ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K+

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR-P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF-P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR-PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR-P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.     

Alteration of 5'-Nucleotidase and Na+, K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler

Abstract: 5'Nucleotidase and Na⁺,K⁺-ATPase are very probably myelin-associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5'nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21-day-old and adult mutants, except Trembler. It was normal in 8-day-old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5'-Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10-day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin-related enzymes and a myelin-specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5'-nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5'-nucleotidase increased (42% and 190% of the control in 60-day-old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.     

Degradation of the P0, P1, and Pr, Proteins in Peripheral Nervous System Myelin by Plasmin: Implications Regarding the Role of Macrophages in Demyelinating Diseases

Activated macrophages secrete a variety of neutral proteinases, including plasminogen activator. Since macrophages are implicated in primary demyelination in the peripheral nervous system (PNS) in Guillain-Barré syndrome and experimental allergic neuritis, we have investigated the ability of plasmin and of conditioned media from cultured macrophages, in the presence of plasminogen, to degrade the proteins in bovine and rat PNS myelin. The results indicate that (a) the major glycoprotein P0 and the basic P1 and Pr proteins in PNS myelin are extremely sensitive to plasmin, perhaps more so than is the basic protein in CNS myelin; (b) the initial product of degradation of P0 by plasmin has a molecular weight higher than that of the "X" protein; (c) large degradation products of P0 are relatively insensitive to further degradation; and (d) the neuritogenic P2 protein in PNS myelin is quite resistant to the action of plasmin. Results similar to those with plasmin were obtained with conditioned media from macrophages and macrophage-like cell lines together with plasminogen activator, and the degradation of the PNS myelin proteins, Po and P1, under these conditions was inhibited by p-nitrophenylguanidinobenzoate, an inhibitor of plasmin and plasminogen activator. The results suggest that the macrophage plasminogen activator could participate in inflammatory demyelination in the PNS.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Characterization of Developmentally Regulated cAMP/Ca2+-Independent Protein Kinases from Dictyostelium discoideum

Cell-free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate-associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S-300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE-Sephacel and heparin-Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE-Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa.     

Development and Applications of a Solid-Phase Radioimmunoassay for the P0 Protein of Peripheral Myelin

This is the first report of a quantitative radioimmunoassay for PO. The assay uses antigen-coated plastic microwells, with antibody binding detected by 125I-labeled protein A. Either peripheral myelin proteins or purified PO may be used as the antigen. Optimal extraction of tissue samples for PO immunoassay requires careful attention to the sodium dodecyl sulfate-to-protein ratio. Sodium dodecyl sulfate interference with antibody binding can be minimized by adding an excess of nonionic detergent and carrier protein to the incubation buffer. This method allows the detection of 0.8 ng of PO (20 ng/ml). Results from this assay showed little or no immunoreactivity in extracts of brain, centra myelin, liver, purified myelin basic proteins, cultured, purified secondary Schwann cells, or membrane preparations from these cells. PO was clearly detectable in Schwann cell cultures from 3- to 4-day-old rats at 12-18 h after dissociation (4% of the level in adult sciatic nerve) and in extracts of one-day-old rat sciatic nerve (2% of the level in adult nerve). Myelin basic protein radioimmunoassays showed that the ratio of PO to myelin basic protein is essentially constant in extracts of sciatic nerve from ne-day-old, four-day-old, and young adult rats. Another result was that PO levels are reduced in the trembler mouse sciatic nerve.     

Pyridoxal-5-phosphate levels in rat brain assayed by a modified method using enzymic decarboxylation of l-[14C] tyrosine

Abstract— The formation of a complex between myelin basic protein and S-100 protein was detected from the change in migration of S-100 protein on immunoelectrophoresis. A degree of specificity for the interaction was shown by two observations: (1) two other pure acidic proteins. III-III-2 and bovine serum albumin, did not show it and (2) complex formation was dependent on specific ions, either Ca²⁺ (10 mM) or Mn²⁺ (1 mM). Mg²⁺, Ba²⁺, and Li⁺ had no effect. Non-specific interactions between S-100 protein and other basic molecules (histones. polylysine) are not dependent on specific ions such as Ca²⁺ and Mn²⁺. The complex was stable at physiological salt concentrations and contained 3 mol of basic protein per mol of S-100 protein. Complex formation was also detected from the alteration of migration rate of S-100 protein in polyacrylamide gels. Serological activity (complement-fixation) of S-100 protein with anti-S-100 serum was reduced in the complex by 30%.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes

Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a "receptor" molecule, perhaps a membrane-anchored protein, for association with the cytoplasmic face of the myelin membrane.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Nerve Growth Factor Induces Na+, K+-ATPase in a Nerve Cell Line

The effects of nerve growth factor (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = 19) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.     

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

Metabolism of Phosphate and Sulfate Groups Modifying the P0 Protein of Peripheral Nervous System Myelin

We have examined the metabolism of phosphate and sulfate groups modifying the P0 protein, the major protein of peripheral nervous system myelin, using an in vitro incubation system. Incorporation of [3H]leucine into the P0 peptide backbone decreased approximately 25-fold between 10 and 90 days of age, a finding reflecting a decreased rate of myelin synthesis in the older animals. In contrast, incorporation of [32P]phosphate into P0 decreased only four- to fivefold, a result indicating that phosphate groups are metabolized independently of the peptide backbone. Developmental decreases in the incorporation of sulfate groups into P0 were similar to those seen for leucine, an observation suggesting that this modifying group is metabolized together with the peptide backbone as a single metabolic entity. The time course of labeling of P0 isolated from the starting homogenate and from myelin was also compared. Results are consistent with sulfation of P0 protein taking place before insertion of newly synthesized P0 into myelin. In contrast, incorporation of phosphate into P0 appears to involve both the newly synthesized pool and the preexisting pool of P0 in myelin. Presumably, entry of phosphate into P0 in myelin involves turnover of preexisting phosphate groups and rephosphorylation by myelin protein kinases. Developmental decreases in the specific activity of P0 phosphate groups in myelin are consistent with the presence of a small, rapidly turning-over pool of phosphorylated P0 (perhaps associated with the axon-myelin interface), which does not increase to the same extent as the marked increase in bulk myelin that occurs during development.     

Hydrophobic Regions in Myelin Proteins Characterized Through Analysis of "Hydropathic"Profiles

Computer-generated "hydropathic" profiles were constructed for graphic comparison of the amino acid sequences for P2 protein, 18.5 kilodalton (kDa) myelin basic protein (BP), and myelin proteolipid protein (PLP). Profiles were also obtained for cytochrome b5, a membrane protein known to be capable of reversible association with lipid bilayers and of a size comparable to that of the myelin BPs. Analysis of the PLP sequence produced profiles generally compatible with the suggestions that PLP has three transbilayer and two bilayer intercalating segments. Profiles for P2 and 18.5 kDa BP were found to contain hydrophilic segments separated by relatively short hydrophobic regions. Whereas hydropathic indices in hydrophobic regions of P2, 18.5 kDa BP, and PLP fall in the value ranges recently reported for cores of globular proteins and intrabilayer domains of membrane proteins, hydrophobic sections of P2 and 18.5 kDa BP have hydropathic indices similar to those in the hydrophobic core (transprotein) regions of globular proteins. None of them are comparable to the region of cytochrome b5 known to anchor that protein in its membrane or to the segments of PLP sequence proposed as intrabilayer domains. This comparison suggests that neither BP has structural characteristics compatible with insertion into the hydrocarbon core of the myelin lipid bilayer, a conclusion that is consistent with a recently published study that identified the bilayer penetrating proteins of myelin with a hydrophobic probe. The above findings suggest an enhancement for some details of myelin architecture and a cautious approach to interpreting data for BP intercalation into bilayers.     

ARE MYELIN PROTEINS SYNTHESIZED IN RETINAL GANGLION CELLS?1

—We studied the incorporation of radioactivity into individual proteins of myelin by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the injection of [³H]tryptophan into the right eye of developing rabbits. We found that the specific activity of basic protein (c.p.m./mg of basic protein) and the specific activity of DM-20 and proteolipid protein (c.p.m./mg total myelin protein applied to the gel) did not approach the ratio predicted by decussation of the fibres of the rabbit optic nerve. The specific activity of Wolfgram protein, however, approached an expected ratio of 15:1. We therefore concluded that myelin basic protein, DM-20 and proteolipid protein were probably not synthesized in retinal ganglion cells.     

Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Immunochemical Characterization of Peripheral Nervous System Myelin 170,000-Mr Glycoprotein

A recently described 170,000-Mr glycoprotein, specific to peripheral nervous system (PNS) myelin, was purified from rat PNS myelin by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to immunize guinea pigs and rabbits. The resultant antisera proved specific for 170,000-Mr glycoprotein by enzyme-linked immunosorbent assay, by immunoprecipitation of the appropriate peptide from solubilized PNS myelin, and by immunoblot analysis of rat PNS myelin. The anti-rat 170,000-Mr glycoprotein antisera cross-reacted with proteins of similar molecular weight in human and bovine PNS myelin, but such proteins were not detected in human or rat CNS myelin or other rat tissues. The 170,000-Mr glycoprotein was also detected by this immunoblot procedure in recently isolated rat Schwann cells but not in those kept in culture for greater than or equal to 3 days. By indirect immunofluorescent microscopy, anti-rat 170,000-Mr glycoprotein antibody bound to rat PNS myelin sheaths but not to other rat tissues. Together, these studies indicate the 170,000-Mr glycoprotein is specific to PNS myelin of several species and that a neuronal influence may be required for its expression by Schwann cells.     

Effects of Ca2+ and polyamines on Na+ and Rb+ influx in excised maize roots

When 1 m M spermidine or spermine was included in an absorption solution which contained 20 m M Na⁺ and 1 m M Rb⁺, Na⁺ influx into excised maize roots ( Zea mays L. cv. Golden Cross Bantam) was reduced. Rb⁺ influx was reduced in the presence of spermidine and uneffected in the presence of spermine when compared with control solutions. When 1 m M Ca²⁺ replaced the polyamines, Na⁺ influx was strongly reduced and Rb⁺ influx was promoted. Rb⁺ influx from 1 m M Rb⁺ solutions which did not contain Na⁺ was also promoted by 1 m M Ca²⁺, but was inhibited by 1 m M spermidine. This Ca²⁺ promotion of Rb⁺ influx could be reversed by 10 times greater concentration of spermidine in the absorption solution. H⁺ efflux from excised roots was inhibited by spermidine when compared with Ca²⁺ or control solutions, however, the plasma membrane ATPase was not inhibited by spermidine. It is concluded that external Ca²⁺ plays two separate roles in membrane function, only one of which can be substituted for by polyamines. The first role, maintenance of membrane integrity, can be substituted for by spermidine or spermine. The second function, maintenance of the Rb⁺ transport mechanism, is Ca²⁺ specific and cannot be substituted for by spermidine or spermine. The results of this study are discussed in terms of electrostatic interactions between the plasma membrane and the Ca²⁺ or polyamines.