Over-Expression of the DM-20 Myelin Proteolipid Causes Central Nervous System Demyelination in Transgenic Mice

Abstract: We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM-20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at ∼3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3–4 months. These symptoms progress in severity with age. Death occurs by 8–10 months. Myelination in 2-month-old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2-month-old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM-20 as normal animals. In 7-month-old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM-20 are greatly reduced relative to the 2-month-old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (>2 yr). Mice with low copy numbers of the transgene (2–4 copies) appear to be unaffected and have normal life spans.     

Intrinsic fluorescence of a non-myelin apoproteolipid and evidence for the existence of conformational flexibility

An extremely hydrophobic protein (Mr = 16000), which in its native form is only soluble in organic solvents and which differs from the myelin proteolipid (Mr = 24000), was purified to homogeneity. Intrinsic fluorescence studies on this apoproteolipid have revealed a large conformational flexibility. In the water-soluble form the emitting residues appear to be buried in a hydrophobic core while in organic solvents they are exposed to the external medium. Structural changes depending on the organic solvent are also observed. The emission characteristics of reconstituted proteoliposomes may be due to the formation of a membrane-linked complex between several proteolipid monomers.     

Purification of the chloroplast-membrane dicyclohexylcarbodi-imide-binding proteolipid by ion-exchange chromatography.

An efficient, mild and rapid procedure is reported for the separation of the dicyclohexyl-carbodi-imide-binding protein of chloroplast membranes from endogenous lipid components. By the use of ion-exchange chromatography the chloroplast proteolipid can be successfully separated from the major part of chlorophyll and other membrane lipids while being retained in a butan-1-ol milieu.     

Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: Reconstitution studies

Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on γ-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid.Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid.The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ 60 mM and 55 μM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 30 μ}\text{M}$ and two $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values, 0.37 mM (at high glutamate concentration) and 3.8 μM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent.In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes.High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane.The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol $\text{N-}\text{ethylmaleimide}$ per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate.Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.     

Studies on Subcellular Fractions Which Are Involved in Myelin Membrane Assembly: Isolation from Developing Mouse Brain and Characterization by Enzyme Markers, Electron Microscopy, and Electrophoresis

An extensive scheme for the subcellular fractionation of myelinating mouse brain is presented. Several centrifugation procedures for the separation of membranes involved in myelinogenesis are critically appraised, and guidelines for selection of centrifugation conditions are given. Characteristics of subcellular fractions are presented in the form of electron micrographs; also presented are distribution of RNA and protein; electrophoretic profiles of membrane proteins, and verification of the myelin-specific basic proteins, proteolipid protein, and glycoprotein by the immuno-electroblot technique; and the distribution of eight marker enzyme activities. Myelin-related membranes were found to differ both qualitatively and quantitatively in their complement of myelin-specific proteins. These myelin-containing fractions appear to represent different stages of myelination that coexist in developing mouse brain. These results provide the fundamental methodologies and background information for kinetic radioisotope analysis of intracellular events in the assembly of myelin presented in a companion article.     

Preparation of the proteolipid apoprotein from bovine heart, liver and kidney.

Proteolipid apoproteins have been prepared from heart, kidney, and liver by dialysis in chloroform/methanol against chloroform/methanol, acidified chloroform/methanol, and chloroform/methanol in succession. They are free of lipids (less than 0.05% P; less than 0.1% carbohydrate). They show a high content of non-polar amino acids, methionine, and tryptophan and contain little or no half-cystine. The differ from neural proteolipid apoproteins by absence of half-cystine, and of covalently bound fatty acids. As recovered from chloroform/methanol solutions, they are soluble in chloroform/methanol and insoluble in water, but a water-soluble form can be prepared by changing the solvent from chloroform/methanol to water in a stream of nitrogen. The chloroform-methanol-soluble form and the water-soluble form are interconvertible. ORD and CD spectra of all proteolipid apoproteins indicate 60-70% alpha-helix content in chloroform/methanol solution and 20-30% alpha-helix in water solution. Sodium dodecyl sulfate gel electrophoresis resolves proteolipid apoprotein into two major components corresponding to ca. 12 000 and 34 000 daltons. With sodium dodecyl sulfate/urea numerous bands appear, with a major one at 30 000 daltons and 8 to 10, ranging downward to 2500. For comparison, neural proteolipid apoproteins also show numerous bands with a major one at 25 000. The marked chemical and physical similarities among all proteolipid apoproteins studied suggest a common role in membrane structures.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

The DM-20 proteolipid is a major protein of the brain. It is synthetized in the fetus earlier than the major myelin proteolipid (PLP)

By studying highly purified CNS proteolipids, we have shown that DM-20 proteolipid, which was considered, until now, to be a minor brain proteolipid is, in fact, almost as abundant as the Major Myelin Proteolipid known also as Proteolipid Protein (PLP). DM-20 proteolipid is even the major brain proteolipid in young foetuses. It is only during myelinisation that the "Proteolipid Protein" increases rapidly and becomes equivalent in weight to DM-20 proteolipid. This study raises the question of the particular function of DM-20 proteolipid.     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF PROTEINS 1

Abstract— Partially purified myelin from brains of 17-day-old rats was separated into 4 subfractions on a discontinuous sucrose gradient by virtue of heterogeneity in density and particle size. The protein composition of each subfraction was determined by densitometry following separation of proteins on polyacrylamide gels in buffers containing sodium dodecyl sulphate. The major proteins studied included two basic proteins, proteolipid protein, the major high molecular weight protein (W) and a group of high molecular weight proteins. The percentage of high molecular weight proteins decreased sequentially from fraction D to A, that of the W protein remained constant, while relative amounts of the two basic proteins increased. Proteolipid protein concentration also increased as a percentage of the total protein from fraction D to B, but the uppermost fraction. A, had a markedly lower amount than fraction B. At 1 h after intracranial injection of [³H]leucine, the specific radioactivity of the basic and proteolipid proteins decreased from fraction D to B, with proteolipid protein in fraction A again anomalous (specific radioactivity higher than expected). These results are consistent with (but do not prove) a precursor-product relationship for individual proteins from denser to lighter subfractions, with the exception of myelin subfraction A. Experiments involving time staggered injections of a [¹⁴C] and later a [³H] labelled amino acid gave data which demonstrated that the W and basic proteins were added simultaneously (or with delays of much less than 20 min) to all of the subfractions, while proteolipid protein was added sequentially, from lower to upper fractions on the gradient. This double isotope technique also confirmed our previous observations that proteolipid protein shows a lag in entry into myelin compared to basic protein.     

A LOW MOLECULAR W eight GLYCOPROTEIN ASSOCIATED WITH ISOLATED MYELIN: DISTINCTION FROM MYELIN PROTEOLIPID PROTEIN

Abstract— A low molecular weight glycoprotein has been demonstrated in myelin isolated from immature rat brains. Both short term and long term fucose incorporation studies have identified this protein in the proteolipid protein region of a sodium dodecyl sulfate, polyacrylamide gel. A 1.7-2.1 fold increase in radioactive fucose in this glycoprotein relative to the major myelin glycoprotein was seen after long term fucose incorporation (21 days) compared to short term incorporation (18–22 h). The demonstration that this fucose-labelled protein is distinguishable from that of proteolipid protein was achieved by a variety of independent techniques. One technique involved a comparison of ether-ethanol extracted, freshly isolated, myelin with myelin extracted with chloroform-methanol. Treatment of isolated myelin with chloroform-methanol results in the solubilization of the proteolopid protein and its subsequent absence on gel electrophoresis while, in contrast, an enhancement of fucose label was observed in the same region of the polyacrylamide gel. Another procedure involved the electrophoretic separation of the radioactive fucose peak from that of proteolipid protein by employing a continuous, phosphate buffered, gel system. Finally carbohydrate analysis by gas-liquid chromatography of a partially purified proteolipid protein fraction did not reveal significant amounts of carbohydrates which are characteristic of glycoproteins. The identification of this minor glycoprotein comigrating with proteolipid protein indicates, therefore, a greater complexity associated with the purified myelin membrane than has been previously demonstrated.     

Phosphate transport in yeast mitochondria: purification and characterization of a mitoribosomal synthesis dependent proteolipid showing a high affinity for phosphate.

It is possible to obtain from yeast mitochondria a proteolipid able to bind phosphate, by two different procedures. One of them, generally used for lipid extraction, leads to the preparation of a more active crude proteolipid. This crude proteolipid has been purified by various chromatographic procedures and the active fraction, in phosphate binding, is always associated with cardiolipin. Its molecular weight seems to be close to 10000. The phosphate binding shows ligand saturation behavior and is inhibited by arsenate and N-ethylmaleimide; succinate is noninhibitory. This protein seems to be dependent on the mitoribosomal synthesis since it is not present in mitochrondria of mutant "petite colonie" and its amount largely decreases in mitochondria from yeast grown in the presence of chloramphenicol. It is possible to extract a proteolipid from the oligomycin sensitive ATPase, showing the same activity and properties. The hypothesis that this proteolipid acts as a part of the Pi carrier and constitutes the oligomycin-sensitive ATPase complex is discussed.     

The same 15 kDa proteolipid subunit is a constituent of two different proteins in torpedo, the acetylcholine releasing protein mediatophore and the vacuolar HATPase

Using the monoclonal antibody 15KI, we have studied, at the cellular and subcellular levels, the distribution of a 15 kDa proteolipid, identified as the subunit of mediatophore, a presynaptic membrane protein able to release acetylcholine when activated by calcium. Aside from the electric lobe, the antigen distribution in the brain of Torpedo paralleled that of the synaptic vesicle antigen SV2 and did not appear to be related to that of acetylcholine and choline acetyltransferase. The 15 kDa proteolipid antigen was therefore present in all nerve endings and not restricted to cholinergic ones. At the ultrastructural level, on cholinergic nerve endings, the antigen was detected associated to synaptic vesicles and, to a lesser extent, to the presynaptic plasma membrane. Indeed, considering the high sequence homology between the mediatophore subunit (Birman et al., 1990) and the proteolipid subunit of the vacuolar type H⁺ATPase, a major enzyme constituent of synaptic vesicles, this distribution was not surprising.

To determine whether antibody 15KI recognizes the vacuolar type H⁺ATPase, we chose a non neuronal cell type which possesses a high content of this enzyme, the kidney proton secreting epithelial cells. Indeed, antibody 15KI intensely labelled the apical plasma membrane of mitochondria rich epithelial cells in kidney tubules. A high density of the antigen was also found associated to intracellular membrane structures such as lysosomal multivesicular bodies, both in kidney epithelial cells and in electromotoneurons. The 15 kDa proteolipid antigen was associated with other vacuolar H⁺ATPase subunits in kidney membranes which was not the case in presynaptic plasma membranes. This illustrates that the 15 kDa proteolipid antigen is a constituent of two different protein complexes, which exhibit very different functional properties.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Electrophoretic behavior of the H+-ATPase proteolipid from bovine heart mitochondria

The proteolipid subunit of H⁺-ATPase was labeled by [¹⁴C]N,N-dicyclohexylcarbodiimide in bovine heart mitochondria. The radioactive labeling was followed using various systems of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). When using discontinuous SDS-PAGE (Laemmli, U.K., 1970,Nature (London)227, 680–685) a monomeric (Mr 7600±1500) and a dimeric form (Mr 17,800±1200) of the proteolipid were detected, while only the monomeric form was found on urea (8 M) containing gels (SDS-PAGE according to Laemmli; or Swank, R. T., and Munkers, K. D., 1971,Anal. Biochem. 39, 462–477). When using SDS-PAGE with Na-P_i buffer (Weber, K., and Osborn, M., 1969,J. Biol. Chem. 244, 4406–4442), only a dimeric form of the proteolipid (Mr 15,000±1000) was detected. Experimental data indicate that the different patterns of proteolipid separation are related to the presence of the two distinct proteolipid conformations in the SDS solution.     

Differentiation and Death of Premyelinating Oligodendrocytes in Developing Rodent Brain

Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20–positive processes that cover 80-μm domains in the cortex and 40-μm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, ~20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.     

Stoichiometric relation of protein components in cerebral myelin from different species

Abstract— In cerebral myelin from man, ox, rabbit, guinea pig and chicken, the amounts of proteolipid protein, basic protein and the fraction of further protein components were found to be present in a fixed ratio of 5·0: 3·5: 2·0 by weight. The molecular weights of 25,000 and 35,000 as obtained for the basic protein and proteolipid protein might indicate that cerebral myelin contains one molecule of basic protein per molecule of proteolipid protein. This fixed ratio of protein components was found to be changed in myelin from the PNS and in cerebral myelin from rat and carp, with their exceptional basic proteins. Using the polyacrylamide-gel electrophoresis it was possible to demonstrate that a homogeneous structural protein (the Folch-Lees proteolipid protein) constitutes about 50 percent of the total amount of myelin proteins in all species studied. An attempt was made to correlate myelin protein and lipid patterns from various species.     

Phosphorylation of multiple sites in a 15,000 dalton proteolipid from rat skeletal muscle sarcolemma, catalyzed by adenosine 3',5'-monophosphate-dependent and calcium/phospholipid-dependent protein kinases

This study reports a partial characterization of a 15,000 dalton (15 kDa) proteolipid present in rat skeletal muscle sarcolemma. The proteolipid is phosphorylated by both cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases, displays an isoelectric point (pI) of 5.9, and can be extracted from sarcolemma by acidified chloroform/methanol (2:1) or non-ionic detergents. Phosphoamino acid analysis and tryptic fingerprinting of the phosphorylated proteolipid indicate that both cyclic AMP- and calcium/phospholipid-dependent protein kinases predominantly phosphorylate serine residue(s) on a single tryptic peptide. Additivity experiments and thermolytic fingerprinting demonstrate a minimum of two distinct phosphorylation sites on the proteolipid, the phosphorylation of which is independently catalyzed by cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases in vitro. This sarcolemma proteolipid, which appears to be identified to a sarcolemma protein previously reported to be phosphorylated upon addition of insulin in a GTP-dependent manner (Walaas, O., Walaas, E., Rye-Alertsen, A. and Horn, R.S. (1979) Mol. Cell. Endocrinol. 16, 45-55), therefore represents a possible membrane target for those neuronal and hormonal stimuli which can regulate cyclic AMP-dependent or calcium/phospholipid-dependent protein kinase activities in skeletal muscle.     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

An electrophoretic analysis of proteolipids from different rat brain subcellular fractions

Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller $\text{Y}{}_0$ value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.