Hartwig Cleve

Solubilization of human erythrocyte membrane glycoproteins and separation of the MN glycoprotein from a glycoprotein with I, S, and A activity☆

Abstract Human erythrocyte membrane glycoproteins are recovered in high yields in the aqueous phase after extraction of the membranes with a mixture of chloroform-methanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase reveals the presence of three main glycoprotein components, glycoproteins I, II, and III, and one additional periodic acid-Schiff positive component, without contamination by other membrane proteins. Their apparent molecular weights correspond to 58 000, 37 000, 24 000, and 50 000, respectively. The blood group antigens I, Ss, and MN are recovered in the aqueous phase in high yields and with marked increase in their specific activities. The aqueous phase also contains moderate amounts of A and B and small amounts of H antigenic activities. Further separation of the glycoproteins of the aqueous phase was achieved by gel filtration on Sephadex G-100 columns in the presence of 1% dodecyl sulfate. The M and N antigenic activities were found in glycoprotein I. N activity as tested with a rabbit antiserum was also observed in glycoprotein III. The highest activities for A, I, and S antigens were found in glycoprotein III. The chloroform-methanol phase does not contain glycoproteins; only the blood group antigens A, B, and H are found in this phase. The interphase, which contains most of the membrane proteins except the main glycoproteins, shows only weak ABH, I, Ss, and MN activities. The data indicate that the major glycoprotein of the human erythrocyte membrane, glycoprotein I, is the so-called MN glycoprotein, and that the I and S antigenic activities are associated with glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 000. Glycoprotein III is also associated with the A antigenic activity.

Solubilization and comparative analysis of mammalian erythrocyte membrane glycoproteins

Abstract SDS-acrylamide gel electrophoresis reveals the presence of a major glycoprotein in human, ox, horse, swine, and sheep erythrocyte membranes. Their apparent molecular weights differ among the various species. The major glycoproteins and additional minor glycoproteins can be recovered in the aqueous phase after extraction of the membranes with a mixture of CHCl3-CH3OH at room temperature. The extracted glycoproteins remain in the supernatant after centrifugation at 100,000 g for 60 min. in Tris-EDTA buffer. These glycoprotein preparations possess high activities for the phaseolus vulgaris phytohemagglutinin receptor, the infectious mononucleosis heterophile antigen, and the myxovirus receptor. Their specific activities and yields differ markedly from species to species.

Genetic studies on the deficiency of ß2-glycoprotein I of human serum

Summaryβ2-glycoprotein I concentrations have been determined in human sera by the radial immunodiffusion technique. In 260 healthy individuals a bimodal distribution was observed. In 94% of sera concentrations ranged from 16 to 30 mg/100 ml with a mean value of 21.3±3.6 mg/100 ml. In 6% of sera lower concentrations were found: x=10.0±1.3 per 100 ml. The results of a study of 38 families with 79 children suggest that β2-glycoprotein I concentrations are controlled by a pair of autosomal co-dominant alleles called BgN and BgD. Individuals homozygous for the common allele BgN have levels between 16 and 30 mg/100 ml, heterozygous individuals have lower concentrations of 6 to 14 mg/100 ml. Individuals homozygous for the rate allele BgD have deficiency of β2-glycoprotein I. The results in one family, however, did not conform to this simple genetic hypothesis. The concentration of β2-glycoprotein I in serum is also influenced to some extent by non-genetic factors such as sex, age, and various pathologic conditions. The biological function of this glycoprotein is still unknown.

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.

Further family studies on the genetic control of ?2-glycoprotein I concentration in human serum

Summary88 families with a total of 213 children were examined for β2-glycoprotein I serum concentrations. In 74 families parents and children had normal concentrations. In 9 families one of the parents and approximately half of the children had intermediate concentrations. These individuals are presumably heterozygous for a deficiency gene BgD. In these families β2-glycoprotein I concentration appears to be controlled by a pair of alleles which are transmitted as autosomal co-dominants. The results in 5 families did not conform to this genetic hypothesis, since children with an intermediate concentration of β2-glycoprotein I were found whose both parents had a normal concentration of this protein. Non-genetic factors may be responsible for phenotypic variations in the different genetic types.

Properties of the water dissolved membrane proteins of human erythrocytes

Abstract Human erythrocyte membrane proteins were dissolved in water employing a modification of the method of Mazia and Ruby. About 40% of ghost proteins were recovered in distilled water. More than 85% of the water dissolved proteins remained in the supernatant on sucrose density gradient ultracentrifugation, forming one major peak with a sedimentation rate of approx. 3 S. All proteins in the supernatant penetrated a 5% acrylamide gel on electrophoresis with a 5 mM Tris-38 mM glycine buffer system, suggesting that most of the water dissolved proteins are highly dissociated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed about 15 protein components or polypeptide chains in the water dissolved protein fraction. The dissolution of ghost proteins into water is partially selective. Moderate amounts of virus receptor substance and complement inhibitor were detected in the water dissolved fraction while the membrane bound enzymes acetylcholinesterase and (Na + −K + )-dependent ATPase were almost absent. The water dissolved proteins aggregated readily in the presence of low concentrations of electrolytes. This tendency for aggregation prevented further fractionation of the water dissolved proteins by gel filtration or by preparative zone electrophoresis.

Rapid and simple diagnosis of the two common α1-proteinase inhibitor deficiency alleles Pi*Z and Pi*S by DNA analysis

We describe a simple DNA-based method to assign the two common alpha 1-proteinase inhibitor (alpha 1-antitrypsin) deficiency alleles in the Pi-system (Pi*Z and Pi*S). Two sets of mutated primers are used in the polymerase chain reaction (PCR), followed by a restriction enzyme digest of the products. The mutated forward primers create a Taq I site only if the wildtype alleles (mostly M or subtypes) are present and not in the presence of the Pi*Z or Pi*S alleles. The reverse primers are mutated for an invariant Taq I site which serves as an internal control site in order to assure the completion of the restriction enzyme digest. The digested PCR products can be clearly resolved by 2.5% MetaPhore-agarose gel electrophoresis. This simple PCR probing of the most common alpha 1-antiproteinase deficiency alleles can be routinely applied either to samples showing quantitatively decreased alpha 1-antiproteinase values in serum or to blood spots of Guthrie cards used for mass screening purposes. In addition, this method may provide the opportunity for a simple, rapid, and reliable prenatal diagnosis of alpha 1-antiproteinase deficiency in special cases.

Die Verteilung der Gc-Typen und Gc-Allele bei Kranken mit Diabetes mellitus und chronischer Polyarthritis

The distribution of Gc-phenotypes ~nd Gc-alleles has been determined in 104 patients with rheumatoid arthritis and 285 patients with diabetes mel]itus. The distribution was compared with a control of 1879 apparently healthy individuals from the same region. The gene frequencies in the control sample were Gc 1 = 0.7315 and Gc ~ = 0.2685. In patients with rheumatoid arthritis a Gel-frequency of 0.7933 was found, also in patients with diabetes Ge 1 was with 0.7719 more frequent than in the normal control. These differences were statistically not significant. The distribution in the three groups was in agreement with the distribution expected at population equilibrium. Subdivision of the diabetes sample according to the severity of the disease, to late onset of the diabetes, or to the presence or absence of retinopathia diabetiea revealed no statistically significant differences. In diabetes patients with moderate or severe arteriosclerosis of the peripheral vessels Ge 1 was also fomld to be more frequent. However, in the age group below 60 years the increased frequency of the Gc 1 allele in thesepatients differed significantly from the distribution in a normal control. Analysis of further samples of patients with different types of arterio-sclerosis with and without diabetes is suggested.

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.

Biochemical characterization of the β-chain variant haptoglobin Marburg

Summaryβ-chains were isolated from two individuals heterozygous for the β-chain mutant haptoglobin Marburg. Total amino acid composition and tryptic peptides were compared with β-chains from common haptoglobin types. ββMb chain preparations are characterized by the presence of β-chains with an atypical electrophoretic migration rate and by at least three, possibly four additional peptides in their tryptic digests. It is probable that haptoglobin Marburg is the result of an mutational event other than a single base substitution.