Subfractionation of hepatic endosomes in Nycodenz gradients and by free-flow electrophoresis. Separation of ligand-transporting and receptor-enriched membranes.

The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5'-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5'-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.     

Adenyl cyclase of oxyntic cells its association with different cellular membranes

The adenyl cyclase of the oxyntic, or acid-secreting, cells of bullfrog gastric mucosa has been found to be a membrane-bound enzyme. A method has been developed to isolate the adenyl cyclase rich membrane fractions in a hypotonic medium containing dithiothreitol, which has been found to protect the hormonal resposivenes of the adenyl cyclase.Highest specific activity of adenyl cyclase was localized in a light membrane fraction which also had abundant K⁺-stimulated ATPase and K⁺-stimulated $\text{p-}\text{nitrophenyl}$ phophatase and very low cytochrome $\text{c}$ oxidase activty. The three gastric secretagogues tested, namely histamine, pentagastrin and methylcholine, significantly stimulated the adenyl cyclase activity of the light membrane fraction.After treatment with 10 mM Mg⁺ further subfractionation of the light membrane fraction on a sucrose density gradient yielded light membrane subfraction 1, light membrane subfraction 2 and light membrane subfraction 3 in order of increasing densities. The three subfractions had different enzymatic and chemical properties. Adenyl cyclase activity has been found to be distributed in all three subfractions. However, the hormonal responsiveness of the three fractions was quite different. Light membrane subfraction 2 could be stimulated by all three secretagogues, light membrane subfraction 1 by histamine and methylcholine, while light membrane subfraction 3 was refractory to all three secretagogues. On the basis of the cholesterol to phospholipid molar ratio, RNA content, glycoprotein content and the enzymatic data it is suggested that light membrane subfraction 1 and light membrane subfraction 2 are of general plasma-membrane type, while light membrane subfraction 3 is largely of cytoplasmic origin.     

Fractionation of liver plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.     

ISOLATION AND CHARACTERIZATION OF THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was studied after isolation of a vesicle fraction and of vesicular subfractions by means of differential and density gradient centrifugations. The different fractions were examined electron microscopically by negative and positive staining; their content in protein and phospholipid and their ability to bind Ca⁺⁺ were determined. After homogenization, differential centrifugation yielded a "sarcovesicular fraction" (SVF) which was mainly composed of numerous vesicles of different types mixed with fibrous proteins and mitochondrial fragments. This SVF contained 2% of the protein and 25% of the phospholipid of the initial tissue extract. It had a high Ca⁺⁺ binding activity that was preserved for several days by storage in the presence of oxalate. After centrifugations of the SVF on sucrose density gradients, two vesicular subfractions were obtained which were characterized by different sedimentation rates, isopycnic banding, morphology, and composition in protein and phospholipid. (a) The low-density subfraction (ρ 1.10–1.12) contained a heterogeneous population of membranous structures: thick- and thin-walled vesicles, tubular formations, triads, and plasma membranes. Its content in protein and phospholipid was very low. (b) The high-density subfraction (ρ 1.13–1.17) was a very pure subfraction composed only of thin-walled vesicles. Its content in phospholipid was high and the ratio of phospholipid-phosphorus to protein was about 20. The calcium-binding activity found in the total SVF was recovered only in this latter homogeneous subfraction. The origin of these two subfractions from the SR is discussed.     

Hepatic Golgi fractions resolved into membrane and content subfractions

Golgi fractions isolated from rat liver homogenates have been resolved into membrane and content subfractions by treatment with 100 mM Na2CO3 pH 11.3. This procedure permitted extensive extraction of content proteins and lipoproteins, presumably because it caused an alteration of Golgi membranes that minimized the reformation of closed vesicles. The type and degree of contamination of the fractions was assessed by electron microscopy and biochemical assays. The membrane subfraction retained 15% of content proteins and lipids, and these could not be removed by various washing procedures. The content subfraction was contaminated by both membrane fragments and vesicles and accounted for 5 to 10% of the membrane enzyme activities of the original Golgi fraction. The lipid compositions of the subfractions was determined, and the phospholipids of both membrane and content were found to be uniformly labeled with [33P]phosphate administered in vivo.     

Ethanolamine Base-Exchange Reaction in Rat Brain Microsomal Subfractions

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).     

Role of LDL subfraction heterogeneity in the reduced binding of low density lipoproteins to arterial proteoglycans in cynomolgus monkeys fed a fish oil diet.

Previous studies using cynomolgus monkeys have shown that isocaloric substitution of dietary fish oil for lard reduced the in vitro binding of plasma low density lipoproteins (LDL) to arterial proteoglycans (PG) (Edwards, I.J., A.K. Gebre, W. D. Wagner, and J. S. Parks. 1991. Arterioscler. Thromb., 11: 1778-1785). The purpose of the present study was to determine whether all LDL subfractions were equally affected by the type of dietary fat with regard to PG binding and to identify compositional changes in LDL subfractions that might relate to the differential in PG binding. Two groups of cynomolgus monkeys (n = 5 each) were fed atherogenic diets (40% calories as fat; 0.26 mg cholesterol/kcal) containing 20% of calories as egg yolk and 20% as either lard or menhaden fish oil. LDL were isolated from plasma by ultracentrifugation and size exclusion chromatography and subfractionated by density gradient centrifugation. Three density ranges of LDL subfractions were collected from the gradients for determination of chemical composition, apoE and apoB content by ELISA, and binding to arterial PG in vitro. The d 1.015-1.025 g/ml subfraction contained 39 +/- 8% of the LDL cholesterol in the lard group but only 7 +/- 3% for the fish oil group. Values for cholesterol distribution were opposite for the d 1.035-1.045 g/ml subfraction, 8 +/- 1% versus 41 +/- 8%, respectively. Similar trends were noted for the distribution of apoB. For the lard group, LDL binding to arterial PG increased with decreasing density (i.e., increasing size) of the subfractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte.

1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.     

Morphologic and biochemical heterogeneity of lysosomes]

By means of isopycnic centrifugation in the continuous density gradient of sucrose two subfractions of lysosomes were isolated from rat liver homogenates: a "light" one (with the floating density p=1.13) and a "heavy" one (p=1.24). Electron microscopic, enzymatic and electron microscope enzymatic analysis of the isolated subfractions showed that the "light" subfraction consisted mainly of newly-formed primary lysosomes, while the "heavy" one was presented by secondary lysosomes. Parallel biochemical investigations demonstrated a considerable enzymatic heterogeneity of the two lysosomal subfractions: the "light" subfraction was characterized by a high specific activity of acid DNase, acid RNase and beta-galactosidase, and by almost total absence of beta-glucosidase activity, while the "heavy" one was characterized by a high specific activity of beta-glucosidase, beta-glucuronidase and beta-N-acetylglucosaminidase. Possible causes of enzyme heterogeneity of rat liver lysosomes are discussed.     

Changes in subfraction composition of chicken lysine-rich histone during ontogenesis]

Lysine-rich histone isolated from different chicken tissues was separated electrophoretically into 4-5 subfractions. The subfrations reffered to as 1, 2, 3, and 4, occur in each the tissue studied, erythrocyte lysine-rich histone containing an additional subfraction 1a. F1 histone from mitotically active tissues (intestinal mucosa, thymus, testes) has a higher content of subfraction 2, while the same histones from mototically inactive tissues (liver, heart, brain) contain an elevated amount of subfraction 3. F1 histone isolated from liver, brain and heart of 21-day embryo has much more of subfraction 2, than the same histone of adult animal. During the chicken development from hatching till maturation the content of subfraction 2 in these organs decreases, and the content of subfraction 3 increases. The rate of this change in liver corresponds to the rate of DNA synthesis. In F1 histone of erythrocytes the content of subfraction 4 falls down during the post hatching ontogenesis.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.     

Study of the relationship between the subfraction composition of histone F1 and the type of tissue in birds.

The subfraction composition of lysine-rich histone has been studied with the aid of polyacrylamide gel electrophoresis. The subfraction compositions of the histone F1 of several tissues from the chicken, pigeon, and titmouse have been compared. The histone F1 from the tissues investigated consists of four or five subfractions of similar number and electrophoretic mobility (1, 1a, 2, 3, and 4). In the different avian species each subfraction varied its mobility independently of the others. The chicken tissues investigated can be divided into two classes, depending on the relative concentration of subfractions 2 and 3 (A and B): Class A (subfraction 2 is smaller than 3) includes the brain, liver, skeletal muscle, heart, muscular layer of the stomach, and pancreas, and class B (subfraction 2 is larger than 3) includes the intestinal mucosa, thymus, and testes, as well as the liver, heart, and pancreas from a 21-day embryo. Such a division of the tissues corresponds to the varying rate of their cellular renewal. In a parallel examination of the relative concentrations of the individual subfractions in the same tissues from the three avian species it has been found that the relative concentration of subfractions 3 and 2 is increased in the skeletal muscles, heart, brain, and liver, that subfraction 2 is increased in the intestinal mucosa, that subfractions 4 and 3 are increased in the pancreas, and that subfractions 1, 1a, and 4 are increased in the erythrocytes. The results obtained may be interpreted as a consequence of some relationship between the subfraction composition of histone F1 and the type of tissue of the source.     

Enzyme distribution and transport activities in electrophoretically isolated fractions of the muscle sarcotubular system

A simple electrophoretic method is introduced allowing to isolate five fractions of skeletal muscle ST-system vesicles. In a previous study differences in lipid content, 3H-ouabain binding and in presence of triads in individual fractions (Lehotsky et. al. 1986) were analysed. In the present study biochemical characterization was extended, and (in accordance with previous results) major differences were observed to exist between fraction 1 and fractions 3 and 4. SDS-PAGE showed that fractions 3 and 4 were enriched in a protein with m.w. 100 kD, these fractions showing the highest specific activities of (Mg2+ + Ca2+)-ATPase and oxalate-supported Ca2+-uptake; activities of Mg2+-ATPase and surface membrane marker enzymes were the lowest in these fractions. On the other hand, in fraction 1 the highest activities of Mg2+-ATPase and marker enzymes of the surface membrane were observed together with a decreased content of the 100 kD protein and activities of Ca2+ transport. It could be concluded that the method is suitable to differentiate between relatively pure SR (fractions 3 and 4) and fractions rich in sarcolemma or T-tubules components (fractions 1 and 5).     

Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells

Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.