Bibliothek

Notizen: Abstract: Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 ± 0.17 μm2 from normal myelin and 3.71 ± 0.28 μm2 for diseased myelin. Vesicle size was reduced to 4.08 ± 0.21 μm2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2 during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 ± 0.28 to 5.40 ± 0.31 μm2 Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 ± 0.193 × 10−8 compared with -6.093 ± 0.202 × 10−8 m2 s−1 V−1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 ± 0.151 × 10−8 m2 s−1 V−1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m−1 respectively. The phase transition temperature determined by wide angle x-ray diffraction studies was 63°C for normal myelin vesicles and 43°C for MS myelin vesicles. The presence of Ca2 did not affect the phase transition temperature, whereas EDTA decreased it to 40°C in the case of normal myelin vesicles. In the case of MS vesicles, Ca2 increased the phase transition temperature from 43 to 53°C, whereas EDTA had no effect. From these data, we conclude that both the surface properties (surface charge) and the physical structure of the bilayer are affected in MS. The Ca2 concentration of the vesicles from normal myelin was slightly elevated over that of vesicles from MS myelin. The Mg2 concentrations were not significantly different.

Notizen: Abstract: Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of ∼25–30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157–170 〉 120–130 〉 24–33. Because all three peptides contained two Cit residues, the greater reactivity of 157–170 suggested structural factors may modulate the activity. With S49, an antibody raised against peptide 69GSLPQKSQ-RSQDEN84 (rat sequence), a region in which Cit was not found and therefore is identical for both C-1 and C-8, showed greater reaction with C-1 than C-8, further supporting a role for conformational factors. Because the major difference between C-1 and C-8 is the presence of Cit in the later, conformational changes in this region of MBP must have been induced by Cit located even at some distance in the linear sequence of amino acids.

Notizen: Abstract: Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components, C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-I3 was ∼10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-I3 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of p-structure in a previous study. In that study, C-1, which contained about 13% & structure before phosphorylation, increased to −40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstmted that the β structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidy groups of Arg5 and Arg9. On the other hand, phosphorylation of C-4, which was already −34% P-structure, was twice as fast at Ser7 as for C-I, suggesting that the preexistence of 8-structure in the molecule facilitated phosphorylation at this site. In previous studies phosphorylation of C-I was shown to decrease vesicle aggregation, which we concluded to be the result of electrostatic repulsion between the phosphate on the protein and that of the lipid. The present data suggest that phosphorylation of some specific site may be playing an important role in the conformation of the protein.

Notizen: Abstract: Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two-thirds of the total amount applied to a CM-52 cellulose cation-exchange column was recovered in the unbound fraction of the column; the remaining one-third bound to column and was eluted as a single OD280 peak. This bound material did not show the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55–60 kDa), which constituted most of this protein fraction and a low molecular weight protein (∼ 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment-mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods. Good reactivity, comparable to that found with human MBP was observed in the enzyme-linked immunosorbent assay with an antibody (S-53) raised in rats against the synthetic peptide GSLPQKAQRPQDEN. The fraction that bound to the CM-52 cellulose column and was eluted with a salt gradient corresponded in net charge to component 5 of the bovine or human MBPs as deduced by alkaline gels.