Fred A. Garver

Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase

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 CNPI 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.

Comparative immunochemical studies of primate hemoglobins.

The antigenic properties of a number of chromatographically purified primate hemoglobins were compared to those of normal human hemoglobin using a sensitive radioimmunochemical procedure. The degree of inhibition of the antigen-antibody reaction with heterologous hemoglobins appeared to be related to the structural similarity of these proteins to the normal human hemoglobin immunogen. With the exception of the baboon hemoglobin, the antigenicity of the hemoglobins paralleled the phylogeny of the primates. The gorilla and chimpanzee hemoglobins were antigenically identical to normal human hemoglobin, whereas the gibbon and orangutan hemoglobins were substantially more variable. Of the Old World monkey hemoglobins examined, the baboon produced lower inhibition values, suggesting a greater degree of structural dissimilarity than other Cercopithecoidea hemoglobins, which is compatible with a greater rate of evolutionary change occurring in this protein. Using the known amino acid sequences of human and other primate hemoglobins, we have attempted to identify antigenic determinant areas of the proteins.

Structural characterization of γ1-heavy chain disease protein BAZ: Heterogeneity of the constant region

Abstract γ 1 -heavy chain disease protein (HCD) BAZ has a molecular weight of 60,000 daltons and contains a substantial quantity of carbohydrate (14%) in comparison to normal γ-chains which is distributed as follows: sialic acid (5%), fucose (1.7%), hexoses (3.5%), glucosamine (1.7%), and galactosamine (2.0%). Dansylation of the protein indicates that it has a blocked N -terminal amino acid, whereas treatment of the protein with hydrazine liberates glycine as the C-terminal amino acid. Thus, both amino and carboxy terminal residues appeared to be homologous to normal γ-chains. The amino acid composition of the protein also shows a markedly lower half-cystine content in comparison to other γ 1 -chains, indicating that the protein lacks interchain disulfide bridges connecting the heavy chains due to a deletion encompassing the hinge region. Amino acid sequence analysis of the C-terminal octadecapeptide of protein BAZ demonstrated a previously unrecognized amino acid substitution in the constant region. It contains a substitution of glycine for alanine at position 431. This exchange may conceivably represent another allotypic variant of IgGl proteins.

Generation of a Monoclonal Antibody Specific for Hb G-Philadelphia [α268(E17)Asn→Lysβ2] and Development of an Immunoassay

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the α268(E17)Asn→Lysβ2 substitution of Hb G-Philadelphia. Hybridomas were produced by fusion of RBF/DnJ immune splenic lymphocytes with FOX-NY murine myeloma cells and selected in adenine-aminopterin-thymidine (AAT) medium. Culture fluids were screened by ELISA for antibody reacting with Hb G-Philadelphia but not Hb A. One such culture was cloned by limiting dilution, expanded and injected into pristane-primed, cyclophosphamide-suppressed BALB/c mice for ascites production. An enzyme-linked immunoassay was developed by conjugating hemoglobin in hemolysates or purified hemoglobins to the plastic surface of wells of a microtitre plate. The ascites fluid containing the Hb G-Philadelphi a Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzi-dine), a deep blue color developed, signifying a positive reaction. We a...

Chronic lymphatic leukemia evolving into chronic myelocytic leukemia

A diagnosis of chronic lymphatic leukemia (CLL) was made in an 83‐year‐old man on the basis of marked lymphocytosis (131.1 × 109/L) and infiltration of the marrow (77%) by small lymphoid cells. The hemoglobin was 11.8 g/dL and the platelet count was 427.5 × 109/L. With minimal and unsustained treatment, abrupt resolution of the lymphocytosis occurred with reciprocal emergence of a classic picture of chronic myelogenous leukemia (CML). During the hematologic evolution, blood lymphoid cells were isolated for surface marker, cytogenetic, and functional studies. E‐rosette receptors, Fcreceptors, and membrane immunoglobulins were found on only 7.5%, 7.4%, and 10.0% of these cells. These cells also failed to express a CLL‐associated antigen we have detected on ≈︁ 95% of the cells of all CLL patients studied, regardless of cell phenotype. In addition, the patients lymphoid cells responded poorly to leucoagglutinin (LPHA) stimulation, showed increased spontaneous metabolic activity and exhibited the Philadelphia chromosome. These observations suggest that the patients transient initial lymphocytosis was not due to CLL, but perhaps represented myeloid precursors in circulation prior to terminal differentiation.

Detection and quantitation of the fetal hemoglobin variant Hb F-Malta-I in adults.

A variant of fetal hemoglobin (Hb F-Malta-I) has been detected and quantitated in adult blood with a sensitive radioimmunoassay employing monospecific anti-sera. The concentration of Hb F-Malta-I was 0.002–0.05%, with an average value of 0.011%. The ratio of Hb F-Malta-I/Hb F in adults was about 4.8%, compared to a ratio of about 27% in the newborn. Since the F-Malta-I variant is a product of a mutated Gγ locus, which is one of the nonallelic structural genes directing the γ chain synthesis, its presence in blood of adults shows that the synthesis of this gene is not completely suppressed after birth, as was previously suggested.

Identification and Quantification of Hb C With an Enzyme-Linked Immunosorbent Assay

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique beta 6 GLU----LYS substitution in Hb C. Next, an antibody to rabbit gamma-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. For quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit gamma-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. Of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 +/- 5.7% by ELISA and 25.1 +/- 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 +/- 10.1% by ELISA and 24.7 +/- 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 +/- 4.2% by ELISA and 85.0 +/- 6.6% by RIA. Subjects with Hb C beta(+)-thalassemia had 66.5 +/- 3.7% Hb C as measured by ELISA and 63.5 +/- 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.

Immunochemical properties of abnormal hemoglobins C-harlem (β6Glu → Val, β73 Asp → Asn), S (β6Glu → Val), korle bu (β73Asp → Asn), vancouver (β73Asp → Tyr), and mobile (β73Asp → Val)

Abstract Antibodies were made to three mutant hemoglobins, each containing different single amino acid substitutions at β 73 : Hb Korle Bu ( Asp → Asn ), Hb Mobile ( Asp → Val ), Hb Vancouver ( Asp → Tyr ); and to one mutant hemoglobin, Hb C-Harlem, containing two substitutions in the β chain (gb6 Glu → Val , as in Hb S, and β 73 Asp → Asn , as in Hb Korle Bu). The antiserum to Hb C-Harlem contained two antibody populations, each specific for one mutant amino acid residue. The antiserum to Hb Vancouver was completely specific for this mutant and did not cross-react with Hb Mobile and Hb Korle Bu; however, antiserum to Hb Korle Bu partially cross-reacted with Hb Mobile and to a smaller degree with Hb Vancouver. Antiserum to Hb Mobile exhibited even less cross-reactivity with Hb Korle Bu and C-Harlem and none with Hb Vancouver. These and previous studies indicate the involvement of at least three independent areas in the β chain as antigenic determinant sites. It appears that the three mutants at β 73 elicit the formation of antibodies which have a gradation in their specificity due to the nature of the amino acid sidechain.

Physicochemical and immunochemical properties of γ1 heavy chain disease protein baz

Abstract The physicochemical and antigenic properties of a γ1 heavy chain disease protein (γl HCD BAZ) are described. Protein BAZ is positive for the Glm(1) determinant and gives a reaction of identity with Fc fragment, whether derived from Cohn Fraction II or from an IgG1 myeloma protein. It also gives a reaction of complete identity with the γHCD proteins CRA and ZUC. The mol. wt of the native protein was determined from (1) sedimentation-diffusion, (2) sedimentation-viscosity, and (3) sedimentation-equilibrium measurements to be approx 60,000 daltons. The mol. wt of the unreduced protein in 6 M guanidine hydrochloride was found to be 30,000 daltons, which was identical to that of the reduced-alkylated form in 6 M guanidine. These results established that γ1 HCD BAZ is a noncovalently linked dimer and suggested the possibility of a missing hinge region.

Immunochemical identification of normal and variant hemoglobins after electrophoretic separation and transfer to nitrocellulose membranes

A highly specific method for the conclusive identification of normal and variant human hemoglobins is described in this communication. The method employs the standard electrophoretic technique in combination with the immunoblot technique using monospecific antisera raised in rabbits. Various hemoglobins such as Hb, S, C, A2, and F were separated on an alkaline polyacrylamide gel or a cellulose-acetate membrane, and transferred to a nitrocellulose membrane and the individual hemoglobins were identified by the immunoblot procedure. With this method hemoglobins with similar or identical electrophoretic mobilities can be definitively identified with the use of monospecific antisera.