Interrelation of delta-aminolevulinic acid and brain myelin proteins as affected by benzene and lead

An experimental model of subacute lead and benzene intoxication was induced in rabbits. It increased significantly delta-aminolevulinic acid (14C-ALA) incorporation into CNS myelin. ALA stably fixes on myelin proteins, especially Wolfgram proteins. In spite of this about one half of ALA incorporated into myelin linked with its basic proteins. Lead and benzene intoxication cause changes in the proportion of the basic fractions of myelin proteins. The amount of basic proteins increases, while the amount of proteolipid and Wolfgram proteins decreases. Lead and especially benzene intoxication decreases exogenous ALA insertion into basic proteins and proteolipid proteins, with ALA fixation on Wolfgram proteins remaining unchanged.     

Phosphate groups modifying myelin basic proteins are metabolically labile; methyl groups are stable

Young and adult rats received intracranial injections of [33P]orthophosphoric acid. The time course of the appearance and decay of the radioactive label on basic proteins in isolated myelin was followed for 1 mo. Incorporation was maximal by 1 h, followed by a decay phase with a half-life of approximately 2 wk. However, radioactivity in the acid-soluble precursor pool (which always constituted at least half of the total radioactivity) decayed with a similar half-life, suggesting that the true turnover time of basic protein phosphates might be masked by continued exchange with a long-lived radioactive precursor pool. Calculations based on the rate of incorporation were made to more closely determine the true turnover time; it was found that most of the phosphate groups of basic protein turned over in a matter of minutes. Incorporation was independent of the rate of myelin synthesis but was proportional to the amount of myelin present. Experiments in which myelin was subfractionated to yield fractions differing in degree of compaction suggested that even the basic protein phosphate groups of primarily compacted myelin participated in this rapid exchange. Similar studies were carried out on the metabolism of radioactive amino acids incorporated into the peptide backbone of myelin basic proteins. The metabolism of the methyl groups of methylarginines also was monitored using [methyl-3H]methionine as a precursor. In contrast to the basic protein phosphate groups, both the peptide backbone and the modifying methyl groups had a metabolic half-life of months, which cannot be accounted for by reutilization from a pool of soluble precursor. The demonstration that the phosphate groups of myelin basic protein turn over rapidly suggests that, in contrast to the static morphological picture, basic proteins may be readily accessible to cytoplasm in vivo.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

The Presence of Aldehyde-Reacted Proteins in Normal and Multiple Sclerosis White Matter

The incorporation of tritium from NaB3H4 into the major protein components of myelin and the presence of weak fluorescence emission bands at wavelengths of approximately 440 and 500 nm from sodium dodecyl sulfate-solubilized, delipidated white matter are indicative of the presence of the products of aldehyde reactions with proteins. The incorporation of tritium from NaB3H4 into myelin proteins was confirmed by reaction with purified components of myelin basic protein or with lipophilin, a purified fraction of proteolipid protein. From the extent of tritium incorporation into the purified proteins, it is estimated that approximately 0.2 mol of tritium is incorporated/mol of myelin basic protein and approximately 0.4 mol of tritium/mol of proteolipid protein. There is approximately 50% greater incorporation of tritium into a more degraded, less positively charged form of the basic protein. The incorporation of tritium into normal and multiple sclerosis white matter was compared. There is a small but statistically significant difference in the percentage of the total counts incorporated into the major protein fractions for the two groups, with the multiple sclerosis samples showing a higher percentage of the counts in the Wolfgram protein and a lower percentage in the myelin basic protein compared with the normal samples.     

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.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich fractions.     

Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading

Lack of neurite growth in optic nerve explants in vitro has been suggested to be due to nonpermissive substrate properties of higher vertebrate central nervous system (CNS) white matter. We have searched for surface components in CNS white matter, which would prevent neurite growth. CNS, but not peripheral nervous system (PNS) myelin fractions from rat and chick were highly nonpermissive substrates in vitro. We have used an in vitro spreading assay with 3T3 cells to quantify substrate qualities of membrane fractions and of isolated membrane proteins reconstituted in artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by treatment with proteases and was not associated with myelin lipid. Nonpermissive proteins were found to be membrane bound and yielded highly nonpermissive substrates upon reconstitution into liposomes. Size fractionation of myelin protein by SDS-PAGE revealed two highly nonpermissive minor protein fractions of Mr 35 and 250-kD. Removal of 35- and of 250-kD protein fractions yielded a CNS myelin protein fraction with permissive substrate properties. Supplementation of permissive membrane protein fractions (PNS, liver) with low amounts of 35- or of 250-kD CNS myelin protein was sufficient to generate highly nonpermissive substrates. Inhibitory 35- and 250-kD proteins were found to be enriched in CNS white matter and were found in optic nerve cell cultures which contained highly nonpermissive, differentiated oligodendrocytes. The data presented demonstrate the existence of membrane proteins with potent nonpermissive substrate properties. Distribution and properties suggest that these proteins might play a crucial inhibitory role during development and regeneration in CNS white matter.     

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.     

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

Suppressive Effect of Triethyllead on Entry of Proteins into the CNS Myelin Sheath In Vitro

Incorporation of [¹⁴C]leucine into the myelin sheath was studied in brain stem slices prepared from 22-day-old rats. Individual major myelin proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. There was a time lag before incorporation of the label into proteolipid protein (PLP) and intermediate protein (IP) reached maximal rates. Labelling of basic proteins (BP) and Wolfgram proteins (WP) revealed a much shorter lag in entry. Appearance of radioactive proteins in the myelin sheath was significantly hampered by triethyllead (PbEt₃) added to the incubation medium at micromolar concentrations. Inhibition values were highest in the case of PLP and were closely followed by the values for IP. BP and WP were less inhibited, although incorporation of these proteins into myelin was still suppressed more than was synthesis of total homogenate protein. Thus, myelin-forming cells seem to be unduly vulnerable to the toxin relative to the rest of the tissue. Furthermore, the results indicate an interference of PbEt₃ with certain posttranslational processes involved in furnishing of integral myelin proteins.     

Effects of monensin on posttranslational processing of myelin proteins

Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Uridine Transport and Metabolism in the Central Nervous System

Myelin and myelin-containing (P3) fractions were prepared from human white matter by discontinuous sucrose gradient centrifugation. The myelin isolated from each of the fractions of different densities was morphologically and biochemically distinct. Light myelin fractions consisted of compact, multilamellar myelin, whereas the denser fractions consisted predominantly of loose myelin with fewer lamellae. The amounts of both basic protein and lipophilin (proteolipid protein) were reduced in the denser fractions. In contrast, the high-molecular-weight components were elevated in the dense fractions. The lipid composition was similar in all the fractions studied. Analysis of basic protein by gel electrophoresis at pH 10.6 revealed differences in basic protein microheterogeneity among the fractions. The light myelin fraction was enriched in the more positively charged basic protein components (components 1, 2, and 3), whereas these components were reduced in the denser fractions. Myelin in the dense fractions was enriched in the more modified forms of basic protein (components 6, 7, and 8). The pattern of microheterogeneity was different for basic protein isolated from myelins of a 2-year-old and an adult brain; the former showed fewer components and mainly the most cationic species. On the other hand, the pattern of microheterogeneity of basic protein isolated from the different density gradient fractions was similar for both ages.     

In vivo phosphorylation of myelin basic proteins: single and double isotope incorporation in developmentally related myelin fractions

The phosphorylation of myelin basic proteins (MBPs) was studied in developing mouse brain. Based on our previous work we postulated that phosphorylation of MBPs takes place prior to their appearance in the myelin compartment as well as within the myelin sheath. To further test this hypothesis we utilized a subfractionation protocol that yields brain fractions enriched in myelin membranes of differing developmental stages. Incorporation of radioactive phosphate into MBPs was studied in each of the subcellular fractions. After 5- and 15-min incubations of isotope in vivo the highest specific radioactivities (SAs) of MBPs were found in the least mature myelin fractions. Incorporation of 32P in MBPs was greater into serine residues than threonine residues in all of the subcellular fractions studied. The relative turnover of MBP phosphates was studied in each of the subcellular myelin fractions using a time-staggered, double isotope methodology. The most rapid equilibration of MBP phosphates with the trichloroacetic acid (TCA)-soluble phosphate pool occurred in the most mature myelin fractions indicating that the highest turnover of MBP phosphates occurs in the most mature myelin fractions. The SAs and turnover rates of each of the four commonly observed mouse MBPs (14, 17, 18.5, and 21.5 kDa) were similar in any particular subfraction demonstrating that the MBP phosphotransferase system(s) acts on each of the MBPs in a similar manner.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

Differential effect of colchicine upon the entry of proteins into myelin and myelin related membranes

Brain slices from 20 day old rats were incubated with radioactive aminoacids in the presence and absence of 500 M colchicine and the appearance of labeled proteins in myelin and in a myelin-like fraction (SN₄ fraction) was measured. In the presence of the inhibitor, the entry of proteolipid proteins was decreased to 55% in myelin and to 45% in SN₄ fraction with reference to control values while the entry of basic proteins and other minor protein components was unaffected in both fractions. The synthesis of proteolipid proteins was not affected by the presence of colchicine; moreover, a slight accumulation of these proteins was observed in microsomes. The results suggest that the microtubular system is involved in the transport of proteolipid proteins from their site of synthesis to their site of deposition and that the various types of myelin proteins follow different transport routes to enter into this special type of membrane.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)