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1.
Brain slices prepared from 20-day old rats were incubated with [3H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [3H]palmitic acid into PLP of fraction SN4 (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type ‘precursor-product’ between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [3H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.  相似文献   

2.
Effects of monensin on posttranslational processing of myelin proteins   总被引:13,自引:11,他引:2  
Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label.  相似文献   

3.
—An in vitro system using mouse brain homogenates has been developed to study the synthesis of the myelin basic proteins. Incorporation of [3H]leucine into protein in this system did not require additional energy sources. The system was slightly stimulated by glucose and strongly inhibited by puromycin. Myelin basic proteins were isolated from incubation mixtures by conventional techniques of solvent extraction and column chromatography, and finally separated into the large and small components by polyacrylamide gel electrophoresis in an acetic acid-urea system. Gels were stained, sliced, dissolved, and counted, and relative rates of incorporation of label into the two basic proteins were determined at several ages. The ratio of radioactivity incorporated into the small (S) and large (L) basic proteins, over a 30 min incubation period, was found to increase from 0.97 at 10 days to 1.59 at 21 days and decline thereafter. These data generally agree with earlier studies on the in vivo synthesis of the myelin basic proteins in mice. An interesting feature of the time course was that incorporation of [3H]leucine into the purified myelin basic proteins relative to incorporation into total protein in the homogenate increased almost 2-fold during the course of the 30-min incubation. This suggested that post-translational processing of at least one of the two basic proteins was occurring. To examine this possibility further, experiments were conducted in which incorporation was allowed to proceed for 2–5 min, before being inhibited with puromycin; the incubation was then continued for up to 25 min longer. Although total incorporation was inhibited immediately after puromycin addition, label was found to continue to accumulate in the basic proteins to the extent of 30–100% above controls. These data support the notion that the MBPs are synthesized as precursors and then processed to yield authentic myelin basic proteins and that this processing can occur in vitro.  相似文献   

4.
Incorporation of [14C]leucine into the myelin sheath was studied in brain stem slices prepared from 22-day-old rats. Individual major myelin proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. There was a time lag before incorporation of the label into proteolipid protein (PLP) and intermediate protein (IP) reached maximal rates. Labelling of basic proteins (BP) and Wolfgram proteins (WP) revealed a much shorter lag in entry. Appearance of radioactive proteins in the myelin sheath was significantly hampered by triethyllead (PbEt3) added to the incubation medium at micromolar concentrations. Inhibition values were highest in the case of PLP and were closely followed by the values for IP. BP and WP were less inhibited, although incorporation of these proteins into myelin was still suppressed more than was synthesis of total homogenate protein. Thus, myelin-forming cells seem to be unduly vulnerable to the toxin relative to the rest of the tissue. Furthermore, the results indicate an interference of PbEt3 with certain posttranslational processes involved in furnishing of integral myelin proteins.  相似文献   

5.
6.
Recovery of Proteolipid Protein in Mice Heterozygous for the Jimpy Gene   总被引:1,自引:1,他引:0  
We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.  相似文献   

8.
Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [3H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [3H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.  相似文献   

9.
Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the in vitro incorporation of 14C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.  相似文献   

10.
Synthesis and incorporation of myelin polypeptides into CNS myelin   总被引:17,自引:6,他引:11       下载免费PDF全文
The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.  相似文献   

11.
Mice ranging in age from 16 to 44 days were injected intracerebrally with 3H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of 3H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.  相似文献   

12.
The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.  相似文献   

13.
In a study of a system suitable for investigating long-term effects on brain protein metabolism, we measured amino-acid incorpration into isolated immature brain explants incubated under sterile conditions up to ten days. Measurements of changes in total proteins, total DNA, cell number during the experiments, and 14C-thymidine incorporation measurements indicated no significant net growth; new cell formation was below 5% in a 5-day period; therefore, amino-acid incorporation was mainly due to protein turnover. The rate of incorporation in our immature brain preparation was similar to that of the adult brain in vivo: by ten days about one-half of the tissue protein turned over. The label incorporated was released in subsequent incubations with cold amino acids. Such release occurred in all subcellular fractions examined. Incorporation was fairly stable; at temperatures below 30 degrees C it rapidly declined, but it was not affected when phenylalanine or the branched chain amino acids (leucine, isoleucine, valine) were elevated in the incubation medium. Brief exposure to low amino-acid media had no effect; longer exposure resulted in tissue damage. Our model system indicates that overall brain protein turnover is not sensitive to such variations in the level of most amino acids, which may occur under various conditions. Protein metabolism of the nervous system occurs at a high rate. A recent long-term labeling method (Lajtha, Latzkovits, and Toth, 1976) gave a best fit to incorporation curves by assuming two compartments for adult brain proteins, one of which (about 6%) has a half-life of 15 hr and the other (94%) has a half-life of ten days. The disappearance of protein-bound label with time under conditions in which all proteins were previously labeled indicated that most, possibly all, proteins in brain are in a dynamic state (Lajtha and Toth, 1966). Incorporation of amino acids was found in all proteins and structures that have been studied to date; myelin proteins previously thought less active are also metabolized at a significant rate (Sabri, Bone, and Davison, 1974; Lajtha, Toth, Fujimoto, and Agrawal, 1977). We have fairly extensive information available in addition to turnover studies about the mechanisms of protein synthesis in brain (Roberts, 1971); protein breakdown was also studied in some detail (Marks and Lajtha, 1971). In contrast to our knowledge about protein metabolism under physiological equilibrium conditions, our information about alterations during functional demands or pathological conditions is scanty. Although a significant amount of work has been reported, largely because of technical difficulties the results are difficult to interpret unequivocally. The present report represents our effort to address some of the obstacles: to develop a system in which influences on long-term incorporation can be studied...  相似文献   

14.
Brain slices were prepared from 17-day old rats, and incubated with [3H]glycine or [3H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [3H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.  相似文献   

15.
Abstract— Slices of rabbit spinal cord were incubated with [3H]tyrosine and [35SO4] in the presence of either 5% antiserum to myelin basic protein or 0.21 mM-puromycin. The degree of incorporation of the precursors into the basic protein (BP), the proteolipid protein (PLP) and into sulphatides, as a representative lipid, in isolated myelin was investigated. Anti-BP serum inhibited the incorporation of [3H]tyrosine into BP and PLP from 22 to 46% as compared to controls, whereas puromycin nearly completely inhibited incorporation. The incorporation of [35SO4] into sulphatides was inhibited by anti-BP serum from 20 to 34% and by puromycin from 33 to 65% as compared to controls. These alterations were myelin-specific as shown by the equal or even increased incorporation of the precursors into the homogenates of spinal cord. The results are discussed in relation to the interaction of lipids and proteins in membrane assembly.  相似文献   

16.
Myelin Proteolipid Protein Gene Expression in Jimpy and Jimpymsd Mice   总被引:2,自引:1,他引:1  
Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpy(msd) (jpmsd; myelin synthesis deficient) and compared with that in wild-type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild-type mouse PLP gene. PLP-specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2- and 2.4-kilobase PLP-specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild-type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homogenates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild-type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at less than 10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.  相似文献   

17.
We examined chronological changes of myelin proteins of the brainstem and spinal cord of the twitcher mouse (15, 20, and 30 days old), a murine model of human globoid cell leukodystrophy caused by a genetic deficiency of galactosylceramidase I activity. The yield of myelin was normal until postnatal day 20, whereas galactosylsphingosine (psychosine) accumulated with age in myelin. The protein profiles of myelin and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the myelin remained normal throughout the experimental period. Fatty acylation of proteolipid protein (PLP) was examined in a cell-free system by incubation of myelin with [3H]palmitic acid, CoA, and ATP, and was normal at postnatal day 15, but decreased after postnatal day 20. Decreased fatty acylation of PLP was also observed in the twitcher mouse at postnatal day 20 when the isolated myelin was incubated with [14C]palmitoyl-CoA in the absence of ATP and CoA, or the slices of brainstem and spinal cord were incubated with [3H]palmitic acid. The activity of fatty acid:CoA ligase was reduced in myelin. These data suggest that decreased acylation of PLP in twitcher mouse myelin is probably due to reduced activities for both activation and transfer of fatty acid into PLP and that metabolic disturbance is present in myelin because acylation of PLP has been shown to occur in myelin membrane. Although psychosine (200 microM) inhibited only 17% of the acylation in vitro, it may be responsible for the reduced acylation of PLP in vivo.  相似文献   

18.
The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.  相似文献   

19.
Rats of 20-days of age were injected intracranially with radioactive palmitic acid to study its incorporation into proteolipid protein (PLP) of myelin and myelin subfractions. At short times (120 min), the radioactivity present in PLP was shown to be due to palmitic acid bound to the protein by ester linkages. The specific radioactivity of palmitic acid labeled PLP was identical in all the myelin subfractions except the myelin-like fraction, in which it was lower, suggesting that the entry of the fatty acid into PLP of the different subfractions occurs simultaneously.Experiments using time staggered injections of 14C- and 3H-labeled palmitic acid also showed that entry of the fatty acid into PLP of the various subfractions was simultaneous. These results seem to indicate that the acylation of PLP occurs in the myelin membrane and that synthesis and transport of this protein are events unrelated to the acylation process.  相似文献   

20.
The acylation of myelin proteolipid protein (PLP) and intermediate protein (IP) was investigated in an in vitro system of tissue slices prepared from actively myelinating rat brainstem. The incorporation of [3H]palmitate into the proteins in nine subcellular fractions including myelin and other cellular membranes which are actively involved in the synthesis and intracellular transport of the proteins was measured. More than 80% of [3H]palmitate-labeled proteins were recovered in myelin. The incorporation was highest in the heavy myelin and lowest in the light myelin subfraction. Appreciable acylation was also detected in the myelin-like fraction. On the other hand, the remaining fractions comprising a variety of endo- and ectomembranes, which harbored over 90% of newly synthesized PLP and IP as seen from [3H]leucine labeling showed practically no [3H]palmitate incorporation. The results indicate that the acylation of PLP and IP is a late event in their posttranslational processing and occurs only at their entry into the myelin sheath.  相似文献   

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