Catherine F.C. Macpherson

DIFFERENCES IN THE SUBCELLULAR AND SUBSYNAPTOSOMAL DISTRIBUTION OF THE PUTATIVE ENDOPLASMIC RETICULUM MARKERS, NADPH‐CYTOCHROME c REDUCTASE, ESTRONE SULFATE SULFOHYDROLASE AND CDP‐CHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE IN RAT BRAIN

Abstract— A comprehensive study has been undertaken on the subcellular and subsynaptosomal distribution of a number of markers for subcellular organelles in preparations from rat brain. Although the activity of most enzymatic markers was decreased by freezing and storage at ‐ 70oC, no significant changes were noted in the distribution of these activities. This demonstrates that contamination of brain fractions by subcellular organelles can be accurately assessed after freezing and thawing. A marked discrepancy was noted between the distribution of three putative markers for endoplasmic reticulum. CDP‐choline‐diacylglycerol cholinephosphotransferase (EC 2.7.8.1) activity was mainly limited to the microsomal fraction and was present to a lesser extent in the synaptosomal fraction than the other putative markers for endoplasmic reticulum. Estrone sulfate sulfohydrolase (EC 3.1.6.2) activity demonstrated a bimodal distribution between the crude nuclear and microsomal fractions. However, considerable activity was associated with the synaptosomal fraction. NADPH‐cytochrome c reductase (EC 2.3.1.15) activity sedimented in the microsomal and the synaptosomal fractions. Calculations based on the relative specific activities of the microsomal and synaptic plasma membrane fraction indicated that the contamination of the synaptic plasma membranes by endoplasmic reticulum was 44.5% (NADPH‐cytochrome c reductase), 38.0% (estrone sulfatase) and 9.0% (cholinephosphotransferase). Since it is believed that virtually all of the synthesis of phosphatidylcholine by cholinephosphotransferase occurs in the neuronal and glial cell bodies, it was concluded that cholinephosphotransferase is a satisfactory marker for the endoplasmic reticulum derived from these sources. The results suggest that NADPH‐cytochrome c reductase and estrone sulfatase may be present in the smooth endoplasmic reticulum system responsible for the fast transport of macromolecules along the axon to the nerve endings as well as in the endoplasmic reticulum of the cell bodies. The possible relation between that portion of the smooth endoplasmic reticulum involved in fast axonal transport and the GERL (Golgi, Endoplasmic Reticulum, Lysosomes) complex discovered by Novikoff and his coworkers (Novikoff, 1976) is discussed.

Prevention of experimental allergic encephalitis in Lewis rats by rat and bovine spinal cord proteins

EXPERIMENTAL allergic encephalitis (EAE) is an inflammatory autoimmunedisease of the central nervous system (CNS) that is mediated by T lymphocytes sensitised to the organ-specific basic protein (BP) of CNS myelin2. Lesions of delayed hypersensitivity develop in the brain and spinal cord when sensitised lymphocytes traverse the walls of CNS blood vessels and react with the target antigen in myelin3. EAE can be prevented by injecting BP in Freunds incomplete adjuvant (FIA) before challenge, suppressed by injecting BP soon after challenge and treated by injections of BP after clinical symptoms appear4. Prevention, suppression and treatment are immunologically specific for BP and can be dissociated from the production of humoral antibody against BP5. The demonstration that pretreatment of guinea pigs with bovine spinal cord protein (BSCP)6,7 prevented the development of EAE8,9 is thus of considerable importance because BSCP is chemically and antigenically distinct from bovine BP and is not encephalitogenic. As pretreatment with SCP induced a state of nonspecific unresponsiveness to bovine BP in the guinea pig, we investigated whether SCP had anti-encephalitogenic activity in the Lewis rat, a highly inbred rat strain used extensively for the study of EAE. We report here that Lewis rats pre-treated with rat SCP (ref. 10) were protected against EAE, as they did not develop the disease when they were subsequently immunised with rat myelin BP in complete Freunds adjuvant (CFA).

The spinal cord protein (SCP): Appraisal of physiological and immunological hypotheses of anti-encephalitogenic action

This invitation to contribute to the Festschrift for Michael Heidelberger has given me an opportunity to present an overview of my recent immunochemical studies on the proteins of the central nervous system. pretreated with bovine gray matter protein and all of the animals that had been pretreated with bovine liver protein developed severe EAE. No histologic lesions were found in the brains of four of the BSCPpretreated animals.

Evidence that rat peripheral myelin does not contain the rat spinal cord protein (RSCP-PN)

Rat spinal cord protein (SCP) from peripheral nerves (RSCP-PN) was not detected in purified rat peripheral nerve myelin by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis or by immunodiffusion analyses using an anti-rat SCP in peripheral nerve (RSCP-PN) serum. The slab gel electrophoretic analyses also revealed that RSCP-PN has an appreciably lower molecular size than the component of rat peripheral myelin that is identified as P2 by its molecular size of 13,600 daltons. Thus, RSCP-PN and rat P2 are unrelated proteins.

Evidence that the bovine spinal cord protein is not an intrinsic component of peripheral myelin

Bovine spinal cord protein from peripheral nerve (BSCP-PN) was detected in the soluble fraction of the initial 0.8 M sucrose homogenate of bovine peripheral nerves by immunodiffusion analyses and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The BSCP-PN in the soluble fraction of the 0.8 M sucrose homogenates was 25% of the BSCP-PN found in the soluble fraction of 0.3 M NaCl homogenates of peripheral nerve. BSCP-PN was also identified in purified bovine peripheral nerve myelin by immunodiffusion analyses and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Densitometry data indicated that the BSCP-PN in myelin decreased from 25% of the total protein to approximately 8% when myelin was extracted with 0.3 M NaCl or 0.05 M HCl. The protein that remained in the BSCP-PN band of the NaCl-extracted myelin was identified as the periodic acid-Schiff II glycoprotein of peripheral myelin. Basic proteins such as BSCP-PN or lysozyme bound to myelin and to NaCl-extracted myelin when they were added to homogenates of myelin in 0.8 M sucrose. Pepsin, an acidic protein, did not bind to myelin under the same conditions. The results suggest that in 0.8 M sucrose, positively charged BSCP-PN released from the cytoplasm by homogenization binds to negatively charged myelin; thereafter, the BSCP-PN-myelin complex remains intact until it is dissociated in media of sufficiently high ionic strength. This interpretation is consistent with the immunohistological studies which demonstrated that BSCP-PN was not in the myelin sheath but was clearly localized in axons and in, or adjacent to, the Schwann cell basement membrane.

Partial characterization of the human anti-encephalitogenic protein: Isolation from spinal cord and spinal nerves

Abstract 1. 1.|The human spinal cord protein (HSCP) is immunochemically closely related to the anti-encephalitogenic bovine spinal cord protein (BSCP) and immunoreactive HSCP is similarly distributed in brain, spinal cord and spinal nerve roots (a peripheral nervous tissue) in the proportions of 1 : 6 : 60. HSCP and protein immunochemically identical to HSCP (HSCP-PN), were purified from frozen spinal cords and frozen peripheral nervous tissue, respectively, by extraction with 0.15 M sodium chloride, carboxy-methyl cellulose (CM-cellulose) chromatography and gel filtration on Sephadex G-50. 2. 2.|Purified HSCP and HSCP-PN formed one band in sodium dodecyl sulfate polyacrylamide gel electrophoretograms and had estimated molecular sizes of 13 700 and 14 700, respectively. The amino acid compositions were similar except that HSCP had 16% glutamic acid and lacked half cystine while HSCP-PN contained 11.9% of glutamic acid and 1.0% of half cystine. 3. 3.|Immunodiffusion analyses with anti-HSCP or anti-BSCP sera revealed that extracts of spinal cord and spinal nerves and purified HSCP and HSCP-PN are composed of immunogenically distinct major and minor forms. The major forms and the minor forms of HSCP and HSCP-PN are identical to each other but share different antigenic amino acid sequences with BSCP. Immunoelectrophoretic profiles of spinal cord and spinal nerve extracts indicated that the major and minor HSCP antigens also existed in two different molecular forms having the electrophoretic mobilities of a β- or a γ-serum globulin. The four different molecular forms were present in purified HSCP-PN, but only the forms with γ-electrophoretic mobility were present in purified HSCP.

Two integral antigens shared by the synaptic membranes and endoplasmic reticulum of rat brain: Separation by differential extraction

Abstract Two major brain membrane antigens designated X1 and X2 were found to be shared by rat brain synaptic membranes (RSM) and rat brain endothelial reticulum (RBER) by immunodiffusion analyses with IgG solutions prepared from the sera of rabbits hyperimmunized with RSM and RBER. The antisera were absorbed with the soluble fraction of rat brain (RBE) which removed antibodies directed against antigens in the soluble fraction of brain. Antigens X1 and X2 are considered to be integral components of the RSM and RBER membrane matrixes because they were not removed from the membranes by sequential washing with sodium salts but were extracted with Triton X-100 or butanol. Sequential treatments of RSM and RBER with different concentrations and volumes of Triton X-100 yielded Tx1a, Tx1b and Tx2 extracts; Tx1 solutions contained antigens X1 and X2 while Tx2 solutions contained only antigen X2. Treatment of RSM or RBER with butanol rendered both antigens soluble in water. The concentrated aqueous solutions of X1 and X2 were stable for several months in 0.5% Triton X-100. Anti-X1 antisera were prepared by absorbing anti-RSM sera with RBE and RSM-Tx2 extracts.

Isolation and further characterization of the anti-encephalitogenic rat spinal cord protein from brain, spinal cord and peripheral nerves.

Abstract After fresh rat nervous tissue was delipidated with acetone, all the immunoreactive rat spinal cord protein (RSCP) in brain (RB-SCP), spinal cord (RSCP) and peripheral nerve (RSCP-PN) could be extracted with 0.05 M sodium acetate buffer, pH 5.0. The extracts were purified by absorption on CM-52 cellulose at pH 5.0 and by gel filtration on Sephadex G-50. At this stage, RSCP-PN formed one band when analyzed by SDS-polyacrylamide gel electrophoresis. In contrast, preparations of RB-SCP and RSCP contained two components of similar molecular size. Only the larger component was present in the specific precipitates formed by mixing anti-RSCP antibody with RB-SCP or RSCP, clearly indicating that the smaller component was a contaminant. Thus, the final purification step for RB-SCP and RSCP consisted of absorption on cyanogen bromide-activated Sepharose-4B coupled to anti-RSCP antibody followed by elution of immunoreactive RSCP at pH 2.5. RB-SCP, RSCP and RSCP-PN isolated from delipidated nervous tissue were identical by immunodiffusion analysis. RSCPs have a molecular weight of 11 000 ± 500, as estimated by gel exclusion chromatography and by SDS-polyacrylamide gel electrophoresis. They have the electrophoretic mobility in agarose at pH 8.6 of a serum β-globulin and an isoelectric point of pH 4.5. RB-SCP and RSCP contain respectively about 17, 24 and 42% of basic, acidic and hydrophobic amino acids. RSCP-PN has a somewhat higher content of acidic amino acids, (31%) and a lower proportion of hydrophobic amino acids (32%). All RSCPs lack cystine and contain only 0.2% of tyrosine. Immunodiffusion analyses showed that at the antibody level there is no immunochemical relation between bovine SCP (BSCP) and purified RB-SCP, RSCP or RSCP-PN. However, there is a protein in rat peripheral nerve that is similar but not identical to BSCP. This protein is not present in extracts of brain or in extracts of spinal cord from which the spinal nerves have been removed.

One-way humoral immune cross-reactivity between bovine spinal cord protein and bovine myelin basic protein

Abstract Whether bovine myelin basic protein (BP) and bovine spinal cord protein (SCP) cross-react at the humoral immune level was assessed with a sensitive solid-phase enzyme immunoassay. We found that a hyperimmune anti-SCP serum reacted strongly with SCP and cross-reacted nearly as well with BP. A hyperimmune anti-BP serum reacted only with BP. Antigenic competition analysis revealed that SCP and BP both inhibited binding of the hyperimmune anti-SCP serum to solid-phase adsorbed SCP and BP, while only BP inhibited binding of the hyperimmune anti-BP serum to solid-phase adsorbed BP. Finally, BP cross-reactivity antibodies were present in early bleedings from rabbits immunized with SCP that had been passed through an anti-BP immunosorbent column. These results clearly show there is a one-way humoral immune cross-reactivity between SCP and BP which goes in the direction of SCP to BP.