Evidence for the Presence of CDP-Ethanolamine: 1,2-diacyl-sn-glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin

Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.     

Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

The Presence of Phospholipase D In Rat Central Nervous System Axolemma

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.     

Evidence for expression of some microtubule-associated protein 1B in neurons as a plasma membrane glycoprotein

Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein.     

Schwann Cell Proliferation Is Accompanied by Enhanced Inositol Phospholipid Metabolism

Inositol phospholipid metabolism during mitogen-induced Schwann cell proliferation has been examined. Addition of axolemma- and myelin-enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four- to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30-70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60-80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 may be one of the initial molecular events in the transduction of the mitogenic signal across the Schwann cell plasma membrane.     

Subcellular distribution of sulfated glucuronyl glycolipids in human peripheral motor and sensory nerves

Sulfated glucuronyl glycolipids (SGGL) have been implicated as important target antigens in patients with demyelinating polyneuropathy and IgM paraproteinemia. Sulfated glucuronyl paragloboside (SGPG), a major species of SGGL, was identified in the subcellular fractions of human peripheral motor and sensory nerves using a simple and quantitative method. SGPG was found to be concentrated in the myelin-enriched fractions of both motor and sensory nerves (1.3±0.3 and 1.5±0.4 µg/mg protein, respectively), whereas its concentration was 0.9±0.2 and 1.8±0.6 µg/mg protein in the axolemma-enriched fractions of motor and sensory nerves, respectively. Our finding that SGPG is more abundant in the human sensory nerve axolemma-enriched fraction may account for the clinical and pathological observations that the lesions are more heavily concentrated in the sensory nerve than in other parts of the nerve tissues in this disorder.     

Mitogenic Effect of Axolemma-Enriched Fraction on Cultured Oligodendrocytes

An in vitro system has been devised to study the mitogenic effect of axolemma on cultured oligodendrocytes. Addition of axolemma-enriched fraction to cultured oligodendrocytes results in a dose-dependent mitotic response with an 11-fold stimulation at a membrane concentration of 200 micrograms/ml. The interaction between oligodendrocytes and axolemma is specific, as myelin-enriched fraction, astrocyte membrane, and red blood cell membrane showed little or no effect on the oligodendroglial proliferation under similar conditions. In addition, cultured astrocytes were tested with the same axolemma membrane, and no mitotic stimulation was observed. The mitogenicity of AEF membrane on cultured oligodendrocytes is sensitive to heat and trypsin treatment, suggesting that the axolemma mitogen may be a protein.     

Identification of an Axolemma-Enriched Fraction from Peripheral Nerve

Abstract: A method has been devised for the fractiona-tion of whole peripheral nerve. The procedure utilizes differential centrifugation and separation on a linear sucrose gradient (10–40%, wt/wt). A membrane fraction localized between 26% and 29% sucrose was not only enriched for the plasma membrane markers, 5'-nucleotidase and acetylcholinesterase (AChE), but also possessed the highest binding of [³H]saxitoxin, a specific marker for sodium channels. Neurons in the lumbar dorsal roots and ventral horns of rats were injected with [³H]fucose to label glycoproteins associated with the axolemma from sciatic nerve. Fractionation of the labeled nerves demonstrated a coincidence in the distribution of [³H]fucose-labeled material and AChE activity in the sucrose density gradient. The increase in the specific activity of marker enzymes for plasma membrane, sodium channels, and labeled membrane, previously demonstrated to be of axolemmal origin, identified the 26–29% region of the sucrose gradient as enriched for axolemma derived from peripheral nerve.