Preparation and further characterization of the MN glycoprotein of human erythrocyte membranes.

Human erythrocyte membrane glycoproteins were solubilized and recovered in the aqueous phase after extraction of red cell ghosts with a mixture of chloroform and methanol. The major glycoprotein, the so-called MN glycoprotein, was prepared from this phase by gel filtration on Sepharose 4B columns in 6 M guanidine hydrochloride. By SDS-acrylamide gel electrophoresis the MN glycoproteins from the three major genetic types, MM, MN, and NN, were found to be relatively free from minor glycoprotein contaminants. The carbohydrate and amino acid composition did not reveal significant differences between the three MN genotypes. The specific activities of the purified glycoproteins were determined for inhibition of agglutinating anti-M and anti-N rabbit antisera and for inhibition of myxovirus hemagglutination. It was, furthermore, established that the purified MN glycoproteins contain a receptor for Phaseolus vulgaris phytohemagglutination. The red cell MN glycoprotein inhibits partially lymphocyte stimulation induced by unfractionated Phaseolus vulgaris phytohemagglutinin; the red cell MN glycoprotein does not influence the lymphocyte stimulation induced by purified Phaseolus vulgaris mitogen.

The Hurler syndrome: a study of cultured lymphoid cell lines.

Lymphoid suspension lines have been established from three patients with the Hurler syndrome and four normals. The Hurler lines can be distinguished from normals by (a) staining characteristics, (b) increase in total cellular mucopolysaccharide content, and (c) increase in dermatan sulfate. Hyaluronic acid is absent in cultured lymphoid cells from normal persons and patients with the Hurler syndrome. The availability of biochemically marked suspension cultures should prove useful for enzymatic studies as well as for further elucidation of this clinical syndrome.

The relationship between 2 -microglobulin and immunoglobulin in cultured human lymphoid cell lines.

beta(2)-microglobulin was detected on the cell surface and in the medium of human lymphoid cells established in long-term culture. The secretion of beta(2)-microglobulin was relatively uniform when different cell lines were compared, whereas IgG production varied widely. kappa- and micro-membrane antigens were modulated by specific antibody; beta(2)-microglobulin was not modulated. Anti-kappa and anti-micro antisera had no effect on the expression of membrane beta(2)-microglobulin, nor had anti-beta(2)-microglobulin antiserum any effect on the expression of kappa- and micro-membrane antigens.

Characterization of cystic fibrosis factor and its interaction with human immunoglobulin.

Cystic fibrosis factor activity (CFFA), assayed as the ability to stop oyster ciliary movement, was present in serum-free medium from actively growing cystic fibrosis skin fibroblast cultures. CFFA was associated with a low molecular weight, negatively charged molecule that contained no uronic acid and was heat and pH labile. When CFFA-positive media were mixed with human IgG1, the CFFA was chromatographically displaced and emerged with the IgG1 fraction on column chromatography. Experiments in which various immunoglobulins were added to CFFA-positive culture media and then incubated with specific anti-immunoglobulins suggested that CFFA binding was class specific for human IgG, subclass specific for IgG1 and IgG2, and occurred with intact unaggregated heavy chains but not with kappa- and lambda-light chains, or Fab, Fc, and F(ab')(2) fragments. The serum protein beta(2)-microglobulin, which has structural homology to IgG, also bound CFFA.

Isolation and partial characterization of the major glycoproteins of horse and swine erythrocyte membranes.

The major glycoproteins of horse and swine erythrocyte membranes were isolated and examined chemically and immunologically. The major glycoprotein of horse erythrocyte membranes had a molecular weight of 33 000 and consisted of 46.2% protein and 53.8% carbohydrate, of which 9.4% was hexose, 10.1% hexosamine and 33.7% sialic acid. This glycoprotein was associated with activity for the infectious mononucleosis heterophile antigen. There were two different major glycoproteins in swine erythrocyte membranes. One major glycoprotein had a molecular weight of 46 200 and consisted of 34.2% protein and 65.8% carbohydrate, of which 18% was hexose, 19% hexosamine and 27.2% sialic acid. This glycoprotein had phytohemagglutinin (Phaseolus vulgaris) binding activity. The other glycoprotein had a molecular weight of 29 000 and consisted of 50.4% protein and 49.6% carbohydrate, of which 6.4% was hexose, 7.0% hexosamine and 36.3% sialic acid. This glycoprotein had weak or absent phytohemagglutinin binding activity.

Isolation and partial characterization of two minor glycoproteins from human erythrocyte membranes.

Two minor glycoproteins GP-II and GP-III, were isolated from human erythrocyte membranes and characterized chemically and immunologically. The chemical composition of GP-II and GP-III was similar: GP-II consisted of 81% protein and 19% carbohydrate of which 4.9% was hexose, 5.4% hexosamine and 7.8% sialic acid. GP-III consisted of 76% protein and 24% carbohydrate of which 7.6% was hexose, 7.2% hexosamine and 8.1% sialic acid. The amino acid composition of GP-II and GP-III was also similar. GP-II and GP-III, however, differed in chemical composition from the MN glycoprotein. GP-II and GP-III were associated with the blood group activities Ss, I and A, but not with the MN antigens. GP-III had higher blood group activities per mug of protein than did GP-II. The specific activities for the Ss blood group antigens were increased 3-10-fold by purificantion of GP-III from the aqueous phase of chloroform methanol extracts.

Sequence and organization of the human vitamin D-binding protein gene.

The structure and organization of the human vitamin D-binding protein (DBP) gene has been determined. The gene is composed of 13 exons and 12 intervening sequences. With the help of the polymerase chain reaction (PCR) introns were amplified using exon-specific oligonucleotide primers, and were sequenced after subcloning; the exon/intron borders were determined. The introns 2, 5, 7, 9 and 10 were sequenced completely; the introns 1, 3, 4, 6, 8, 11 and 12 were sequenced in part. We designed intron-specific primers for the amplification of each exon by the PCR-method. This permits the analysis of mutational and function-related sites. By comparison with the genes for human albumin and alpha-fetoprotein the gene for DBP/GC is confirmed as a member of this multigene family. The location of the introns in the coding region of the human DBP-gene is identical with the position of the introns in the rat DBP-gene.

The polymorphic human chaperonin protein HuCha60 is a mitochondrial protein sensitive to heat shock and cell transformation.

The HuCha60 protein, a polymorphic protein on two-dimensional gels of human lymphocytes, is found to be structurally and functionally related to the Escherichia coli groEL gene product: The structural homology is evident from the N-terminal amino-acid sequence analysis and from the immunological cross-reactivity with an antiserum against the E. coli groEL gene product. The functional homology is suggested by the heat sensitivity and the growth dependence of this protein. Both genetic variants of the HuCha60 occurring on the two-dimensional protein pattern of lymphocytes, the common "a" variant and the rare "b" variant, are strongly enhanced after heat shock. The expression of the HuCha60 in resting or normally growing cultures human cells is in general low, whereas in mitogen-stimulated cells or transformed cell lines the synthesis of the HuCha60 is strongly enhanced. After cell fractionation and subsequent two-dimensional gel electrophoresis and immunoblotting, the HuCha60 has been found to be mainly expressed in mitochondria. In the cytosol fraction two different molecular weight forms of the HuCha60 have been observed with low expression. Also in the nuclear fraction, HuCha60 is present in low concentration.

Sequence of a putative human housekeeping gene (HK33) localized on chromosome 1.

A gene (HK33) localized on human chromosome 1 has been detected by crossreaction of its fusion protein with a monospecific antiserum directed against human vitamin-D-binding protein (hDBP; group-specific component). Its cDNA sequence analysis showed no evident homologies neither to the sequence encoding hDBP nor to any other sequence. The largest cDNA clone of 3.2 kb includes a 897-bp coding region and a large 3' untranslated region with at least four polyadenylation sites. Further cDNA amplification using PCR demonstrated a total cDNA length of approx. 3.7 kb. Northern blot analysis revealed signals at about 2.2-2.5 kb and 4.0 kb, the shorter transcripts representing mRNAs using one of the two polyadenylation sites at about 2.0 kb. Synthesis of the 299-amino-acid polypeptide (33 kDa) in the bacterial host, with subsequent Western blot analysis, verified the sequence-specific recognition by the hDBP-specific antiserum. The search of protein databanks revealed no homology of HK33 to any known sequence. Since the gene is transcribed in all cells and tissues tested so far, it is a strong candidate for another housekeeping gene.

PI polymorphism in Israel: report on six Jewish population groups.

A total of 1148 Israeli Jews was typed for PI and divided by areas of origin into six groups: Eastern Europe (n = 236), Central Europe (n = 156), Rumania (n = 158), Bulgaria (n = 215), North Africa (n = 229), and Middle East (n = 154). Frequencies of PI*M1 (0.74-0.77) in Jews of European countries were higher than 0.68 in Jews of N. Africa and 0.62 in Jews of the Middle East. PI*M2 frequencies were correspondingly lower in European Jews: 0.11-0.14 vs 0.19 and 0.20 in non-European Jews. PI*M3 frequency range was 0.07-0.11 in European Jews and was highest in Middle Eastern Jews (0.17). PI*Z was found in one MZ individual. PI*S was low (less than 0.01) except in Sephardi Jews of Bulgaria and N. Africa (0.016 and 0.015). A rare variant, PI*Elemberg, was observed in five individuals from different countries of origin. The present results are in accord with those of a previous study on some Israeli Jewish groups and on some other Middle Eastern groups.

[Vitamin D-binding protein in human plasma. Analysis of genetic variation]. / Das Vitamin-D-bindende Protein des menschlichen Plasmas. Zur Analyse genetischer Variabilität.

The vitamin-D-binding protein (DBP) also known as group-specific component (GC) is described. This transport protein for vitamin D and its metabolic derivatives has genetic variants which are disclosed by electrophoretic procedures or by isoelectric focusing. Three common and one hundred rare alleles are known. The biochemical, molecular and population genetics are briefly outlined as an example of inherited variability in man.

Two-dimensional electrophoresis of human lymphocyte proteins: two-dimensional polymorphisms and paternity testing.

Genetic polymorphisms of seven human lymphocyte proteins, analyzed by two-dimensional electrophoresis, were evaluated in respect to their suitability for paternity testing. Current data of an enlarged family and population study for five proteins (p23, p30, p40, p60, p66), already described for a smaller population sample of Southern Germany, are presented together with evidence for a new polymorphic protein (p42), recently observed in our survey. These six proteins occurred in isoelectric focusing as two different variants, acidic (a) and basic (b). The genetic basis of the protein variations was ascertained (i) by the presence of homozygous and heterozygous phenotypes, (ii) by the Mendelian mode of transmission of the variants as allelic gene products within 17 families and (iii) by the demonstration of a gene-dosage dependence comparing the spot intensities in homozygous and heterozygous phenotypes. For quantitative data, laser densitometric scanning of the protein spots followed by computer-assisted quantitative evaluation of the spot intensities was performed. The allele frequencies of the polymorphic protein were calculated from the phenotype distributions within a sample of 56 unrelated individuals from Southern Germany. Gene frequencies of the common alleles ranged between 0.991 and 0.518. To discuss the suitability of the two-dimensional polymorphisms for paternity testing the theoretical exclusion probabilities were assessed for seven polymorphic proteins observed in our population sample, the six polymorphisms with two alleles described here and a further polymorphism (p75) with six alleles. For five proteins (p23, p40, p42, p66 and p75) we found sufficiently high values for the theoretical exclusion probabilities, ranging from 10% to 34%.(ABSTRACT TRUNCATED AT 250 WORDS)

The group specific component/vitamin D binding protein (GC/DBP) system in the analysis of disputed paternities.

The group-specific component (GC) was discovered in 1959, and in the same year a vitamin D binding protein (DBP) in human plasma was found; however, their identity was established as late as 1975. In the GC/DBP system three common alleles, GC*1F, GC*1S, and GC*2, determine six GC phenotypes: 1F, 1S, 2, 1F-1S, 2-1F and 2-1S, these common alleles having been found in all human populations studied. In addition, more than 120 GC variants have been discovered, with varying frequencies in different populations. The distribution of the common GC phenotypes and the presence of rare GC variant phenotypes render the GC/DBP system useful for the analysis of disputed paternities.

Genetic polymorphism of a lymphocyte protein (p75) with six different alleles studied by two-dimensional electrophoresis: qualitative and quantitative data.

The genetic polymorphism of a human lymphocyte protein (p75) was studied by two-dimensional electrophoresis within 17 families and, in addition, 22 unrelated individuals from Southern Germany, resulting in a total of approximately 100 individuals. The cytosolic and membrane proteins from cell lysates of phytohemagglutinin stimulated and [3H]leucine labeled lymphocytes were separated by two-dimensional electrophoresis. The p75 protein with an approximate molecular weight of 75,000 occurred in six variants with slightly different isoelectric points and/or apparent molecular weights. Three common variants (a, b, and c) and three rare variants (d, e and f) could be distinguished. Among the approximately 100 individuals studied we observed 15 different phenotypes, three homozygous (p75-a, -b, -c) and 12 heterozygous (p75-ab, -ac, -bc, -ad, -ae, -be, -bf, -cd, -ce, -cf, -de, -df) phenotypes. The genetics of the p75 protein variations was ascertained by family studies and quantitative computer analysis. We were able to show a Mendelian mode of inheritance of the variants within the families and a gene-dosage dependence of the protein spots in homozygous and heterozygous phenotypes. The data allowed us to assume a polymorphic protein p75 determined by six alleles on a autosomal gene locus. The allele frequencies were calculated from the phenotype distribution within 56 unrelated individuals. The gene frequencies of the three common alleles ranged between 0.38 and 0.22 and the gene frequencies of the three rare alleles ranged between 0.01 and 0.07.