Frederick Wolfgram

A new proteolipid fraction of the nervous system. I. Isolation and amino acid analyses.

THIS investigation was undertaken to examine the effect of the pH of the tissue homogenate on the ability of chloroform-methanol (C-M) to extract neural proteolipids. It became apparent that the problem is complicated by the fact that acidification sometimes renders simple proteins soluble in polar organic solvents. LEVINE (1954) and DELAVILLE, DELAVILLE and DELAVILLE (1954) showed that serum albumin precipitated with trichloroacetic acid is soluble in methanol and acetone. MICHAEL (1962) demonstrated that this solubility is not due to the trichloroacetic acid itself since HCl will also solubilize albumin in these solvents. MICHAEL found that this solubility is not a unique property of albumin since insulin, gelatin, globin and protamine are also solubilized upon acidification. Brain proteins, too, are extracted into ethanol acidified with HC1 (OTSUKI and GEIGER, 1963). LEBARON and ROTHLEDER (1960) found a ‘viscous protein’ that can be solubilized in chloroform-methanol-HCl (C-M-HCI) from the C-M insoluble residue of white matter. While the present work was in progress, GAITONDE (1961) observed an increase in the soluble proteins of the pooled acetone, chloroform-ethanol (1 : 1, v/v) and diethyl ether extracts from rat brain proteins precipitated with trichloroacetic acid. LEBARON and LEES (1962) also found an increased amount of C-M-soluble protein in trichloroacetic acid precipitates of bovine white matter. Neither of these investigators separated this protein from the Folch-Lees proteolipid and they pointed out that its solubility in the solvents may have been due to the acidic conditions rather than to attached lipids. In a more recent study, however, GAITONDE (1 963) without additional evidence, designated this fraction as proteolipid (‘Proteolipid 2’). This report deals with the repetition, extension and re-evaluation of some of the above-mentioned experiments concerning the solubility of proteins in acidified organic solvents. Evidence is also presented to show that there exists in white matter and in peripheral nerve a new trypsin-digestible proteolipid that is extracted into C-M on HCl acidification of the tissue homogenate and which was not demonstrated in the originalinvestigation of FOLCH and LEES (1951) or in any subsequently published work. A preliminary report of this investigation has been published (WOLFGRAM, 1963).

MYELIN TYPES WITH DIFFERENT PROTEIN COMPONENTS IN THE SAME SPECIES1

Abstract— Myelin was isolated from bovine optic nerve, cerebral white matter, spinal cord white matter and peripheral nerve (intradural spinal roots). The freeze‐dried myelin completely dissolved in phenol‐formic acid‐water (14:3:3, w/v/v), and acrylamide gel electrophoresis of the myelin proteins was performed with this solvent. Qualitative and quantitative differences were observed in the myelin proteins from the various regions of the CNS. Myelin of peripheral nerve contained proteins that are apparently unique to it and which are not found in the myelin of the CNS.

Amyotrophic lateral sclerosis: effect of serum on anterior horn cells in tissue culture.

A high proportion of diluted serums of patients with amyotrophic lateral sclerosis were toxic to the anterior horn cells of the mouse in tissue culture. This is not a general cytotoxicity, since apparently only the neurons were killed. Serums from other degenerative neurological diseases were inactive.

Spectrophotometric assay for choline acetyltransferase

Abstract A rapid and simple spectrophotometric assay for choline acetyltransferase is described. The method employs 4,4′-dithiodipyridine to measure the coenzyme A produced by the enzymic reaction. The conditions of the assay are described. The results are compared with those obtained by the radiochemical assay of the enzyme.

Purification and some properties of choline acetyltransferase (EC 2.3.1.6) from bovine brain.

Abstract— Choline acetyltransferase (ChAc) has been isolated and highly purified from the caudate nuclei of bovine brains. The procedure involved: (1) making an acetone‐ and chloroform‐insoluble powder from the tissue; (2) treating the powder with aqueous buffer and chromatographing the extractable soluble proteins on an organomercurial‐sepharose column; (3) removing impurities by passage through columns of DEAE‐cellulose and hydroxyapatite; and (4) separation of the heme‐containing protein from ChAc by denaturing the former with a mixture of chloroform and n‐butanol. The purified ChAc was essentially homogeneous as judged by polyacrylamide gel electrophoresis and exhibited a pH optimum at about pH 7. The partially purified ChAc dissociated into two non‐identical subunits when chromatographed with a dilute buffer on Bio‐gel A. It did not dissociate when a more concentrated buffer was used. The purified ChAc dissociated on the Bio‐gel A even in the presence of a high salt concentration. The dissociation was accompanied by a great loss of enzymatic activity, and we concluded that the presence of other proteins tends to prevent the dissociation of ChAc on gel filtration.

ACTIVATION, INHIBITION AND AGGREGATION OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6)

—Mercuric chloride, silver acetate and cupric sulphate (0·1 mm) completely inhibited purified choline acetyltransferase from bovine caudate nuclei. At the same concentration cadmium chloride and zinc acetate gave a 50 per cent inhibition. Potassium and sodium salts more than doubled the enzymatic activity while creatinine hydrochloride more than tripled it. Guanidine hydrochloride was less effective than creatinine hydrochloride but more effective than KCl and NaCl. Sodium chloride and creatinine hydrochloride had a synergistic effect on the enzyme.

Demyelinating antibodies in multiple sclerosis.

No. 26 Demyelinating antibodies in

What if multiple sclerosis isn't an immunological or a viral disease? The case for a circulating toxin.

The literature contains studies on many causes of demyelination: Vascular occlusion —exogenous toxins—allergic reactions—and virus diseases. There remains, however, a small group of conditions which seems to have been ignored almost completely in discussions of cerebral demyelination. These consist of brain damage apparently resulting from some toxin liberated within the human body and not obtained from the outside.

Serum linoleic acid in multiple sclerosis

Total serum fatty acids were analyzed in 30 patients with multiple sclerosis and in 33 controls. No significant decrease in linoleic acid was found in the patients with multiple sclerosis. A disturbance in linoleic acid metabolism is not inevitably associated with this disease.

THE AMINO ACID COMPOSITION OF CENTRAL AND PERIPHERAL NERVE NEUROKERATIN

THE WORD ‘neurokeratin’ was coined by EWALD and KUHNE (1877) to describe the myelin protein resistant to digestion with gastric and pancreatic juices. It has remained a subject of interest ever since. There have been several investigations of its elemental and amino acid conipostion (ARGIRIS, 1907; NELSON, 1916; BLOCK, 1932, 1951). Traditionally neurokeratin has been characterized by its resistance to proteolytic enzymes (usually pepsin or trypsin) and its insolubility in organic solvents and dilute acids and bases. FOLCH and LEES (1951) showed that the chloroform-methanol soluble proteolipids of nervous tissue, which they discovered, are also trypsin and pepsin resistant. Furthermore, LEBARON and FOLCH (1956) and FOLCH and LEBARON (1959) demonstrated that after removal of the proteolipids with chloroform-methanol there still exists a trypsin-resistant protein (TRPR) in the insoluble residue. This TRPR has phosphorus, inositol, and peptides bound to it and these ‘phosphatido-peptides’ can be removed from the protein by treatment with chloroform-methanol-HCI (200: 100: 1, by vol.). It was clearly established then by these investigations that neurokeratin prepared by the traditional methods is a mixture of proteolipid protein and TRPR. The question thus arises as to whether the protein moieties of proteolipid and TRPR are similar or not. The earlier amino acid analyses of neurokeratin (BLOCK, 1951) provide no answer since they were made on neurokeratin prepared by the methods in use before the discovery of proteolipids. The amino acids of bovine proteolipid protein from white matter were analysed by FOLCH and LEES (1951). WOLFGRAM and ROSE (1961) repeated the analysis of the proteolipid from this tissue and also determined the amino acid composition of the proteoIipids of human CNS white matter and human and bovine intradural spinal roots. It was pointed out that the proteolipids from these diverse sources have remarkably similar amino acid patterns. TRPR is a satisfactory term to describe the non-proteolipid protein of the CNS resistant to proteolytic enzymes. However, in the peripheral nervous system, TRPR is misleading in that collagen is also a TRPR (FOLCH, LEES and CARR, 1958; WOLFCRAM and ROSE, 1961). For this reason we propose to designate our fractions as neurokeratin, objectionable as the word may be, and to redefine it as the chloroformmethanol insoluble, trypsin-resistant protein of nervous tissue freed of collagen and phosphatido-peptides, E X P E R I M E N T A L Most of the methods used have been described previously (WOLFCRAM and ROSE, 1961). All of the experiments reported here were done on bovine centrum ovale white matter or bovine intradural