Studies on Mv red cells. II. Immunochemical investigations

The properties of the Mv antigen, a low incidence receptor of the MNSs blood group system, were investigated by serological tests with protease treated red cells and inhibition assays with glycoproteins or peptides from normal and Mv erythrocytes. Our data demonstrate that the Mv receptor represents an allelomorphic form of the 'N' antigen on the Ss sialoglycoprotein, rather than variant of the M receptor on the MN sialoglycoprotein. Anti-Mv plus -N (serum Arm.) reacts with the N, 'N' and Mv antigens, whereas anti-Mv (serum Arch.) is specifically directed against the latter receptor.     

Genetic variants involving the major membrane sialoglycoprotein of human erythrocytes. Studies on erythrocytes of type Mk, Miltenberger class V and Mg.

1. Membranes from erythrocytes heterozygous for the Mk and Miltenberger Class V (Mi.V) condition and membranes from erythrocytes homozygous for the Mg condition were studied by polyacrylamide-gel electrophoresis by using the periodate/Schiff stain binding of radioiodinated lectins and labelling with lactoperoxidase. 2. Both the Mk and Mi.V conditions are associated with a decreased content of the major blood-group-MN-active sialoglycoprotein. 3. An unusual blood-group-M-active membrane component was found in Mi.V cells of appropriate genotype. No comparably component was found in Mk erythrocytes. 4. The Mg antigen appears to result from a modification of the MN-active sialoglycoprotein found in normal cells. Our results suggest that the Mg sialoglycoprotein contains fewer sialotetrasaccharides than does the normal sialglycoprotein. This may result from changes in the amino acid sequence of the protein. 5. The results are discussed in relation to differences in the antigenic properties of Mk, Mi.V and Mg cells and their possible influence on the structure of the surface of each of these cells.     

Abnormal blood-group-Ss-active sialoglycoproteins in the membrane of Miltenberger class III, IV and V human erythrocytes.

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.     

The Dantu erythrocyte phenotype of the NE variety. I. Dodecylsulfate polyacrylamide gel electrophoretic studies

Red cell membranes from patient NE, Mr. Dantu and 16 additional Black individuals, positive for the low-frequency MNSs-system antigen Dantu, were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. The content of the major, blood group M- or N-active sialoglycoprotein (glycophorin A, GP A) was found to be decreased by about 57%. The blood group S- or s-active sialoglycoprotein (GP B) was decreased by about 51% in membranes from proven Dantu/U heterozygotes and not detectable in those from patient NE and other Dantu+U- individuals. Donor NE was shown to exhibit the genotype Dantu/u. Dantu-positive cells exhibit a proteinase-resistant GP B-GP A hybrid with an apparent molecular mass of 29 KDa whose intramembraneous and cytoplasmic domains were shown to be similar to those of GP A. The molar hybrid: GP A ratio in all cells was found to be about 2.4: 1, indicating that the NE variety of the Dantu phenotype is much more frequent than the Ph or MD types. The significance of an additional minor 'new' component (molecular mass 21 KDa) in Dantu+ membranes and the minor component J (molecular mass 22 KDa) occurring in normal and Dantu+U+ GP preparations, but not in those from Dantu+U- cells, has not been resolved. The apparent molecular mass of the anion channel protein (band 3) in all cells of the NE variety was shown to be decreased by about 3 KDa, due to a shortening of carbohydrate chains. This suggests that the hybrid, just like GP A, might form a complex with band 3.     

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.     

Studies on Papanicolaou staining. III. Quantitative investigations of orangeophilia and cyanophilia

This paper provides data derived from the visible light absorbance spectra of Papanicolaou stained epithelial cells from the uterine cervix. Twenty-four types of spectra have been considered, namely, those derived from orangeophilic and cyanophilic nuclei and cytoplasms of superficial, intermediate, parabasal and dysplastic cells, and cells of carcinoma in situ and invasive carcinoma. Wavelengths of maximum absorbance and peak absorbances are tabulated. The proportions of bound orange G, eosin Y, aluminum-hematein and light green SF yellowish have been calculated. For the majority of cell types, dyebinding differences between orangeophilic and corresponding cyanophilic substrates were statistically significant. CIE coordinates were calculated from absorbance spectra; again differences between organeophilic and cyanophilic cells were statistically significant in most cases. Although the designation of cells as orangeophilic or cyanophilic is made on the basis of cytoplasmic coloration, the nucleus is also usually orangeophilic or cyanophilic. These nuclear differences are real and not due to the effects of over- and underlying cytoplasm.     

Glutamic-pyruvic transaminase (GPT) in non-human primates,I. Its activities and electrophoretic variation in red blood cells

Glutamic-pyruvic transaminase (GPT) in red cells of 25 species of non-human primates was investigated. There were significant differences in red cell GPT activities among species. Some species in the Prosimiae and the Ceboidea have high red cell GPT activities, while the others of these families examined have low activities. In contrast, red cell GPT activities were too low to be detected in the Cercopithecoidea and the Pongidea. The intraspecific variation of GPT zymograms was observed in Aotes trivirgatus by starch gel electrophoresis.     

Studies on the reaction between human growth hormone and oleic acid using polyacrylamide gel electrophoresis.

Through the work of U. J. Lewis and E. V. Cheever (1967, Endocrinoloyg81, 1338–1348) and U. J. Lewis, E. V. Cheever, and B. K. Seavey (1968, J. Biol. Chem.243, 260–267) it has been known for a number of years that human growth hormone (hGH), and many other proteins, reacts with unsaturated fatty acids to give rise to species with enhanced electrophoretic mobility. In view of the possible importance of this reaction in the genesis of charge isomeric protein artifacts, and for the understanding of hGH as a system of multiple isomers with distinct, and in some cases enhanced, specific activities, the nature of this reaction was investigated further. It was found that (1) the positions of oleic acid and growth hormone on polyacrylamide gel electrophoresis (PAGE) are coincident, indicating that the reaction leads to binding of the fatty acid to the protein: (2) the increment in molecular net charge on growth hormone is proportional to the molar ratio between the reactants, oleic acid and hGH; (3) the binding is noncovalent since it reverses under conditions of isoelectric focusing; (4) the reaction product has a molecular size indistinguishable from that of the reactant, hGH, by the criteria of “quantitative” PAGE (however, the reaction product exhibits an elevated negative molecular net charge in PAGE at pH 10.2); (5) the apparent isoelectric points of the hormone and its reaction products with oleic acid are indistinguishable in isoelectric focusing; (6) the interaction does not seem to involve the carboxyl charges on oleic acid since it is independent of ionic strength; (7) a noncovalent hydrophobic interaction with the protein is indicated since the range of electrophoretic mobilities exhibited by the hGH-oleic acid complex is smaller in the presence of benzyl alcohol in the gel than that exhibited by controls in it absence; (8) the reaction does not seem to involve free radical derivatives of the unsaturated fatty acid since it is not altered when the polyacrylamide gel is in a nonoxidative state; (9) the effect of the reaction with oleic acid on the tryptophan spectrum reflects only nonspecific interaction of the hormone-concomitant tryptophan perturbation.     

A rapid method for the detection of trypsinogen and chymotrypsinogen after electrophoretic separation of pancreas extract on polyacrylamide gel

Several methods have been described for the visualization of proteolytic activity on electropherograms obtained with starch (1,2), agar and agarose (3–6), paper (7), and cellulose acetate (8–11) as supporting media. In most of these reports casein was used as a (nonspecific) substrate. In only one case (11), the authors used substrates specific for trypsin and for chymotrypsin.The proteins in a pancreas extract could be satisfactorily separated by electrophoresis on polyacrylamide gel and we looked for the possibility of localizing proteolytic activities in this gel.Recently (12,13) methods for the direct localization of proteolytic activity in polyarcylamide gels were published, but no attempt was made in these articles to distinguish between trypsin and chymotrypsin. In this paper we will describe a method for the detection of Tg¹ and ChTg after their activation in the polyacrylamide gel. The method allows a rapid and reliable localization of the two proenzymes.