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The PO protein of the myelin of chick sciatic nerve was isolated and purified by propanoic acid extraction of peripheral nervous system (PNS) myelin, delipidation, Sepharose CL-6B chromatography in the presence of sodium dodecyl sulfate (SDS), and preparative SDS-polyacrylamide gel electrophoresis (PAGE). Approximately 15% of the PO protein in the sciatic nerve myelin was recovered in a homogeneous state. The purified protein monomer has an apparent molecular weight of 32.1K as determined by gel electrophoresis. The PO protein undergoes extensive aggregation during exhaustive dialysis and freeze-drying and yields stable dimers, trimers, and tetramers. The aggregation of the PO protein after freeze-drying is independent of the presence of a reducing agent (2-mercaptoethanol) in the solubilizing medium. The PO protein is a glycoprotein. The amino acid composition of the chick PO protein indicates a definite species difference when compared with mammalian PO proteins although the NH2-terminal isoleucine residue seems to have been retained during evolution.

Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy. The exchange rates of amide protons, stabilized by the hydrogen bonds of the three alpha-helices of the headpiece, could be determined quantitatively. The exchange rates in these helices showed repetitive patterns of about three to four residues. A correlation with the position of the amide proton in the interior or the exterior of the alpha-helix of the protein was found. The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R. et al., J. Mol. Biol. 182, 179-182 (1985)).

The ATP-hydrolyzing activity of membranes prepared from human erythrocytes depends on the presence of Mg2+ ions. Maximum activity is observed when the concentrations of Mg2+ and ATP are equal. The pH–rate profile of the enzymatic reaction shows a maximum at pH 7.8. The ATP-hydrolyzing activity has decreased to half the maximal activity at pH 6.3 and 9.8, respectively. The enzyme is inhibited by sulfhydryl reagents like p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide. Membranes that are titrated with NaOH or treated with p-CMB lose up to 30% of their protein content. This loss is reversed when Mg2+ or Mg2+ plus ATP is added. However, neither Mg2+ nor Mg2+ plus ATP detectably alters the rate of reaction between the sulfhydryl groups and specific reagents, nor protects the ATP-hydrolyzing activity from inhibition by p-CMB. The amount of protein which dissociates from the membrane increases with pH. The curve describing this extraction has a sigmoid shape with an inflection point at pH 10.2. Maximum solubilization is obtained at pH 11.2. Thus the results indicate that sulfhydryl groups and also the structure of the membrane are important for the ATP-hydrolyzing activity.

Membrane protein preparations were obtained by n-butanol extraction of salt-free aqueous suspensions of human erythrocyte ghosts. The solubilized protein contained 4.5% carbohydrate, including glucosamine and galactosamine, 1.7% sialic acid, and 0.2% phosphorus. Gel electrophoresis indicated the presence of a large number of proteins in the solubilized fraction. Of the phosphorus present 15% could be extracted with chloroform–methanol (2/1) and was shown to consist of phosphatidylserine and some phosphatidylinositol. A further 65% of the phosphorus was extracted with chloroform–methanol–HCl (200/100/1) and this extract was shown to consist principally of diphosphoinositide and triphosphoinositide. The remaining protein-bound phosphorus, representing 0.03% of the protein, could not be separated from the protein. Following treatment with the organic solvents the protein was resolubilized. The carbohydrate and sialic acid concentrations and the gel electrophoretic pattern were not altered. Following incubation of erythrocytes with inorganic 32P, the polyphosphoinositides were rapidly labelled. The phosphoprotein was also rapidly labelled but to a lesser extent. The phosphatidylinositol and phosphatidylserine were very poorly labelled.

The major /S-V-acetyl-D-glucosaminidase component in human blood plasma has been isolated. Final purification is 195-fold with 14% yield. Purity is confirmed by gel electrophoresis and isoelectric focusing. The enzyme has an isoelectric pH of 4.73 and apparent molecular weight of about 105,000 from sedimentation equilibrium centrifugation and gel chromatography. This value remains unchanged after re-Halifax, N.S., Canada.

The alpha 2-macroglobulin (alpha 2-M) was purified from the plasma of normal individuals and from that of cystic fibrosis patients. The proteins exhibited identical optical properties. Both proteins have an absorbance coefficient of A = 1060 g . cm-2 at 280 nm. The circular dichroism spectra are identical and indicate about 45% beta-sheet structure and almost no alpha-helix. The spectra of solutions at pH 8.0 do not change when trypsin is added. The fluorescence spectra of the alpha 2-M measured at pH 8.0 have contributions by tyrosine and tryptophan residues. The fluorescence intensities are identical and are enhanced about 30% when trypsin is added in 2:1 molar ratios.