Separation and identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels.

A method is presented for the separation and detection of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels and by immunoblotting. The gel staining procedure is a modification of a method used to demonstrate enzyme activity on blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. The results show that immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase can be separated under equilibrium conditions on isoelectric focusing gels with an expanded alkaline pH range after solubilization in a mixture of nonionic/zwitterionic detergents and urea. Enzymatically active 2',3'-cyclic nucleotide 3'-phosphodiesterase focused as two closely spaced bands at pIapp 8.1 and 8.8, respectively, while 2',3'-cyclic nucleotide 3'-phosphodiesterase immunoreactivity was detected as four distinct bands at pIapp 4.2, 7.4, 8.8, and 9.3 and a diffuse band at pIapp 7.9-8.2. By two-dimensional separation these five bands showed molecular weights of about 43-47 kDa, i.e., corresponding to reported values for immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase. Since enzyme activity is associated with only two of the bands, nonspecific and artifactual banding due to, e.g., detergent micelle formation, is unlikely.

Degradation products of myelin proteins in a light CNS subcellular fraction.

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from heavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.

Isoelectric focusing of membrane proteins: high resolution separation of myelin proteins.

A mixture of the nonionic detergent Triton X-100, the zwitterionic detergent 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), 9M urea and carrier ampholytes was found comparable to media containing sodium dodecyl sulfate in the capacity for solubilization of myelin proteins, including the highly hydrophobic proteolipid protein. The solubilized sample was incorporated into the polymerization mixture before moulding an ultrathin gel, with heat convection characteristics allowing a high wattage to be applied, thus allowing fast separation with high resolving power. Since the most basic protein in myelin focuses at a pH greater than 10, fast separation is essential in order to minimize decay of the cathodic end of the pH gradient.

Marchi-positive myelinoid bodies at the transition between the central and the peripheral nervous system in some vertebrates.

The CNS-PNS (central nervous system-peripheral nervous system) transitional region of cranial and spinal nerve roots in some vertebrate species was analysed with respect to the occurrence and the distribution of myelinoid Marchi-positive bodies. Both cranial and spinal nerve roots contained more Marchi-positive bodies in their CNS than in their PNS segments. An accumulation of Marchi-positive bodies was usually noted just central to the CNS-PNS borderline. Comparisons between calibre spectra and Marchi index in the cat revealed a particularly high number of Marchi-positive bodies in nerve roots with a high content of myelinated fibres with diameters greater than or equal to 5 microns. Marchi-positive bodies were absent in CNS tissue lacking myelinated nerve fibres. CNS borderline internodes measuring between 200 and 300 microns in length were noted in fibres as thick as 15 microns in feline S1 ventral and dorsal roots. The general picture was similar in all analysed species. Noteworthy however, was the small difference in number of Marchi-positive bodies between CNS and PNS tissue in Xenopus. The chicken contained many myelinoid bodies of similar size and texture as the Marchi-positive bodies but without the Marchi-positive staining properties. The results show that normally occurring Marchi-positive bodies in the CNS are more numerous along paranodal segments than along mid-internodal segments of myelinated nerve fibres and thus support the hypothesis that Marchi-positive bodies are preferentially derived from paranodal myelin.

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.

Acid phosphatase activity at nodes of Ranvier in alpha-motor and dorsal root ganglion neurons of the cat.

Acid phosphatase (AcPase) activity in feline alpha-motor and dorsal root ganglion (DRG) neurons was analysed histochemically by light and electron microscopy. The occurrence and distribution of the AcPase activity expressed within the axon differed depending on neuron type and distance from the cell body. Both in alpha-motor and DRG neurons, AcPase-positive bodies of various morphological categories were observed mainly at nodes of Ranvier, where they were more frequent distal than proximal to the nodal midlevel. In the peripherally located processes of both neuron types, most of the larger AcPase-positive bodies were associated with the paranodal axon-Schwann cell network. In the centrally located processes the AcPase-positive bodies were situated in the constricted axon segment and the adjacent paranodal axoplasm. Both in motor and DRG axons, AcPase-positive bodies were more frequent at the spinal root level than at a level central to the PNS-CNS borderline. The observations indicate that lysosomes (i.e. AcPase-positive bodies) constitute part of the intra-axonal system of organelles in normal, large, myelinated alpha-motor and DRG axons of the cat. Lysosome-mediated degradation of retrogradely transported endogenous and exogenous materials may be extensive in normal peripherally directed neuronal processes. The study also suggests a difference between PNS and CNS parts of the same axon with regard to the local turnover of lysosomal organelles.

Peroxidase activity at CNS nodes of Ranvier and in initial axon segments of lumbosacral alpha-motoneurons after intramuscular administration of horseradish peroxidase.

The occurrence of peroxidase activity in central (CNS) and peripheral nervous system (PNS) parts of alpha-motor axons was studied by light and electron microscopy in adult cats after injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. The intrafunicular parts of the axons were virtually free of HRP-positive bodies except at a few nodes of Ranvier. Most of these nodes were weakly HRP-positive and contained, irrespective of a survival time between 25 and 48 h, only a few HRP-positive bodies randomly scattered in the nodal axoplasm. In contrast to this and as described elsewhere (J. Neurocytol., 15 [1986] 253-260), the nodal regions of alpha-motor axons at the level of the ventral root showed strong and characteristic accumulations of HRP-activity. The initial axon segments and adjoining axonal parts contained many HRP-positive bodies. We conclude that the CNS and the PNS parts of an alpha-motor axon differ with regard to the way nodal regions interact with retrogradely transported HRP. Possible mechanisms behind this difference are discussed.

Isolation of myelinoid Marchi-positive bodies from normal rabbit spinal cord.

Normal rabbit spinal cord was homogenized in sucrose and fractionated by centrifugation in a sucrose density gradient system slightly modified after the Norton-Poduslo method for the isolation of myelin. The following fractions were recovered: the fraction floating on 0.32 M sucrose, the myelin fraction at the 0.32 M/0.85 M interface and the pellet. After fixation in glutaraldehyde the fractions were subjected to Marchi staining, a histochemical method used for the demonstration of degenerating myelin. The floating fraction was enriched in Marchi-positive bodies as compared to the homogenate while the myelin fraction and the pellet contained low amounts. No esterified cholesterol was found in the floating fraction. Since histochemical and electron microscopical studies have shown that Marchi-positive myelinoid bodies in the normal CNS are associated with node-paranode regions our results indicate a possibility to isolate and biochemically characterize a presumably closely myelin-related fraction of known anatomical origin. The absence of esterified cholesterol in the floating fraction shows that biochemical or biophysical properties other than a content of esterified cholesterol may give rise to a positive Marchi reaction.

Peroxidase activity at nodes of ranvier in lumbosacral ventral spinal roots and in the PNS-CNS transitional region after intramuscular administration of horseradish peroxidase.

The axoplasm of nodes of Ranvier in feline lumbosacral ventral spinal roots was analysed by light and electron microscopy 18-168 h after the injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. Three main HRP distribution patterns were distinguished at PNS nodes (bordered by Schwann cells only) of large fibres transporting HRP. The type A pattern, characterized by a distal accumulation of HRP-positive bodies and a proximal system of vesiculotubular membrane profiles. The incidence of this type of node was highest at relatively short survival times. The type B pattern, which appeared somewhat later, resembled the type A node with the addition of a disc-like, proximal accumulation of HRP activity. The type C pattern which contained scattered HRP-positive bodies and delicate strands of membraneous profiles, dominated 72 h after injection. The number of HRP-positive PNS-CNS borderline nodes (bordered by both Schwann cells and glial cells) was less than 5% of the corresponding value in the same fibres of the ventral root proper. A highly segregated state of the axoplasm of PNS-CNS borderline nodes was noted only in two cases. The observations indicate a functional difference between nodal axoplasm at the PNS-CNS borderline and nodal axoplasm in the PNS part of the alpha motor neuron.