Susceptibility of the Wolfgram proteins and stability of 2',3'-cyclic nucleotide 3'-phosphodiesterase of rat brain myelin to limited proteolytic digestion.
The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.
D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism; D009185:Myelin Proteins / Q000378:metabolism*; D009186:Myelin Sheath / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D004676:Myelin Basic Protein; D009185:Myelin Proteins; C028312:Wolfgram proteins; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; C571339:Cnp protein, rat; D010447:Peptide Hydrolases; D014357:Trypsin