Myelin

Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19-21-day-old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10-15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi-disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin-sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.     

The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.     

ACID-LABILE (AMIDE) NITROGEN METABOLISM OF BRAIN PROTEINS

When the TCA-insoluble fraction of samples of rat brain was extracted with organic (lipid) solvents, a soluble protein fraction was obtained which was metabolically active in the release and uptake of ammonia. The total nitrogen content of this fraction increased during aerobic incubation of slices of cerebral cortex and in brain samples after electrical stimulation of the rats, but under these conditions the content of protein-bound, acid-labile (amide) nitrogen decreased. Treatment of rats with intraperitoneally injected camphor caused a decrease in the content of acid-labile nitrogen both in the proteins extracted into lipid solvents from the TCA precipitate of brain tissue samples and in the residual, washed TCA precipitate. By contrast, after treatment of rats with pentobarbitone, the content of proteinbound, acid-labile nitrogen increased in the lipid solvent extract but decreased in the residual, washed TCA precipitate. After electrical stimulation of rats, the content of acid-labile nitrogen in proteins of subcellular particles isolated from homogenates of brain exhibited dissimilar changes from control level: a pronounced decrease in the crude nuclear fraction but no change or a slight increase in the crude mitochondrial and in the combined microsomal and supernatant fractions. When slices of rat brain were incubated aerobically in the presence of glucose, ammonia was transferred from free amino acids to the acid-labile (amide) nitrogen of protein. Evidence was obtained indicating the participation of aspartate and purine nucleotides in this transfer.     

DISTRIBUTION OF RIBOSOMAL MATERIAL IN INCUBATED AND ELECTRICALLY STIMULATED CEREBRAL SLICES

Abstract— The distribution of ribosomal fractions has been examined in fresh cerebral cortex tissue and in slices maintained in vitro both with and without electrical stimulation. The electrical stimulation used was of a type that has previously been shown to diminish amino acid incorporation into protein.
Membrane-bound and free fractions were obtained and the ratio of their RNA contents were, for the control tissue 3, and for the electrically stimulated tissue 1 , 8. Electrical stimulation was found to decrease the Mg²⁺ binding affinity of the free. fraction but was without effect on the bound fraction. Stimulation was also found to increase the leakage of soluble protein and RNA from the tissue and its accumulation in the incubation medium.     

Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

PHOSPHORYLATION OF NUCLEAR PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER IN VITRO

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ-³²P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [³²P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14 m -NaCl-soluble, 0.25 n -HCl-soluble (mainly histone) and acidic phenol-soluble proteins, NaCl-soluble protein showed the highest phosphorylation while HCl-soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol-soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ-³²P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.     

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age-related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain-1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin-associated glycoprotein decreased with age. Levels of the oligodendrocyte-specific proteins 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin/oligodendrocyte-specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age-related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain-1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain-1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain-1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.     

Relationship between plasmin-trypsin-inhibitory and sialyltransferase activities

Previously we have shown that the measurable soluble sialyltransferase (STase) activity released into the medium during the incubation of rat jejunal slices was dependent upon the presence of a heparin-binding fraction (HBF) from heat-inactivated serum or a trypsin-binding protein (TBP) isolated from HBF. Both HBF and TBP were able to inhibit trypsin and plasmin. The measurement of galactosyltransferase (GTase) activity which was also released in incubations was not dependent on HBF or TBP. The present study is directed towards further exploring the relationship between STase activity and protease inhibitory activity. Heat-inactivated serum from turpentine-treated rats (HTS), had higher plasmin-trypsin-inhibitory (HTS) activities compared to heat-inactivated serum from control rats (HCS). When HTS was used to supplement jejunal incubations, there was a 25–40% increase in the measurable STase activity in the incubation medium compared to similar incubations carried out in buffer alone. In contrast, with HCS the increase was 10–15%. During incubations with hepatocytes, STase activity detected in the incubation medium was increased with the incubation buffer was supplemented with HTS compared to incubations supplemented with HCS. Serum antiproteolytic activity was higher in turpentine rats compared to controls. Incubation of serum at 37°C led to a progressive decrease in plasmin-trypsin-inhibitory and STase activities. TBP a plasmin and trypsin inhibitor was able to prevent the decrease in STase activity. Overall, serum STase activity was higher in the turpentine treated rats. In contrast, GTase activity in serum as well as that detected in the medium during jejunal and hepatocyte incubations was not dependent on protease inhibitory activity. The results show that there is a relationship between soluble STase and plasmin-trypsin-inhibitory activities.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS IN VITRO

Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein-bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM-NH₄Cl or 40 mM-malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5-4 μmol of NH₃ released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein-bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH₃. Proteins isolated from cerebral cortical slices incubated with L-[U-¹⁴C]glutamic acid or L-[U-¹⁴C]glutamine contained 105 (±0.095) per cent of the total ¹⁴C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0-22 to 0-42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM-¹⁵NH₄Cl or L-[amide-¹⁵N]glutamine or both. In the amide N of protein-bound glutaminyl residues the atoms per cent excess ¹⁵N ranged from 007 to 0-42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the ¹⁴C studies. Simultaneous analyses of free pool NH₃ and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry.