Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

Hyperimmune antisera to chromatographically purified hemoglobins F and A2 were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A2 and/or F present in hemolysates of individuals with beta-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were commpared to values obtained by microcolumn chromatography for the measurement of Hb-A2 or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 mug of these hemoglobins.

Protein NEI, which has a molecular weight of 23500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt-precipitation and purified by ion-exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion-exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides ; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous.Protein NEI belongs to subgroup I1 of the A-chains. It has a series of subgroup specific exchanges and a subgroup specific deletion a t position 96. Protein NEI differs from all other A-chains of subgroup I1 by possessing a sixth cysteine residue. This additional cysteine residue a t position 88 is so far unique, as it is adjacent to an invariant cysteine a t position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue a t position 93, one of the hypermutable areas of the variable part. Furthermore we conclude by comparison with other A-chains that all completely elucidated A-chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.Structural studies with monoclonal immunoglobulins have provided the first insight into the genetic mechanism of antibody formation [l--51. Since the molecule is divided into a variable and a constant part, the genetic control of the immunoglobulins is more complicated than with other protein families.There is, however, an increasing amount of evidence, that the extreme heterogeneity of the variable parts, which is responsible for the specificity of the antibody, is genetically determined. Crucial in this respect is the high order of regularity which governs the amino acid exchanges and deletions of the variable parts. These linked amino acid exchanges (subgroup and subsubgroups) can only be explained by an evolutionary process [3,6--81.One of the proteins which has contributed to the understanding of this variability is Bence-Jones protein NEI, a A-type L-chain of subgroup 11. The primary structure of this protein has been reported previously in a short communication

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.