Structural Considerations
The Composition of Nervous Membranes J. F O L C H - P I McLean Hospital, Belniont, and Department of Biochemistry, Harvard Medical School, Boston, Mass. ( U S A . )
The literature on the chemistry of membranes is much too vast to allow a meaningful review within the space and time available for this presentation. Therefore, instead of attempting such an impossible task, the subject matter of this presentation will be limited to the discussion of four groups of compounds which occur mainly, if not exclusively, in the nervous system and all of which are clearly identifiable as membrane constituents. These compounds are gangliosides, proteolipids, polyphosphoinositides and neurokeratin. Gangliosides are neuronal components; proteolipids, polyphosphoinositides and neurokeratin are myelin components. Since even our information on these compounds - and specially the sum total of our uncertainties about them - is much too vast for adequate presentation in the time available, the following comments will bear mainly on those aspects of their chemistry that are specially pertinent to their function as membrane constituents. GANCLIOSIDES
In 1941, Klenk isolated from brain a new aniino acid to which he gave the name of
iieuruminic acid. In 1942, he described a group of new brain glycolipids that were
characterized by the presence of neuraminic acid and which were otherwise constituted by a lipid moiety, presumably a ceramide, and a carbohydrate moiety presumably consisting of one or more monosaccharides. He named them gangliosides because their distribution in the tissue suggested that they were components of ganglion cells. Subsequent work by Klenk and other workers established that neuraminic acid was present in gangliosides usually as an N-acetyl derivative, and that it was identical with siulic acid which had been isolated by Blix from submaxillary mucin a few years previously (Klenk, 1936); that the carbohydrate moiety of gangliosides contained hexosamine(s) in addition to neutral sugars; and, finally, especially after the introduction of thin layer chromatography, that gangliosides comprised a large number of closely related chemical compounds. The chemistry of gangliosides has been the object of recent authoritative reviews, to which the reader is referred for a detailed discussion of the subject (Svennerholm, 1964; Ledeen, 1966). In summary, gangliosides are complex glycolipids consisting, Referenrrs p. 13-14
2
J. F O L C H - P I
according to Kuhn and Wiegant (1963), of a lipid moiety in the form of a ceramide, and of a carbohydrate moiety in the form of a tetrasaccharide, as follows: galactose (1 -3)N-acetylgalactosamine( 1-4)gaIactose( I -4)glucose( 1- 1)ceramide
To this backbone are attached, I , 2 or 3 sialic acid residues constituting respectively mono-, di- or trisialogangliosides. Variations in the carbohydrate moiety have been
reported and at present upward to 12 different gangliosides have been recognized. In addition, the ceramide moiety, although consisting mainly of sphingosine and stearic acid, contains also higher and lower homologs of sphingosine, and a host of different fatty acids, thus multiplying several times the number of individual gangliosides that occur in nature. In parallel with this chemical work, it was observed that, although gangliosides were extracted from brain tissue with conventional lipid solvents, they were easily soluble in water as undialyzable solutes. In aqueous solutions they appeared to be monodisperse (Folch et al., 1951), with an apparent molecular weight which was first computed, from ultracentifuge data, as being 250 000 and which by other methods of measurement employing different parameters has been given values ranging from 180 000 to 400 000. This observation led to the assumption that a physically homogeneous high molecular weight compound was being dealt with, and to it was given the name of strandin (Folch et al., 1951). With advances in the chemistry of gangliosides it became apparent that strandin was a polymeric form of gangliosides, and even a critical micellar concentration of 0.015 % was suggested (Howard and Burton, 1964). Since it had been observed that preparations of gangliosides contained small amounts of polypeptides (Folch et a / . , 1951; Folch and Lees, 1959) or proteins, it has been also suggested that these presumed protein contaminants might play a part in determining the remarkable homogeneity of the micellar solutions of strandin (Rosenberg and Chargaff, 1956). That gangliosides are, at least in part, membrane components appears to be a reasonable assumption on the basis of their distribution in the nervous system, of their rate of accumulation during brain development (Folch, 1955), of histochemical evidence (Diezel, 1959, and of their distribution among subcellular fractions of brain tissue (Wolfe, 1961 ; Wherrett and Mcllwain, 1962; Seminario et al., 1964; Burton et al., 1964; Eichberg et al., 1964; Spence et a/., 1964). In addition, a consideration of some of the properties of gangliosides clearly points to them as being exceptionally well designed as membrane constituents: the presence of the carboxyl group of sialic acid which permits binding with organic and inorganic cations, the presence of the lipophilic groups of the ceramide and of the hydrophilic groups of the carbohydrate moiety which permit interaction with many different substances including proteins, lipids, and many small molecule substances, and the ability to form micelles of fairly uniform size. Indeed, it is not surprising that gangliosides have been implicated by many workers in different membrane functions : cation transport (Mcllwain, 1962), acetylcholine release at the presynaptic membrane, synaptic inhibition, receptor function for serotonin (Burton et al., 1964), for tetanus toxin (Van Heyningen, 1963), which parallel the well established function of sialic acid as a viral receptor in red
COMPOSITION O F NERVOUS MEMBRANES
3
blood cells, just to mention a few highlights in a considerable literature dealing with possible functions of gangliosides. We will close this brief survey by discussing the interaction of gangliosides with sodium, potassium, calcium and magnesium, and an apparent effect of the presence or absence of polypeptide on the behavior of the resulting complexes. As expected, all four cations combine with gangliosides, presumably by simple electrostatic bonds. These combinations are reversible, and each cation can displace the others from combination with the ganglioside, the divalent cations being more effective than the monovalent. However, the calcium-ganglioside complex is much less polar than either the free ganglioside or the complexes of ganglioside with the other cations (Quarles and Folch-Pi, 1965). Thus, when these salts of gangliosides are dissolved in the biphasic system chloroform : methanol water 8 : 4 : 3, v/v/v, free ganglioside, and its sodium, potassium or magnesium salts remain in the upper (polar) phase. On the other hand, the calcium complex will remain in the upper phase at low and at high concentrations of calcium ions, but at intermediate concentrations of these ions, it will partition into the lower, least polar phase. This effect of calcium appears to require the presence of small amounts of other lipids, sulfatides being especially effective in this action. In addition, the presence of polypeptide will tend to produce an accumulation of calcium gangliosidate at the interphase. All these interactions illustrate the dramatic changes that may occur in the physical properties of gangliosides, hence on their possible behavior as membrane constituents, and they also point to a possible crucial influence of the presence of small amounts of polypeptides on ganglioside properties. The effect of polypeptides does not appear to be a general protein property. Since the concentrations of calcium that effect the change in polarity of the ganglioside fall in part within the physiological range of concentrations of calcium, it is clear that the observations on the model employed may have implications for the behavior of gangliosides in vivo.
+
Proteolipids, polyphosphoinositides and neurokeratin These three groups of substances are closely related biochemically and anatomically. As will be detailed below, these are myelin constituents and, since myelin itself is formed by the infolding of the plasma membrane of the satellite glial cells around the axons, it is obvious that myelin components are membrane components by definition. In addition, polyphosphoinositides are constituents of both proteolipids and neurokeratin and proteolipids and neurokeratin appear to be very closely related. Since polyphosphoinositides are components of both proteolipid and neurokeratin, it might be pertinent to review highly their history. In 1941 Folch and Woolley (1942) reported the occurrence of inositol as a constituent of brain lipids. Subsequent work resulted in the isolation of an inositol-rich lipid fraction (Folch, 1949) which appeared to have as constituents, inositol diphosphate, glycerol and fatty acids in integral molar ratios and to which the name diphosphoinositide was given (DPI). Later work, using chromatographic techniques, showed, that besides diphosphoinositide, there was a triphosphoinositide (Dittmer and Dawson, 1961 ; Brockerhoff and Ballou, 1961), References p. 13-14
4
J. F O L C H - P I
and that, in fact, the latter might well be the most abundant of the two, DPI possibly being derived by partial dephosphorylation of TPI. Proteolipids - The name proteolipid was introduced in 1951 by Folch and Lees to designate substances consisting of a protein moiety and a lipid moiety and characterized by a complete insolubility in water and solubility in some organic solvents, especially in chloroform : methanol mixtures. The name is intended to emphasize that proteolipids are lipoproteins which behave like lipids. The original observation that led to the discovery of proteolipids was that chloroform : methanol extracts of brain, presumably freed of nonlipid material by water washing, contained protein material (Folch and Lees, 195I). This protein material remained in chloroform through successive water washings, i.e., it was not only soluble in chloroform but insoluble in water. The protein material could be obtained by simply taking to dryness the extract, and extracting the residue with chloroform : methanol. Apparently, in the course of drying the protein underwent some rearrangement that resulted in the loss of its original solubility in chloroform : methanol. As a consequence, the protein remained as an insoluble residue. It contained 14 % N, 1.75 % S and, after acid hydrolysis, 91 % of its nitrogen could be recovered as free amino acids. Its amino acid composition revealed a preponderance of monoamino-mono-carboxylic acids, a high concentration of methionine and cysteine (or cystine) and a relatively small concentration of acidic and of basic amino acids. The material was resistant to the action of trypsin, pepsin, papain and erepsin. Later, it was found to be hydrolyzable by pronase. Distribution of proteolipids. - Although especially abundant in nervous tissue, proteolipids are also found in a wide variety of animal and vegetable tissues. Bovine tissues contain the following amounts of proteolipid protein (mg/g tissue weight) : heart, 3.5; kidney 2.0; liver, 1.6; lung, 0.95; uterus, 0.6; biceps, 0.4. In spinach chloroplasts they represent 2-4 % of dry weight (Zill and Harmon, 1961). These values are only indicative because the yields obtained may have been incomplete. In the nervous system, proteolipids are found at highest concentration in white matter (20-25 mg/g wet tissue) and at about 1/5 this concentration in gray matter. They are present in peripheral nerve at only 1/20 to 1/80 the concentration in white matter (Folch et al., 1958), which may well indicate a qualitative difference between peripheral and central myelin. They are absent from fetal brain and their appearance and progressive accumulation is concurrent with myelination (Folch, 1955). In a study of 28 different anatomical areas of the human nervous system, Amaducci (1962) has observed marked and consistent differences from one anatomical area to another. He has shown that the highest concentration of proteolipids occurs in central white matter, with 1/5 to 1/10 as much in gray matter, and only 1/20 to 1/80 as much in peripheral nerve. Within this general pattern the concentration of proteolipids decreases progressively from cerebral white matter to spinal cord white matter, with cerebellar white matter showing an intermediate value. In spinal cord itself, the concentration of proteolipids appears to decrease in the anterolateral columns
COMPOSITION OF NERVOUS MEMBRANES
5
from rostra1 to caudal levels, while no such gradient is found in the posterior columns. The anterior and posterior spinal roots contain proteolipids at substantially lower concentrations than are found in spinal cord white matter, but this amount is still several times that present in peripheral nerves. The concentration of proteolipids found in gray matter from various areas shows no clear pattern. On the basis of all these observations it had been assumed that, in the central nervous system, proteolipids were in part myelin components. Later work on isolated myelin has established that this is the case and that proteolipids are the main protein found in myelin (Autilio, 1966). In gray matter and in non-neural tissues, especially in heart, proteolipids have been traced to mitochondria at least in part. Ptirifcation of ,i.hite matter proteolipids - Hitherto proteolipids have been obtained from tissue only by extraction with chloroform : methanol. From the extracts thus obtained proteolipids have been prepared in various states of relative purity by the “fluff” method, by emulsion-centrifugation, by dialysis, or by chromatography. The “fluff” method (Folch and Lees, 1951), the one originally used for the preparation of proteolipids, is based on the tendency of proteolipids to concentrate at interfaces. The chloroform : methanol extract is allowed to equilibrate with at least five-fold its volume of water; a biphasic system consisting of a chloroformic phase and an overlying water-methanol phase is eventually obtained. The proteolipids are in part concentrated at the interface as a fluff and, in part, in the chloroformic phase. By further handling, proteolipids A and B are obtained from the fluff, and proteolipid C from the chloroformic phase. These preparations contain from 20 to 70 % protein, and are, otherwise, purely operational concentrates of proteolipids. The emulsion-centrifugation procedure (Folch e t a ] . , 1959) is based on the difference of density between free lipids and proteolipids. A washed chloroform-methanol extract of white matter is taken to dryness in vacuum. The resulting residue is emulsified in 30-fold its weight of water, and the emulsion is centrifuged at 4600 g for 1 h. The supernatant is decanted, the residue suspended in the same amount of water as before and the new suspension centrifuged as before. The whole cycle is repeated twice more. The third and fourth supernatants are water clear or only slightly opalescent. The residue from the fourth centrifugation is again suspended in the same volume of water as before, and the new suspension centrifuged at 200 g for 10 min. The supernatant is decanted. The residue is soluble in chloroform containing small amounts of methanol and water. It is a crude proteolipid preparation that contains approximately 30-40 % protein, 40 % phosphatides, and 12-15 % each of cerebrosides (including sulfatides) and cholesterol. This crude preparation can be purified further by extracting in succession, twice with 70-fold its weight of ethyl ether, and twice with 80-fold its weight of ethanol. The final residue represents a total preparation of white matter proteolipids with the composition given in Table I. Preparatiori ofproteolipids by dialysis - Since proteolipids are high molecular weight compounds, they can be separated from free lipids by dialysis in organic solvents. Murakami et a/. (1962) used dialysis in the purification of brain heart proteolipids Rrfcrmrrs p. 13-14
TABLE I A V E R A G E C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D F R A C T I O N S P R E P A R E D BY D I F F E R E N T M E T H O D S
(Unless otherwise noted, all components are expressed at % of the respective fraction)
Procedure and Fraction
Fluff method Proteol. A Proteol. B Proteol. C Emulsion-Centrifugation Crude Concentrated Dialysis Chromatography I I1 111 Water-soluble proteolipid protein
Yield, fresh tissue
(wig)
Proreolipid protein
Phosphatides
Cerebrosides
Cholesterol
E 1 em/'% ar 280 mp
( 7);
( %)
I %I
( %)
12-20
5-15
50 20
traces traces traces
5-1 3
7 1 0.2
6 - 8 9 -11 10.5- 13.5
20 10 10
3540 55-65
25-30
a 5 0 25-30 20
35-45 55-65 70
40 25 20-25
4
95 95 95
2- 5 2 2
-
-
13-14 13-14 13-14
99-100
1
-
1622
4 7
30
3- I
2- 5
COMPOSITION O F NERVOUS MEMBRANES
3.0
I II 'I
2.5
2
g
01
I
2.0
+ U t
7 5 2 5 4 5 J: 7 0 3 0 6
80 20 3
8515l\! I
t
'
I I I
60409
/I
7
1.5
v,
z
LL]
n
a
1.0
0 k-
CL
0.5 0
5
10
15
20
25
30
TUBE NUMBER (each 5 m l )
Fig. 1 . Chromatography of proteolipids obtained by the emulsion-centrifugation method on a silicic acid column. A 10 mm inner diameter column packed with 4 g silicic acid was used. It was loaded 1 2 8 0 mp = 8.1. The ratios on the upper line express the prowith 69 mg proteolipids Elcm portions of chloroform, methanol and water of the eluting mixture. - Optical density at 280 mp; _ _ _ _ amount of P.
and Thompson et al. (1963) have applied it to the purification of myelin proteins. In the case of brain white matter, the proteolipid and free lipid mixture obtained from a washed lipid extract, or partially purified proteolipid preparations are dissolved in chloroform-methanol 2 : I and the solution placed in a cellophane dialysis tubing previously washed with water and with chloroform-methanol, and dialyzed against the solvent mixture. The system is shaken gently, the diffusate is changed daily and the dialysis allowed to proceed until the diffusate is free of solutes. Usually 7 days suffice. The composition of such preparations is given in Table I.
Chromatography of proteolipids - Matsumoto et al. ( 1 964) have chromatographed the concentrated proteolipid preparations prepared by the emulsion centrifugation procedure, on silicic acid columns. The details of such a chromatographic run is given in Fig. 1. It shows that the first two peaks obtained are free lipids, with little or no protein, and that they are followed by three peaks consisting mainly of protein. It is noteworthy that the last protein peak can only be eluted by chloroform-methanol 1 : 1 containing HCI. This last fraction shows solubility properties different from those of the starting preparation in the sense that in the biphasic system chloroform-methanolwater 8 : 4 : 3 (v/v/v), the original proteolipid is found quantitatively in the chloroformic phase, whereas the proteolipid recovered from the last chromatographic Rrferencrs p. 13-14
8
J. F O L C H - P I
fraction has a definite partition between the two phases, the methanolic-water phase containing about 1/5 as much proteolipid as the chloroformic phase. Properties and composition of proteolipids - All proteolipid preparations described above are soluble in chloroform or in mixtures ofchloroform with methanol and water. They are completely insoluble in water and in aqueous solutions and in the biphasic system chloroform-methanol-water 8 : 4 : 3 (v/v/v), they will concentrate quantitatively in the chloroform phase. All the proteolipid preparations have been found to be resistant to the action of pepsin, trypsin, papain and erepsin. This resistance is not due to the presence of lipids, because it is found in the water-soluble proteolipid protein ( v i ) which is free of lipids, and in the insoluble denatured proteolipid protein described below. The only enzyme that attacks proteolipids is pronase, although the extent of this susceptibility has not been determined exactly. The chemical composition of the various proteolipids is given in Table I. Composition given for proteolipids A, B and C is merely indicative because both yield and composition vary widely according to the exact conditions followed in preparation. The other methods of preparation yield more consistent products. An important fact illustrated by this table is that the amount of lipids in proteolipids may vary from 60 % (in crude emulsion-centrifugation proteolipid) to less than 5 % in the three chromatographic fractions without any change in general solubility properties. Proteolipid protein - It has been isolated as an insoluble material by drying from solutions in biphasic systems (Brockerhoff and Ballou, 1961) or by exposure toalkaline pH’s at certain ionic strengths (Webster and Folch, 1961). At pH 8 or 9, proteolipids can split, with liberation of free lipids and, of protein, as an insoluble material, provided the medium contains ions at sufficient concentration. At pH 8.8 between ionic strengths 0.001 and 1 .O the proportion of proteolipid split is proportional to the logarithm of the ionic concentrations; this fact suggests that the mechanism of splitting is by ionic competition. These insoluble proteolipid proteins still contain small amounts of lipids. The lipid content can be reduced by extraction with hot chloroform : methanol; the lipid most firmly bound to the protein appears to be a polyphosphoinositide mainly triphosphoinositide. It can be removed only with chloroform : methanol acidified with HC1 to 0.04 N concentration (Pritchard and Folch-Pi, 1963). The amino acid composition of the different proteolipids has been estimated repeatedly by different methods and in different laboratories with wholly concordant results. These are that the amino acid patterns of the different white matter proteolipid preparations are identical or so similar as to be indistinguishable from each other. Table I1 gives the amino acid composition of preparations obtained by emulsion centrifugation of the chromatographic parallel fractions obtained from them, and the water soluble proteolipid (v.i.). For more meaningful comparison, serine, the concentration of which varies with the amount of phosphatidyl serine present, has been oomputed uniformly at 6 % of total amino acids on a molar basis; methionine and
P
T A B L E I1 A M I N O A C I D C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D P R E P A R E D B Y DIFFERElvT M E T H O D S
(Results expressed as
Amino Acids
Leucine Isoleucine Valine Glycine Threonine Serine Proline Aspartic Acid Glutamic Acid Histidine Arginine Lysine Tyrosine Phenyl Alanine Alanine Methionine Half cystine
Washed total Lipid extract
11.2 7.6 9.5 8.3 11.5 (1KO)* 2.2 2.9 2.4 2.7 2.2 7.4 3.0 8.0 9.0 (6.0)**
Crude Proteolipid
o’,
of total a-amino acid N recovered from acid hydrolysates) Concentrated Proteolipid
I
Chrontatographic Fractions II
III
Water Soluble Proteolipid Protein
11.1 5.9 7.3 9.7 8.4 (7.3)* 2.7 3.7 4.5 2.1 1.7 6.9 4.8 8.3 11.0
11.4 4.9 6.7 10.7 8.3 (7.4)* 3.1 4.0 5.8 1.9 2.0 3.8 4.9 8.3 12.0
11.8 5.3 6.7 9.6 9.0 (5.5)* 3.0 4.3 4.3 1.7 2.7 3.9 4.4 8.6 13.3
11.1 4.9 6.5 10.5 9.0 (5.2)* 2.3 3.9 3.8 1.8 3.1 4.3 4.9 8.2 13.5
11.4 4.8 6.4 10.8 8.8 (5.0)* 2.4 3.9 4.2 2.0 3.2 4.4 4.9 8.2 13.2
11.1 4.9 6.9 10.3 8.5 (8.5)* 2.8 4.2 6.0 1.8 2.6 4.3 4.6 7.9 12.5
(6.0)**
(6.0)**
(6.0)**
(6.0)**
(6.0)* *
(6.0)**
* Serine computed uniformly at 6 % of total Amino Acids. ** Methionine and half cystine computed at 6 % of total Amino Acids.
c)
0
=! 0
2:
0
-I
Z
m
w < 0
10
J. F O L C H - P I
half-cystine have also been computed jointly at 6 %, a concentration consistently found when these amino acids are estimated independently. Tryptophan, which is destroyed by acid hydrolysis, is not included. From optical density values at 280 mp, it can be computed to amount to 3 % or higher. It can be seen that the amino acid composition is indistinguishable from preparation to preparation and very similar to the composition of total chloroform-methanol soluble protein. The same holds true for proteolipid A, B and C, and proteolipids separated by dialysis. However, in the latter case, 10 to 15 % of the protein is found in the diffusate. This dialyzable fraction is protein and not a simple mixture of amino acids. The amino acid composition of the dialyzable material is different from that of the undialyzable proteolipid in that the former contains relatively more glycine, alanine and perhaps threonine. This may represent a real difference or a differential loss of amino acids in the course of hydrolysis because of the presence of a large concentration of lipid in the diffusate. The amino acid pattern of proteolipids has the following features: (a) A relative scarcity of acidic and of basic amino acids; aspartic and glutamic amount jointly to less than 10 % and arginine, lysine and histidine amount jointly also to less than 10 %; (b) A wealth of methionine and half-cystine, as is to be expected from the high concentration of sulfur in proteolipid protein (1.75 %); (c) A relative abundance of the so-called non-polar amino acids, i.e., amino acids that when combined in a peptide chain, offer only non-polar groups to the medium; leucine, isoleucine, valine, glycine, proline, phenylalanine and alanine amount to 57-58 % of total amino acids. If tryptophan is added, over 60 % of amino acids are non-polar ; (d) The relatively high concentration of tryptophan as indicated by the high optical density at 280 mp. On the basis of the least abundant residue, proteolipid protein is computed to comprise 125 amino acid residues of an average size of 100, which gives a minimal molecular weight of 12,500 for the protein moiety of proteolipids (Folch-Pi, 1959). Water-solubleproteolipidprotein - If proteolipids are dialyzed in chloroform : methanol containing HCI to 0.04 N concentration, and then the composition of the outer phase is slowly changed to pure water by gradually decreasing the organic solvent content of the successive outer phases, the retentate is found to consist of protein essentially free of lipids (Tenenbaum and Folch, in press). This preparation has an amino acid pattern indistinguishable from that of the starting proteolipids. It is soluble in acidified aqueous solutions and in chloroform. Apparently, it is the result of a conformational change of the original proteolipid protein (Zand, 1966). The dramatic change in solubility properties is concomitant with the removal of triphosphoinositide. Organization of the proteolipid molecule - Although no complete model of the proteolipid molecule can yet be formulated, it is clear that the lipids in proteolipids exist in different types of binding. Triphosphoinositide is almost certainly bound by an
COMPOSITION O F NERVOUS MEMBRANES
11
electrostatic bond. Other lipids, mainly phosphatidylserine, are bound by ionic linkages which can be dissociated by ionic competition. Finally, other lipids must be bound by more labile types of association. Of these three types of bonds, the first two most likely occur in vivo, while the third type most likely represents in vitro associations. The peculiar solubility properties of proteolipids, which remain unchanged even when the lipid content is reduced to 5 % or less, must be explained in terms of the protein moiety. Since the proteolipid molecule must present a non-polar surface, a tertiary structure must be postulated which would bring to the surface the non-polar groups of the amino acids, while retaining their polar groups in the core of the molecule. A possible structure would involve the stabilization of a particular conformation by triphosphoinositide which, being a polyanion, could combine with the cationic charges of the protein, thus orienting them towards the core of the helical structure and leaving an outer surface occupied mainly by non-polar groups. The release of triphosphoinositide concomitantly with the transformation of proteolipid protein to a water-soluble form would be in favor of this explanation. Neurokeratin - The name of neurokeratin was given by Ewald and Kuhne (1874-77) almost a century ago, to the gastric juice-resistant, pancreatic juice-resistant, fraction of brain proteins. The material was obtained by defatting brain tissue by exhaustive extraction with ethanol and ethyl ether, and submitting the defatted residue to the action of gastric juice and of pancreatic juice, in succession. The final product was an insoluble protein material, rich in S (1.7 %) and free of P. On the basis of its distribution in the nervous system, of the increase in its concentration in temporal relationship to myelination, and of some histochemical evidence, it was concluded that neurokeratin was a myelin constituent, and the name was adopted by histologists to designate the protein framework of the myelin sheath. LeBaron and Folch (1956) were able to prepare neurokeratin by a procedure milder than that used by earlier workers. The trypsin and pepsin resistant material obtained from white matter and designated Trypsin resistant protein residue (TRPR) was resistant to the action of proteolytic enzyme, was characterized by general insolubility, and contained 1.7 % S. In brief, it was very similar to classic neurokeratin except for the important difference that it contained about I .7 % P, almost all of which corresponded to polyphosphoinositide, presumably combined in it by an electrostatic linkage. They also showed that the classical procedure for preparation of neurokeratin resulted in the complete destruction of the constituent polyphosphoinositide, thus yielding a P-free product. The amount of polyphosphoinositide (PPI) present in TRPR accounts for the bulk of the PPI of brain tissue. In the original description of proteolipids (Klenk, 1941), it became obvious that there were many similarities between neurokeratin and proteolipid protein: general insolubility, high sulfur content, relationship to the myelin sheath, similar amino acid composition. The suggestion was made that neurokeratin might, in fact, be a product of breakdown of proteolipids. This suggestion was given further credence by References p.
13-14
12
J. F O L C H - P I
the finding that neurokeratin in its “native” state contained polyphosphoinositide in electrostatic combination just as is the case with proteolipid. This suggestion has been both reinforced and complicated by the recent work on isolated myelin. As already mentioned, it has been found that isolated myelin is completely or almost completely soluble in chloroform : methanol. Operationally this means that neurokeratin and TRPR, which are prepared from the chloroform : methanol insoluble fraction of white matter cannot be prepared from isolated myelin, since it yields no chloroform : methanol insoluble fraction. This forces the conclusion that if, indeed, neurokeratin is a myelin constituent, it exists in it in a form that is soluble in chloroform : methanol after isolation of myelin. This would place neurokeratin in the same category as proteolipids. On the other hand, these observations raise the question of the mechanism by which neurokeratin would become insoluble in chloroform : methanol, and why the same thing would not apply to the proteolipids. There is as yet no answer to these questions. Polyphosphoinositides (PPI) - Many facts pertaining to the discussion of PPI have already been mentioned and the following will only complement them and attempt a brief synthesis of our present knowledge on these interesting compounds. PPI are found mainly in combination in TRPR, which accounts for 80 to 90 per cent of white matter PPT, the balance being found mainly in proteolipids. They are clearly myelin TABLE 111 C O M P A R I S O N O F LEVELS O F P O L Y P H O S P H O I N O S I T I D E S I N D E V E L O P I N G R A T B R A I N A N D O F T R Y P S I N RESISTANT PROTEIN RESIDUE
Rat brain
Age
TPI 2 days 4 days 7 days 10 days 16 days 17 days 19 days 34 days 35 days 40 days Adult
(TRPR) IN
DPI
l(g P/g brain 9.2 12.2
Mouse brain TRPR Total 8.5
-
2.7 3.3
-
11.9 15.5
19.4 44.4
5.7 8.7
25.1 53.1
-
-
11.7
54.5
-
42.8
-
D E V E L O P I N G MOUSE B R A I N
-
TRPR
fresh wt. 0.063 0.115 0.19 0.38 0.40 0.67 0.67 -
TRPR-phosphorus p g P/g brain 5.0 9.4 15.2 30.4
-
32.0 53.6 53.6 -
Results for rat courteously supplied by Doctors J. Eichberg and G. Hauser. Results from mouse obtained or computed from Folch, 1955. Rats were decapitated, the head dropped in liquid nitrogen, and the brain removed without thawing. Mice were anesthesized with ether, the brain removed surgically and placed in a weighing bottle in dry ice. The time elapsed between removal of brain from the living body and its freezing was the time required for the actual freezing of the tissue once placed in contact with the chilled glass wall. The good fitting of values for PPI-P for rat with the values for mice should be regarded as fortuitous. The important analogy is the slope of the increase, which is essentially the same in both species.
COMPOSITION O F N E R V O U S MEMBRANES
13
constituents; they are found in myelin at much larger concentration than in other subcellular fractions of white matter (Eichberg and Dawson, 1965); they are found only in very small amounts, if at all, in non-neural tissues. They appear at the time of myelination and they increase in concentration with the gradual accumulation of myelin. Table 111 gives results on this point, courteously supplied by Doctors J. Eichberg and G . Hauser. They show that from 7 days, before myelination, to 34 days, the concentration of PPI-P increased 5-fold ; for comparison, the concentration of TRPR in the mouse at similar ages is given, both as amount of TRPR and as P (Folch, 1955). There is a remarkable analogy between the total amount of PPI-P and of TRPR-P, a fact that, although not unexpected, bears out strongly the myelinic nature of PPI. Numerous observations attest that PPI exhibits a high rate of P turnover, a fact in sharp contrast with the generally low level of metabolic activity of other myelin components. As yet, no evidence has been forthcoming relating this high metabolic activity of PPI-P to neural function. The marked neural character of PPI, their high metabolic activity, suggests that they must play some crucial role in nerve tissue. What this role is can only be established by further work. ACKNOWLEDGEMENT
The original work described in this discussion was supported by Grants NB-00130 and NB-02840 of the National Institute of Neurology and Blindness, National Institutes of Health. REFERENCES AMADUCCI, L. (1962) The distribution of proteolipids in the human nervous system. J. Neurochem., 9, 153-160. AUTILIO, L. (1966) Fractionation of myelin proteins. Fed. Proc., 25, 764. BLIX,G . (1936) The carbohydrate groups of the submaxillary rnucin. Z. Physiol. Chem., 240.43-54. BROCKERHOFF, H . A N D BALLOU,C. E. (1961) The structure of the phosphoinositide complex of beef brain. 1.Biol. Cherri., 236, 1907-1911. BURTON,R. M., HOWARD,R . E., BAER,S. AND BALFOUR, Y. M. (1964) Gangliosides and acetylcholine of the central nervous system. Biochirn. Biophys. Acta, 84, 441441. DIEZEL, P. M. (1955) Bestimrnung der Neuraminsaure im histologischen Schnittpraparat. Narurwiss., 42, 487-488. DITTMER, J. AND DAWSON, R. M . C. (1961) The isolation of a new lipid triphosphoinositide, and rnonophosphoinositide from ox brain. Biochem. J., 81, 535-540. EICHBERG, J. AND DAWSON, R. M. c. (1965) Polyphosphoinositides in myelin. Biochent. J., 96, 644650. EICHBERG, J., WHITTAKER, V. P. A N D DAWSON, R. M.C . (1964) Distribution of lipids in subcellular particles of guinea-pig brain. Biocheni. J., 92, 91-100. EWALD,A, AND KUHNE,W. (1874-1877) Verharrdl. Naturhist.-Meif.,1, 457. FOLCH,J., (1949); Brain diphosphoinositide, a new phosphatide having inositol rnetadiphosphate as a constituent. J. Biol. Cheni., 177, 505-519. FOLCH,J., ARSOVE, S. AND MEATH, J. A. (1951) Isolation of brain strandin, a new type of large molecule tissue component. J . Biol. Chert?.,191, 819-831. FOLCH,5. AND LEES,M. (1959) Studies on the brain ganglioside strandin in normal brain and in Tay-Sachs’ disease. Arner. J. Dis. Child., 97, 730-738. FOLCH,J. (1955) Composition of the brain in relation to maturation. Biochernisrry of rhe Developing Nervous System, H. Waelsch, Editor, Academic Press, New York, p. 121.
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A N D WOOLLEY, D. W. (1942) Inositol, a constituent of a brain phosphatide. J . Biol. Chem., 142,963-964. FOLCH,J. AND LEES,M. (1951) Proteolipids, a new type of tissue lipoproteins - their isolation from brain. J. Biol. Chem., 191,807-817.
FOLCH,J.
FOLCH,J., LEES,M. AND CARR,S. (1958) Studies of the chemical composition of the nervous system. Exp. Cell Res., Suppl., 5, 58-71. FOLCH,J., WEBSTER, G . R. AND LEES, M. (1959) The preparation of proteolipids. Fed. Proc., 18, 228. FoLcH-Pi, J., (1959); etudes Rkentes sur la chimie du cerveau et leur rapport avec la structure de la gaine myklinique. Exp. Ann. Biochim. Med., 21,81-95. HOWARD, R. E. AND BURTON,R. M. (1964) Studies on the ganglioside micelle. Biochini. Biophys. Acia, 84,435-440. KLENK, E. (1941) NeuraminsBure, das Spaltprodukt eines neuen Gehirnlipoids. Z. Physiol. Chem., 268,50-58. KLENK,E. (1942) Uber die Ganglioside, eine neue Gruppe von Zuckerhaltigcn Gehirnlipoiden. Z. Physiol. Chem., 273, 76-86. KUHN,R.AND WiEcAND-r, H. (1963) Constitution of ganglio-N-tetraose and the ganglioside GI. Chem. Ber., 96,866-880. LEBARON,F. N. AND FoLcii-PI, J. (1956) The isolation from brain tissue of a trypsin-resistant protein fraction containing combined insolitol, and its relation to neurokeratin. J . Neurocliem., 1, 101-108. LEDEEN, R. (1966) The chemistry of gangliosides: A review. J . Amer. O i l Chemists' SOC.,43,57-66. LOWDEN, J. A. AND WOLFE,L. S. (1964) Studies on Brain Gangliosides 111. Evidence for the location of gangliosides specifically in neurones. Canad. J . Biocheni., 42, 1587-1594. MATSUMOTO, M., MATSUMOTO, R. AND FOLCH-PI,J. (1964) The chromatographic fractionation of brain white matter proteolipids. J . Neurochem., 11, 829-838. MCILWAIN, H. (1962) New factors connecting metabolic and electrical events in cerebral tissue. In Ulirasirticture and Metabolism of the Nervous System, Research Publication Association Research Nervous Mental Disease, XL. Williams and Wilkins Co., Baltimore (page 43). MURAKAMI, M., SEKINE, H. AND F U N A H A S H I , s. (1962) Proteolipid from beef heart muSCk-'. Application of organic dialysis to preparation of proteolipid. J. Biochem., 51, 431435. PRITCHARD, E. G . AND FOLCH-PI, J. (1963) Tightly bound proteolipid phospholipid in bovine brain white matter. Biochim. Biopliys. Acia, 70,481483. QUARLES, R. A N D FoLcH-PI, J. (1965) Some effects of physiological cations on the behaviour of gangliosides in a chloroform-methanol-water biphasic system. J . Neurochem., 12,543-553. ROSENBERG, A. AND CHARGAFF, E. (1956) Nitrogenous constituents of an ox brain mucolipid. Biochim. Biophys. Acia. 21, 588-589. SEMiNARio, L. M., HREN, N. A N D GOMEZ,G. J. (1964) Lipid distribution in subcellular fractions of the rat brain. J. Neurochem., 11, 197-209. SPENCE, M. W. AND WOLFE,L. S. (1964) The isolation of a ganglioside-rich membrane fraction from new-born rat brain. Sixth Intern. Congr. Biochem., New York, Abstr., V-,5118, 418. SVENNERHOLM, L. (1964) The gangliosides. J. Lipid Res., 5, 145-155. TENENBAUM, D., AND FOLCH,J., (1966); The prepraration and characterization of water-soluble proteolipid protein from bovine brain white matter. Biochim. Biophys. Acta, 115,141-147. THOMPSON, E.B., KIES, M. W. AND ALVORD,JR. (1963) Isolation of a n encephalitogenic phospholipid-protein complex by dialysis of myelin in organic solvents. Biochem. Biophys. Res. Comm., 13, 198-204. VAN HEYNINGEN, W. E. (1963) The fixation of tetanus toxin, strychnine, serotonin and other substance by gangliosidc. J. Cen. Microbiol., 31,375-387. WEBSTER, G. R. AND FOLCH,J. (1961) Some studies on the properties of proteolipids. Biocliini. Biophys. Acia, 49, 399-401. WHERREIT, J. R. AND M C ~ L W A IH. N , (1962) Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material. Biochem. J., 84,232-337. WOLFE,L. S . (1961) The distribution of gangliosides in subcellular fractions of guinea-pig cerebral cortex. Biocheni. J., 79, 348-355. WOOLLEY, D. W. A N D GOMMI, B. W. (1964) Serotonin Receptors: V, Selective destruction by neuraminidase plus EDTA and reactivation with tissue lipids. Narure, 202, 1074-1075. ZAND,R. (1966) Physical chemical studies on the solution properties of bovine brain white matter proteolipids. Fed. Proc., 25, 736. Z u , L. P. AND HARMON,E. A. (1961) Chloroplast proteolipid. Biochim. Biophys. Acta., 53, 579-58 I.
COMPOSITION O F NERVOUS MEMBRANES
Monday afternoon
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DISCUSSION
KATZMAN: I t has been many years since Dr. Folch-Pi characterized and identified the inositol phosphotides and proteolipids and identified the polymer form of inositol phosphate, and it is very exciting to me to see that all these materials are still in the forefront of research. It is most interesting, as Dr. Folch-Pi reported, that the triphosphoinositides, which are so actively turning over, are major constituents of the proteolipids. I would like to ask Dr. Folch-Pi specifically whether it can be demonstrated that in the isolated purified myelin the triphosphoinositides turn over so rapidly. In other membranes where there is a rapid turnover of phosphoinositides, it seems that only a small fraction of phosphoinositide is turning over, and the bulk is not so active. FOLCH-PI:1 don’t think that the triphosphoinositides are especially prominent. The monophosphoinositides, as I am sure you are aware, have a different function from the other inositides and they have a different distribution. Perhaps they are connected with synaptic membranes. In relation to the polyphosphoinositides in myelin, I am not aware that the precise kinetics of a single component’s turnover has been measured. KATZMAN: Has it been shown that the phosphate that is turning over in the purified myelin is specifically the phosphate of the triphosphoinositides? FOLCH-PI: Dawson and others have actually shown the incorporation of P-32 and they have isolated various cell fractions.
MANDEL:There is a turnover of triphosphoinositol in the myelin sheath, but this is quite low compared to the turnover of phosphoinositides in other parts. What is peculiar is that the highest turnover of phosphoinositides is of cardiolipin in the myelin sheith. FOLCH-PI:There is some indication of turnover of cardiolipins, which are the polyanions in mitochondria, but the bulk of the polyphosphoinositides is definitely in the myelin. In isolated proteolipids, LeBaron and Hauser showed years ago that phosphoinositides had a very high turnover of phosphate there. LAJTHA: Can make you any statement on the composition of the proteolipid fractions of the various particulate fractions, and on differences between gray matter and white matter. What I am really driving at is whether you can make any statement about the differences in the composition of the various membranes, whether membranes of glia versus neuronal membranes or particulate membranes. CSAKY:I note that you have isolated the protein from the proteolipids. Could you describe the properties of this fraction, particularly whether you think that this is a typical structural protein? Does it have a high molecular weight? Does it consist of long, thread-like particles? Does it have very high viscosity? Does it respond in its physicochemical properties very readily to ions, and things like that? FOLCH-PI:Some of those measurements have actually been made, although not all of them. However, they are more indicative than exact. Taking the question of molecular size, for example, by the technique of the least abundant component, we get a molecular size of the order of about 12,00015,000. By other physicochemical measurements, for example, sedimentation, diffusion or light scattering, we get somewhat wider scatter with a minimum value of about 20,000 or 30,000. All of these measurements are, of course, done in organic media, since these compounds are insoluble in water. Therefore I would consider every value that we obtained rather comparative only. By Sephadex L-20, molecular size is more between 30,000 and 100,000. Most likely a low polymer formation occurs. We didn’t measure viscosity, again, for the reason that we would have to do that in chloroform and therefore it would have doubtful meaning. But there are people who have done that, for example, Dr. Zahn in Ann Arbor, and Dr. Onkley. We have some information about the tertiary structure, using nuclear magnetic measurements, and in chloroform we get a highly helical structure of about 85 per cent. When the proteins are passed into the water phase, an unfolding of the helixes occurs and they become random coiled.
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CSAKY:But would you be willing to say that it could be considered a s a structural protein of the myelin sheath? FOLCH-PI:I think there is very little doubt about that, although I would not make a statement on all membranes. In heart the bulk of the proteolipids are found in mitochondria, and I would think that they must be the components of the structural membranes. The amino acid turnover is very low in these components so that they show a chemical stability, and although not identical, the amino acid composition is fairly similar to Green’s so-called protein isolated from mitochondria. LAJTHA: You don’t think that the lack of turnover is only apparent and is d u e to lack of permeability, that is, the precursor amino acid that you add from the outside doesn’t penetrate to the inside of the myelin sheath, and therefore it is not incorporated. FOLCH-PI:Now we are talking about the mechanism of the stability or how it is actually obtained. It is possible that it is only an apparent one, as you say, but this is very real as far as the living body is concerned. TOWER:Perhaps we are making a mistake when we are talking about fhe structural protein. Wc certainly are dealing with tissues, and the brain in particular, that have many, many membranes with different functions. Our laboratory has some preliminary results which indicate that in membranerich fractions, subfractionated from cerebral microsomal fractions, there is a protein fraction (not necessarily a single protein, although it comes out on a column in a single peak) which exhibits an amino acid composition of about 30 per cent glutamyl plus aspartyl residues. This, theoretically, at least, provides a set of very high negative charges on these molecules (not provided by phosphates in this case but by carboxyl groups), and since this represents a major portion of the proteins of the endoplasmic reticulum, which, in turn, many of us consider may have an important role in transport, it poses some very interesting possibilities. COXON:Dr. Folch-Pi referred to some interactions between ions and the lipids that he is studying. Can he make any statements about selective affinities as between calcium and magnesium and potassium and sodium? FOLCH-PI:There is certainly a difference between the divalent ions and the monovalent ions. The divalent ions have about 500 times higher affinity; something of that order. This is, of course, just a comparative figure. This may be specific for gangliosides, and the carboxyl group of the sialic acid may have an important role. Calcium there certainly displaces sodium, etc. A very interesting point here is that the calcium salt of gangliosides is mainly non-polar, while the magnesium salt is very polar and the polarity changes very much according to the calcium concentration of the medium with which the ganglioside is in contact, and where such shifts happen is around the physiological levels of thc ions. If calcium is somehow sequestered and the ganglioside is facing a relatively low calcium concentration or there would be high magnesium there, there would be a part of the membrane which would be rather lipophobic, but as soon as more calcium came, and I don’t want to speculate how this would occur, that part of the membrane would become much more lipophilic. Therefore, one could postulate this as part of the mechanism of the actual movement of macromolecules in the membrane. This is attractive because it seems to be reversible and doesn’t particularly require energy, and the concentrations required are within the physiological range. The size, from molecular weight measurements, is about 70 Angstroms of these compounds, which well fits within the usual structural arrangement as we picture the membranes. DOBEING: I wonder if you could make any statement about the turnover of these particles that we are discussing, especially since they are often buried beneath several layers, of, for example, proteolipids which, in themselves, do not turn over very rapidly? Is it possible to account for all the turnover as being the turnover of only the exterior part of the sheaths? FOLCH-PI:Now we are in the field of pure speculation. Amaducci did a very careful study several years ago, trying to correlate proteolipids with myelin structures. He not only confirmed what was already known that peripheral nerve contains very little proteolipids, but from a number of structures, such as the optic nerve and the corpus callosum, the brachial plexus and the sciatic nerve, he could
COMPOSITION O F NERVOUS MEMBRANES
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correlate very well with the sum of the circumferences of the fibers. If you take into account that myelin is usually about half the thickness of the fiber that it surrounds, the myelin is usually related to the surface of the fiber. The smaller the fiber, the greater the sum total of the circumference is, of course, per unit area of the brain. And then he had a very good correlation all the way from the corpus callosurn, which has about 27 parts of proteolipids per loo0 of wet weight basis, down to the brachial plexus which has less then 1 per cent. That could then mean that in the myelin spiral proteolipids would not be distributed uniformly but the distribution somehow would be correlated with the circumference. This could be done by two ways, by having either more or less in the outer turn or the inner turn of the spiral. If you now then equate proteolipids and triphosphoinositides, then you can say that the triphosphoinositides are not equally distributed, and if you postulate that they are richer in the outer turn of the spiral, then you would have the triphosphoinositides much more available than other forms. DOBEING: I f one follows the rate of development of myelin by following the accumulation of the various lipid components, their deposition occurs at different times. On Dr. Folch's hypothesis, it should follow that there would be a different timing for the deposition of the phosphoinositides and o f other components, such as, for example, cholesterol. Could this not be studied in small well-defined areas? FOLCH-PI:I don't think all data were really good enough to put weight on such correlations. In general fashion we have tried such measurements but the data are not really good enough to make too many statements definite. I t did follow the general increase in niyelination and so it showed general correlation. Dawson has done quite a number of such measurements but didn't go into very great detail.