This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically-defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of 'normal' white matter in MS than in controls. A gradient of enzyme activity was observed : control white matter-white matter distant from plaqueclose white matter-edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base-line of thin-layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

PREVIOUS reports have described the preparation of proteins from bovine spinal cord, which, when injected into guinea pigs, caused allergic encephalomyelitis ( KIES, ROBOZ and ALVORD, 1956; ROBOZ, HENDERSON and KIES, 1958~). One of these proteins was isolated in a purified form and shown to be homogeneous by ultracentrifugal and electrophoretic measurements (ROBOZ, HENDERSON and KIES, 19586; MACKENZIE, 1958). It was classified, according to its amino acid composition, as a collagen-like protein (ROBOZ, HENDERSON and KIES, 19586). Since its activity accounted for only a small part of the total activity in the spinal cord other methods of extraction were tried (Ronoz and HENDERSON, 1959). Potassium chloride and sodium citrate solutions were used as extraction media, and active protein preparations were obtained. The potassium chloride extract contained about nine or ten proteins as shown by strip electrophoresis. The fractionation of these proteins by the use of ion exchange and continuous preparative electrophoresis methods is the subject of these studies. The work has not been reported previously except for a short account by ROBOZ EINSTEIN, ROBERTSON and DICAPRIO (1960). E X P E R I M E N T A LFor the preparation of the protein extract, bovine spinal cord was used. (This was generously supplied by a local slaughter house, James Allen & Sons, San Francisco.) After removal of the dura mater and blood vessels, the cord was homogenized in a Waring blender at 4'. The homogenized cord was lyophilized, pulverized, and treated with acetone and petrol-ether precooled in dry ice. This procedure resulted in partial removal of the lipids, thereby making the subsequent filtration and centrifugation easier. Treatment with distilled water or sodium acetate solution followed, 1 1. being used to every 100 g of tissue for 24 hr at 4" under constant stirring with a large-bladed propeller, driven at low speed to minimize emulsification and foaming. The water and the sodium acetate extracts had been found in earlier experiments t o be inactive and were not used in these investigations. From the residue the proteins were extracted overnight at 4" under constant stirring with 5 ", KCI in a ratio of 1 I. to every 100 g of fresh tissue. This was centrifuged under refrigeration at 8000 rev/min (10,400 g) and the supernatant solution further clarified by filtration. The KCI extracts were dialysed against dilute NaCl (0.01 M) and brought to 40% saturation with (NH,),SO,. Since the resulting precipitate was inactive, it was discarded. The supernatant liquor was fully saturated with (NH,),SO, and left overnight a t 4 . A flocculent suspension of proteins was formed and decanted

Abstract— The highly basic encephalitogenic protein isolated from bovine spinal cord was studied by various physicochemical methods: The molecular weight was determined by sedimentation equilibrium, by calculation from the data on sedimentation coefficients and intrinsic viscosities, by measurement of intrinsic viscosity in the presence of concentrated guanidine hydrochloride (according to the method of Tanford, Kawahara and Lapanie, 1967), and by exclusion chromatography on Sephadex G‐100 columns. All values obtained were in good agreement and indicated a molecular weight of approximately 18,000–20,000. Studies of sedimentation velocities in the presence and absence of 6 m‐guanidine‐HCl indicated that there was a significant difference in the values of sedimentation coefficients. The same conditions were applied to the measurements of viscosity; the difference was small but significant. These findings and the magnitude of the intrinsic viscosity suggested that this protein was in a disordered configuration. From these data, it is concluded that the protein was apparently monodispersed, in the presence or absence of the denaturing agent. This protein behaved like a polyelectrolyte in neutral aqueous solution. The measurements of optical rotatory dispersion also confirmed that this protein existed in a disordered configuration.

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t a n f o r d , C a l i f o r n i a , 94304; and t h e Department of Neurology, U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o , C a l i f o r n i a , 94122 and t h e Department of Physiology-Anatomy , U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , Calif o r n i a , 94708. A b s t r a c t Immuno-electron microscopic s t u d i e s of t h e l o c a l i z a t i o n of e n c e p h a l it o g e n i c b a s i c p r o t e i n demonstrate i t s p r e s e n c e i n t h e major d e n s e l i n e of t h e myelin s h e a t h . S t a i n i n g could only b e achieved where t h e myelin w a s p a r t i a l l y disruDted due t o a f a i l u r e of t h e l a b e l l e d antibody t o p e n e t r a t e t h e i n t a c t myelin s h e a t h . This f i n d i n g s u p p o r t s t h e view t h a t t h e a n t i g e n i c d e t e r m i n a n t s of t h e b a s i c p r o t e i n a r e occluded i n v i v o .or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14For personal use only.