Abstract
Two guinea pig myelin basic protein preparations which differed markedly in their contents of high pH electrophoretic or chromatographic forms were studied in an attempt to elucidate the causes of their microheterogeneity. Both total preparations and components isolated therefrom were examined for their amino acid compositions, NH2-terminal and COOH-terminal residues, total phosphorus contents, amd contents of phosphamino acids. The results showed that the five components differed sequentially by a single charge and that the microgeterogeneity arose as a result of secondary modifications of a single secies (Component 1) Of basic protein. Two modifications were demonstrated; viz. phosphorylation of serine and threonine and loss of COOH-terminal arginine. These two modifications were insufficient to account completely for the observed microheterogeneity; an additional cause, deamidation, was postulated. From the relationship between the number of components present in the total basic protein, the phosphorus and phosphoamino acid contents of the components, and the changes in relative electrophoretic mobility of the components which accompanied their phosphorylation and dephosphorylation we conclude that in the native basic protein no more than two sites in any polypeptide chain are phosphorylated.
Highlights
In preparation A 83% of the polypeptides in Component 2 were intact, yet they differed from Component 1 by a single charge
Phosphorylated polypeptides in Component 5 consist of (a) chains containing both a phosphoserine and a phosphothreonine residue and (6) monophosphorylated chains with two additional negative charges resulting from arginine and/or amide loss
Our studies have shown that brain phosphokinase converts Component 1 in vitro to Components 3 and 5, but not to Components 2 and 4, and that Components 1 to 5 differ sequentially by a single charge
Summary
Reagents-Escherichia coli alkaline phosphatase-BAPC, carboxypeptidase A-diisopropylphosphorofluoridate, and carboxypeptidase. Determination of Protein-A known dry weight of salt-free, lyophilized preparation A and each of its components were dissolved in a known volume of H,O. Analyses of zero time and Z-hour controls showed no detectable free amino acids and no differences in the electrophoretic patterns of the basic proteins. Reactions-The total basic protein and its components were treated with Escherichia coli alkaline phosphatase as described by Balhorn et al (18) except that 0.05 M NH,HCO,, pH 8.2, was used instead of Tris-HCI. NH,-terminal residues were determined by reaction of the protein with 5.dimethylaminonaphthalene-1-sulfonyl chloride as described by Tamura et al (19) and separation of the dansyl amino acids on polyamide sheets (7.5 x 7.5 cm) by the procedure of Hartley (20). When maximal resolution of’ components for the purpose 01’quantitation was desired, the amount of protein applied to the gels was limited to 50 pg. llnder these conditions repeated electrophoretic analvses of the same sample yielded relative area values agreeing to with”in 5% for well resolved components and 10’; for less well resolved ones
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