Synthesis of proteolipid by subcellular fractions from rat brain

Brain Research, 49 (1973) 135-150

135

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

SYNTHESIS OF P R O T E O L I P I D BY S U B C E L L U L A R FRACTIONS F R O M RAT BRAIN

JOSEPH A. BABITCH* Department of Biological Chemistry and Brain Research Institute, University of California, Los Angeles, Calif. 90024 (U.S.A.)

(Accepted July 28th, 1972)

SUMMARY Mitochondria, microsomes, synaptosomes and myelin were isolated from the white matter plus brain stem of 14-16-day rat brains and incubated in amino acid incorporating systems. Most incorporation into proteolipid was by mitochondria; microsomes and myelin incorporated least. The amino acid composition of the mitochondrial proteolipid showed similarities to that reported for Folch-Lees proteolipid isolated from myelin. These results support the notion that white matter mitochondria may contribute material for myelinogenesis.

INTRODUCTION Though much insight has recently been achieved in understanding anatomical and chemical changes accompanying myelination, the mechanisms of myelinogenesis are still unknown. Following the pioneering isolation of a myelin protein by Folch and Lees l0 study of the synthesis of rat brain myelin protein was begun by Furst et al. 1~. They found that total proteolipid protein had a half-life of 40 days and was therefore metabolized more slowly than other brain proteins. Similar results were obtained by Clouet and Richter 7, Davison s, Gaitonde 13, Greaney 16, and Davison and Gregson 9. Gaitonde 14 reported that proteolipid protein was synthesized as early as the first day after birth. Soon after, Mokrasch and Manner ~1 confirmed these findings using rat brain slices. Klee and Sokoloff17 stated that ribonuclease did not inhibit proteolipid synthesis. Similar results were obtained by Mokrasch 19 but the purity of these subcellular fractions was not demonstrated and no amino acid analyses were mentioned * Present address: Neurobiology Institute, University of G~Steborg, Fack S-400 30, 33 G~Steborg, Sweden.

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J.A. BABITCH-

for the synthesized material, so it could not be identified as any specific proteolipid. The present studies were undertaken to examine in detail the contributions made by various subcellular fractions to myelinogenesis. This report describes the synthesis of proteolipids by purified subcellular preparations. Amino acid analyses suggest that the major site of synthesis of Folch-Lees myelin proteolipid may be the mitochondrion. METHODS

Isolation o f rat white matter plus brain stem. Fourteen- to 16-day-old male and female rats of an inbred Sprague-Dawley strain were decapitated. One hundred rats were used for each experiment. Brains were immediately isolated and placed on moistened filter paper on ice and adhering blood vessels were removed. White matter and brain stem were isolated by removal of the cerebellum, olfactory lobes, corpora quadrigemina, cortical hemispheres, thalamus, hypothalamus, preoptic region, and the basal forebrain. All subsequent procedures were performed at 0-4 °C unless otherwise stated. Isolation ofmitochondria and synaptosomes. The method was adapted from that of Whittaker et al. 3~ with minor modifications. White matter plus brain stem was homogenized on 0.32 M sucrose by 12 up-and-down strokes of a hand held Thomas homogenizer. Enough sucrose solution was added to make the homogenate 1 0 ~ (w/v). The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 1000 x g in a Sorvall SS-34 rotor. The nuclear pellet was discarded and the supernatant was centrifuged for 20 min at 10,000 ~ g. The microsomal supernatant was discarded. The pellet, containing myelin, mitochondria and synaptosomes, was washed twice by rehomogenizing in two-thirds the original volume of 0.32 M sucrose (to a concentration of 15 ~ (w/v) of the original tissue). Ten ml of this solution were layered on a discontinuous sucrose density gradient composed of l0 ml, 1.2 M sucrose: 5 ml, 1.1 M sucrose; and 5 ml, 0.8 M sucrose. This crude mitochondrial preparation was centrifuged in a SW 25.1 rotor in a Beckman model L or model L-2 centrifuge for 1 h at 54,000 × g. During centrifugation impure myelin banded at the 0.32 M-0.8 M sucrose interface and the synaptosomal fraction was dispersed between the 1.2 M and 0.8 M sucrose layers. The mitochondrial fraction pelletted to the bottom of the tube. The synaptosomal fraction was removed with a Pasteur pipette and the impure myelin band was discarded. The volume of the synaptosomal fraction was measured and, assuming that it was 1.1 M sucrose, enough ice-cold distilled water was added to dilute the suspension to 0.32 M sucrose. Then additional 0.32 M sucrose was added to bring it to 2 0 ~ (w/v) of the original tissue. This was centrifuged in the Sorvall SS-34 rotor for 30 min at 20,000 x g. The synaptosomal fraction pelleted after centrifugation. The yields of mitochondria and synaptosomes were generally 2.0-3.0 and 1.5-3.0 mg protein/g wet white matter plus brain stem, respectively. Isolation o f crude and washed myelin. This procedure is essentially that of Autilio et al. 3 except that in the present studies the SW 25.1 rotor was used instead of the No. 21 rotor.

SYNTHESIS OF BRAIN PROTEOLIPID

137

Isolation of microsomes and the p H 5 enzyme fraction. The procedure of Zomzely et al. 34 was followed. The yield of microsomes was 1.0-2.5 mg microsomal protein/g fresh white matter plus brain stem. Electron microscopy of subcellular fractions. Electron micrographs of the mitochondrial, synaptosomal and myelin fractions were prepared by fixing the centrifugal pellets of subcellular preparations at 4 °C by aldehyde fixation (4 ~ paraformaldehyde, 0.5 ~ glutaraldehyde, 0.1 M sodium cacodylate, 0.01 ~ calcium chloride, pH 7.4). The pellets were trimmed so that the final sections would include all pellet layers. Slices were then post-fixed for 30 min with osmium tetroxide (1 9/0 OsO4 and 0.1 M sodium cacodylate, pH 7.4) and dehydrated in a graded alcohol series. Pellet slices were embedded in fresh Araldite, sectioned thinly (silver to pale gold) on a Porter Blum microtome, Model MT-1, collected on uncoated copper grids, stained with lead citrate, and examined in a Hitachi 11 A electron microscope. Electron microscopy was generously performed by Dr. Penelope Coates and Mrs. Olga Fiorello in the laboratory of" Dr. David Maxwell. Electron micrographs of the microsomal fraction were prepared by fixing the centrifugal pellets in 2 ~ glutaraldehyde, 0.1 M phosphate buffer, and post-osmicated for 2 h. Dehydration in a graded alcohol series was followed by embedding in Epon. Thin sections were collected on copper grids coated with Formvar, stained with uranyl acetate and lead citrate and examined in the AEI 801 electron microscope. Electron microscopy was kindly performed by Mr. W. Edwards in the Department of Biochemistry, University of Cambridge, Cambridge, England. Measurement of protein. Protein was determined by the method of Lowry et al. 18 using bovine serum albumin (Pentex, Kankakee, II1., crystallized, Lot 16) as standard. Incubation of synaptosomes. Synaptosomes were incubated by the method of Autilio et al. ~ using 0.5 #Ci L-[G-t4C]leucine and 5.0/zCi L-[G-aH]valine per ml of incubation medium as labels (New England Nuclear, 276 #Ci/mmole and 2.2 Ci/ mmole respectively). After incubation the reaction was stopped by the addition of an equal volume of an ice-cold solution of 3 mM leucine plus 3 mM valine. Synaptosomes were isolated from the incubated mixture by centrifugation at 40,000 × g for 1 h in the Sorvall SS-34 rotor and transferred to a glass homogenizer for proteolipid isolation. Incubation ofmitochondria. The incubation medium contained 0.3 M mannitol, radioactive amino acids, 0.2 mM EDTA, 10 mM Mg e+, l0 mM Tris, 20 mM K +, 170 mM Na +, l0 mM PO4a-, 75 mM a-oxoglutarate, and 20 mM pyruvate, pH 7.4, and was prepared by combining stock solutions (kept frozen at --20 °C until use) in the amounts and order as follows: (a) 0.1 ml (for each ml of final incubation suspension to be prepared) of 100 mM K2HPO4 in 0.3 M mannitol, pH 7.4; (b) 0.2 ml of a combination of 1 mM EDTA, 50 mM MgClz and 50 mM Tris all in 0.3 M mannitol, pH 7.4; (c) 0.5/tCi L-[G-14C]leucine and 5.0/~Ci L-[G-aH]valine; (d) 0.15 ml of 0.5 M sodium a-oxoglutarate in 0.3 M mannitol, pH 7.4; and (e) 0.05 ml of 0.4 M sodium pyruvate in 0.3 M mannitol. Mitochondria were suspended in 0.3 M mannitol to a concentration of 8-10 mg protein per ml and 0.5 ml for each ml of final suspension were added to start the reaction. An aliquot of the mitochondrial suspension was

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reserved for determination of protein content. Mitochondria were incubated at 37 °C with shaking for 1 h with 95 ~o 02-5 ~ CO2 as the gas phase. The incubation was stopped by the addition of an equal volume of ice-cold 3 m M leucine and 3 m M valine followed by centrifugation of the mitochondria at 40,000 ~< g for I h in the Sorvall SS-34 rotor at 2 °C. Incubation ofmicrosomes. The procedure of Zomzely et al. 34 was followed using 0.5 #Ci L-[G-14C]leucine and 5.0/~Ci L-[G-3H]valine per ml of incubation medium as labels. After incubation the reaction was stopped by the addition of an equal volume of an ice-cold solution of 3 m M leucine plus 3 m M valine. Microsomes were isolated from the incubated mixture by centrifugation at 105,000 × g for 1 h in the Spinco No. 50 rotor and transferred to a glass homogenizer for proteolipid isolation. Incubation and purification of crude myelin. Crude myelin was incubated at a concentration of 0.5 -1.5 mg myelin protein/ml by the procedure of Smith and Hasinoff 28 using as label 0.5/tCi L-[G-14C]leucine and 5.0/zCi L-[G-aH]valine for each ml of suspension. After incubation for 1 h at 37 °C with shaking and with 95 ~ 02-5 CO2 as the gas phase incorporation was stopped by homogenization of the suspension with one volume of ice-cold 3 m M leucine and 3 m M valine. After homogenization the suspension was centrifuged at 2 °C in a Sorvall SS-34 rotor for 20 min at 40,000 × g. This homogenization and centrifugation process was repeated twice for a total of 3 times. The purified myelin was then transferred to a glass container for proteolipid isolation. Isolation and preliminary purification of proteolipid. A procedure similar to that of Cattell et al. 6 was adopted. Subcellular fraction pellets were taken up in 40 times their volume of chloroform-methanol (2:1, v/v) at room temperature. After dispersing the pellets the suspensions resulting were stirred at room temperature for 14 h and filtered through sintered glass funnels to eliminate denatured non-proteolipid protein. Any undenatured non-proteolipid protein remaining was removed by washing the chloroform-methanol solution for 5 days first with 0.2 vol. of water and then with daily changes of the upper phase 11. Proteolipid was isolated by removing the aqueous phase, cooling the organic phase to 0 °C and slowly adding 5 vol. of ice-cold diethyl ether with stirring. The resulting suspension was allowed to stand at 0 °C for 20 h. Then the proteolipid precipitate was isolated by centrifuging at --10 °C for 45 min at 2,000 × g. After discarding the supernatant the proteolipid precipitate was further purified as described below. Finalpurification of protein andproteolipid. The purification procedure was that of Autilio et al. 2. After the final ether wash, proteolipids were hydrolyzed for amino acid analysis and proteins were dissolved in 1 ml of NCS Solubiliser (Amersham/ Searle) by heating at 60 °C for 1-2 h and analyzed for radioactivity in a Packard TriCarb Scintillation counter in a toluene-l,4-bis 2-(4-methyl-5-phenoxazolyl)benzene2,5-diphenyloxazole mixture with efficiencies of 7 5 ~ and 15 ~ for 14C and ~H respectively. Amino acid analysis of proteolipid preparations. Proteolipid fractions were hydrolyzed in 6 N HC1 in vacuo at 110 °C for 70 h. After hydrolysis, HC1 was removed in vaeuo and the residue made up to volume with 0.2 M sodium citrate buffer (pH 2.0)

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SYNTHESIS OF BRAIN PROTEOLIPID

141

2B Fig. 2. Electron micrographs of microsomes prepared from 14-I6-day-old rat white matter by the procedure of Zomzely et al. 34. A, The preparation consists primarily of smooth microsomes and free ribosomes or polysomes. No mitochondria or synaptosomes are apparent, x 30,000. B, The multilamellar vesicle in the lower left corner may be myelin, x 75,000.

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J.A. BABITCH

after the addition of D-[l-14C]l-/3-thienylalanine standard. The amino acids were determined with a Beckman Model 120 amino acid analyzer. To reduce hydrolytic losses, 1-3 drops of 5 % phenol were added before hydrolysis and corrections were made when necessary. Fractions of 4-5 ml were taken from the amino acid analyzer effluent with a fraction collector (Vangard, Model 1010) and 15 ml of Aquasol (New England Nuclear) were added to bring the solution into one phase for liquid scintillation counting. Radioactivity was measured in the Packard Tri-Carb Scintillation Counter and quenching corrections were made when appropriate. RESULTS

Purity of subcellular fractions. The crude myelin used for incubations may have contained small amounts of axonal material (Fig. 1). However, mitochondria, synaptosomes, and microsomes were essentially absent as indicated by the very low succinic dehydrogenase and amino acid incorporating activities of the myelin preparations which, in addition, were completely soluble in 2:1 (v/v) chloroform-methanol after their final purification. Further, myelin did not seem to contaminate the other subcellular preparations (Figs. 2-4). Electron micrographs of the microsomal preparation reveal a mixture of smooth microsomes and free polysomes or ribosomes (Fig. 2). A few ribosome-studded membranes remain. Though it is sometimes difficult to distinguish ribosomes from glycogen granules, the high levels of protein synthesis in the presence of ATP and the absence of protein synthesis in the absence of ATP or an ATP generating system both suggest that a major portion of the particles are ribosomes. For the same reasons significant contamination of the preparation by mitochondria or synaptosomes is unlikely, and, in addition, these preparations were free of the mitochondrial marker enzyme fumarase. Further, synaptosomes and mitochondria are absent from the microsomal preparation micrographs and multilamellar membranes indicating possible myelin contamination occur only rarely. Also, microsomes do not seem to significantly contaminate the other subcellular fractions, though cytoplasmic profiles did occasionally appear in the mitochondrial preparation (Fig. 3). The largest amounts of cross-contamination occurred between the mitochondria[ and synaptosomal preparations (Figs. 3 and 4). While contamination of the mitochondria by synaptosomes was somewhat less than 10%, the contamination of the synaptosomal preparation by mitochondria was estimated at 35 % by counts made from the electron micrographs. This contamination of the synaptosomes by mitochondria was probably due to the extra layer of 1.1 M sucrose in the centrifugation gradients which was introduced to decrease synaptosomal contamination of mitochondria. As can be seen in Figs. 3 and 4, myelin was not a significant contaminant of either the mitochondrial or synaptosomal fractions. Incorporation of L-[G-3HTvaline and L-[G-14CTleucine into protein and proteolipid. Microsomal, mitochondrial and synaptosomal fractions from 14-16-day-old rat white matter were prepared as described in the Methods section. Half of each microsomal preparation was incubated in the standard incubation medium. The other half

SYNTHESIS OF BRAIN PROTEOLIPID

143

Fig. 3. Electron micrograph of the mitochondrial fraction prepared by a modification of the procedure of Whittaker et a l Y . The preparation is described in the Methods section. This fraction consists largely of mitochondria though an intact synapse appears above the centre of the picture, x 49,500.

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J . A . BABITCH

Fig. 4. Electron micrograph of the synaptosomal fraction prepared by a modification of the procedure of Whittaker et al. 32. The preparation is described in the Methods section. Synaptosomes (S) are contaminated by some mitochondria (M). A few synaptosomal mitochondria (m) are indicated. × 24,500.

SUBCELLULAR

FRACTIONS (COUNTS/MIN/MG PROTEIN OR

64~ 21 759~ 163) 22708 ± 2720 (423570-4-71057) 70-419 ( 1362.4. 684) 1517.4. 413 ( 400665- 8437) 46-46 ( 6565269) 760 ± 177 ( 156245- 3098) (

14C* into nonproteolipid protehl

* Amino acid analyses were not performed on non-proteolipid protein samples. ** Average of two separate incubations 5- difference from the mean.

Synaptosomes - - inhibitors

Synaptosomes 5- inhibitors

Mitochondria - - inhibitors

154± 27 2105 -4764) 46655 ± 11860 (825825 ~176455) 138538 ( 2461 5980) 19095363 ( 517165- 10126) 8254 ( 1221 ± 449) 808__+ 177 ( 166525- 3127)

Microsomes - - ATP and an ATP generating system Microsomes + ATP and an ATP generating system** Mitochondria ÷ inhibitors (

3H* into nonproteolipid protein

Fraction

79 ± 14 8-4- 2) 661 ± 134 ( 33 ~ 1) 141 5- 85 ( 6 5 ~ 47) 2349 i 4 5 3 ( 823 5-339) 27:E 9 ( 13 ½ 5) 1093 ~:379 (4555-185) (

aH as valine into proteolipid

46-4- 27 3 -4- 2) 303 -4- 34 ( 16.4. 2) l l 7 d _ 94 ( 575- 48) 2431 -4-482 ( 846 5- 344) 6-4- 4 ( 3 5-2 ) 13345-580 (5565-277) (

14C a s leucine into proteolipid

Incubations were all for 60 min. Conditions are described in the Methods section and the text. All values except the complete microsomal incubations are the average of 3 separate incubations ± S.E.M., and numb2rs in parentheses represent total incorporation per incubation. The inhibited (control) incubations contained 1 m M NaN3 plus 0.5 m M dinitrophenol plus 100 pg/ml cycloheximide.

INCORPORATION OF L-[G-3H]vALINE AND L-[G-14C]LEUCINE INTO PROTEIN AND PROTEOLIPiD OF PROTEOLIPID)

TABLE I

7~

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J.A. BABITCH

was incubated without ATP or an ATP generating system and served as control. For the mitochondrial and synaptosomal incubation experiments, controls accounted for half of each preparation being incubated with 1 mM NAN3, 0.5 mM dinitrophenol, and 100 #g/ml cycloheximide. After incubation for 60 min, each sample was separated into proteolipid and non-proteolipid protein components and analyzed for radioactive incorporation and amino acid composition as described in the Methods section. The proteolipid yields averaged 6, 12, 21 mg/g of microsomal, mitochondrial and synaptosomal total protein respectively. Results are shown in Tables I and II. These data demonstrate that the greatest incorporation into proteolipid was by the mitochondrial system, both in terms of total counts incorporated and specific activity of the synthesised material. Though microsomes actively synthesized nonproteolipid protein, they made very little proteolipid. Since the synaptosomal preparation was significantly contaminated by mitochondria, incorporation into proteolipid by synaptosomes may also have been very low. This is supported by the similarity of the mitochondrial and synaptosomal proteolipid amino acid compositions (Table II). In addition to these fractions, 4 crude myelin preparations were isolated, incubated with radioactively labelled amino acids, and purified as described in the Methods section. Values of counts/min/mg proteolipid ranged from 12 to Ill for

TABLE lI COMPARISON OF THE AMINO ACID COMPOSITIONS OF PROTEOLIPIDS ISOLATED FROM INCUBATED RAT WHITE MATTER SUBCELLULAR FRACTIONS WITH BOVINE F O L C H - L E E S PROTEOLIPID

Data are expressed as moles/100 moles total amino acids (assuming 2 mole °~i tryptophan) corrected for hydrolytic losses. Amino acid

Folch Lees proteolipid ~,~

Mitochondria*

Microsomes* *

Synaptosomes*

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half Cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine

4.3 1.9 2.6 4.2 8.5 5.4 6.0 2.9 10.3 12.5 4.2 6.9 1.7 4.9 I 1.1 4.7 7.9

2.6 1.3 2.1 4.6 6.5 7.6 5.4 4.2 9.8 11.0 0.2 5.6 2.4 9.7 14.9 3.0 7.4

3.8 0.4 4.2 7.3 5.5 9.4 8.6 3.8 13.9 10.1 0 6.4 0.3 7.1 10.5 2.6 4.6

2,9 1.1 2.6 5.2 6.2 7.2 5.7 4.1 9.7 10.4 0 6.3 3.0 9.4 14.2 3.3 6.8

• Average of 6 preparations + S.E.M. • * Average o f 5 preparations ~ S.E.M.

± 0.2 ~ 0.3 ~ 0.3 +_. 0.6 ± 0.5 ~ 0.6 £ 0.6 ~ 0.4 ~ 0.8 ± 0.7 i 0.7 -4 0.3 ~ 1.7 ± 0.4 A- 0.8 ~ 0.2 :% 0.2

-L 0.5 i 0.3 i 0.3 ~ 3.2 ± 0.2 ± 3.6 ± 3.9 ~+ 0.2 q 5.7 ~:!~1.7 ~ 1.9 :~ 0.3 -~ 4.4 -~: 3.9 ~ 1.6 ~ 2.0

-L 0.6 + 0.2 i- 0.4 k~ 1.3 ~ 0.6 -}~ 0.3 ± 1.1 J- 0.6 ~ 1.0 ± 1.1 T ± ± t: + i

0.4 2.6 0.9 0.4 0.4 0.6

SYNTHESIS OF BRAIN PROTEOLIPID

147

these myelin preparations indicating that crude myelin preparations were probably incapable of synthesizing proteolipid in vitro. It can be argued on logical grounds that the presence of radioactivity in the mitochondrial proteolipid cannot be due to 'carry-over' of radioactively labelled non proteolipid protein into an unlabelled proteolipid fraction. If that happened then the specific activity of the resulting proetolipid plus protein fraction should be between those of the proteolipid and non-proteolipid protein fractions. This is not the case. The specific activity of the mitochondrial proteolipid is higher than that of mitochondrial non-proteolipid protein (Table I). The same argument applies to the synaptosomal fractions. However, since microsomal non-proteolipid protein does have a higher specific activity than microsomal proteolipid, 'carry-over' may be invoked to argue that incorporation into microsomal proteolipid is actually lower than it seems while tbr mitochondria the proteolipid incorporation is genuine. Since Wolfgram and Kotorii 3~and Tenenbaum and Folch 29used bovine material and the analyses of Gonzales-Sastre15 were uncorrected for hydrolytic losses, direct comparison with the present amino acid composition is not possible. However, the data in Table II show many similarities between the myelin and mitochondrial proteolipids. The amino acid composition of the mitochondrial proteolipid more closely resembled Folch-Lees myelin proteolipid than did the microsomal material, e.g. the sum of the squares of the variations in amino acid composition between Folch-Lees and mitochondrial proteolipids was 74.52 while the same value for Folch-Lees and microsomal proteolipids was 106.31. The sum of the squares of the variations between Folch-Lees and synaptosomal proteolipids was 71.15, essentially the same as for the mitochondrial materal (Table II). The microsomal proteolipid had an amino acid composition which resembled that of microsomal proteolipid from bovine heart z5 in its higher content of arginine, aspartic acid and glutamic acid, and lower content of threonine and tyrosine (Table II). Nevertheless, there were some similarities between the microsomal and myelin materials, perhaps because the highly specialized isolation procedure would be expected to select proteins of a similar nature which could partition into the organic phase in competition with water. Thus only a few proteins can be isolated from any tissue by this method. DISCUSSION

Nuclei, microsomes or myelin itself do not appear to be sites of significant proteolipid synthesis. Incorporation of amino acids into proteins of isolated myelin preparations has not been unequivocally demonstrated. This may be due, in part, to the absence of ribonucleic acid from myelin1. Other workers have found low levels of incorporation of amino acids into nuclear proteolipid and not much nuclear proteolipid19,z°. Similarly, microsomes do not seem to contribute significantly to proteolipid synthesis. Microsomes contain little proteolipid and microsomal proteolipid had only 1-2 ~ of the specific activity of microsomal protein (Table I). The reasons for believing that incorporation was genuinely mitochondrial rather than due to bacteria, lysosomes or other contaminants have been discussed in

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detail elsewhere 24. They need not be repeated here except to point out that all of the reasons presented apply here and, in addition, lack of incorporation in the complete myelin system suggests that incorporation by bacteria was not a problem. The present results extend previous findings by other workerst7,ag-21, a°. These groups found that the rate of incorporation of amino acids into proteolipid of a crude mitochondrial preparation from rat brain parallelled the time course of myelination. Furthermore, amino acids were incorporated predominantly into the interior of the molecule. Additional support for the related nature of myelin and mitochondrial proteolipid comes from the work of Smith 26. Her data indicate a remarkable degree of similarity between the metabolism of the constituents of both myelin and mitochondrial membranes. She also found that the half-life of proteolipid protein was 35 days, which compares well with a half-life of 31 days for mitochondrial membrane proteins 4. The slightly shorter half-life of the mitochondrial membrane proteins may be due to export of certain of these proteins to sites of myelin construction. While transport of a hydrophobic protein from mitochondria through the cytoplasm to myelin might once have been considered unlikely, the isolation of proteolipid protein in a form soluble in both water and chloroform-methanol mixtures 29 mitigates this this objection. Finally, it has been reported that microsomal and mitochondrial membranes (isolated as one 'heavy membrane' fraction) contain sites for synthesis of myelin lipids'ZL Many authors have attempted to identify the radioactive product(s) of mitochondrial synthesis. Most agree that incorporation occurs almost exclusively into insoluble proteins23, al of the inner mitochondrial membrane 2~. It should be remembered that in the present studies most of the label was incorporated by mitochondria into chloroform-methanol insoluble protein (Table I). This chloroform-methanol insoluble material probably contains the only protein(s) into which amino acids seem to be incorporated by liver mitochondria 5. Due to limitations on the amounts of purified proteolipids which could be isolated, the amino acid composition data are not unequivocal as one would hope. The relatively large standard errors in this data cannot eliminate the possibility that the mitochondrial material has an amino acid composition which is only very similar to but not identical with - - myelin proteolipid. Until the amino acid composition of the rat liver material is known, the possibility that mitochondrial proteolipid is one component of Beattie's 'heterogeneous membrane fraction '5 cannot be excluded. Still, if this material is indeed Folch-Lees proteolipid an additional level of complexity is added to our concepts of the functional interrelationships among subcellular organelles in the brain. -

-

ACKNOWLEDGEMENTS

The author would like to thank Dr. S. Roberts and his staff for their help with various stages of this research. This work was supported by U.S. Public Health Service, National Institute of

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