Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 4. Genetic polymorphism of cytosol 100k polypeptide

Summary We describe a genetic polymorphism of a human cellular polypeptide with mol. wt. 100,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, and 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is one of the abundant polypeptides of B-lymphoblastoid cells, T-lymphoblastoid cells, fibroblasts, and HeLa cells. The data indicate that the cytosol polypeptide with mol.wt. 100,000 shows a genetic polymorphism determined by aew autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 100k polypeptide (C100k polypeptide) and C100P, respectively. In a Japanese population, the gene frequencies of C100P ¹ and C100P ² were 0.907 and 0.093, respectively.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Three different electrophoretic types (1-1, 2-1 and 2-2) of a human cellular polypeptide with molecular weight of 31000 have been identified by the analysis of PHA-stimulated peripheral blood lymphocyte proteins using high resolution two-dimensional gel electrophoresis. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The 31k polypeptide appears to be present as a monomer in the cytosol in a wide range of different cell types, including permanent lymphoblastoid cell lines, fibroblasts and HeLa cells. In an individual with the 31k polypeptide type 2-2, the phenotypes of adenosine deaminase and uridine monophosphate kinase were both type 1. These data indicate that the 31K polypeptide is a new polymorphic protein encoded by a new autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 31k polypeptide (C31k polypeptide) and C31P, respectively. In a Japanese population, the gene frequencies of C31P ¹ and C31P ² were 0.940 and 0.060, respectively. The C31k polypeptide type 2-2 appears to be a molecular weight variant as well as a charge variant.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 2. Genetic polymorphism of lymphocyte cytosol 64K polypeptide

Summary We describe a genetic polymorphism of human lymphocyte cytosol major polypeptide with mol. wt. 64,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is predominent in peripheral blood lymphocytes, B-lymphoblastoid cells, T-lymphoblastoid cells, lymph node, and spleen. The polypeptide has not been detected in HeLa cells, fibroblasts, erythrocytes, serum, and cerebrum. Traces of the polypeptide exist in liver, kidney, and skeletal muscle. It is proposed that the polypeptide and its locus be temporarily designated lymphocyte cytosol 64K polypeptide (LC64K polypeptide) andLC64P, respectively. In a Japanese population, the gene frequencies ofLC64P ¹ andLC64P ² were 0.936 and 0.064, respectively. The data suggest thatLC64P is a new locus, product of which shows genetic polymorphism and is associated with the function and/or the structure of lymphocytes.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis

Summary We describe a genetic polymorphism of cytosol polypeptide with mol. wt. of 38,000 detected in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes by two-dimensional gel electrophoresis. Three different electrophoretic phenotypes (type 1-1, 2-1, 2-2) of the polypeptide have been identified in a Japanese population. Family and population studies indicate that three phenotypes are determined by two common alleles at a single autosomal locus. Since the polypeptide is mainly present in cytosol of cells, we propose that the polypeptide be temporarily designated as cytosol polypeptide with mol. wt. of 38,000 (CP 38) and that the gene for CP 38 be designated as CP 38. The gene frequencies of two common alleles (CP 38 ¹ and CP 38 ²) are 0.899 and 0.101, respectively, in a Japanese population. The data on gel filtration of cytosol proteins on a Sephadex G-100 column suggest that CP 38 exists as a dimer in the cytosol. CP 38 was observed in the wide range of different cells, including B-lymphoblastoid cells, adult skin fibroblasts, HeLa cells, and erythrocytes. In 11 out of 72 individuals, the phenotypes of CP 38 were different from those of adenosine deaminase which is similar to CP 38 in subunit size, cell distribution, and allele frequencies. These data indicate that CP 38 is a new polymorphic polypeptide encoded by an autosomal locus.     

Disruption of the quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase by Triton X-100.

(Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. In the presence of Triton X-100 both polypeptides react to form high molecular weight aggregates with apparent molecular weights of 168,000, 200,000 and 260,000. These aggregates arise as a result of disulfide bond formation which results from the autooxidation of sulfhydryl groups on the two polypeptides of NaK ATPase. These data are discussed in light of studies aimed at determining the size and subunit structure of membrane proteins with Triton X-100.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis

Summary We describe a genetic polymorphism of cytosol polypeptide with mol.wt. of 20,000 detected in lymphocytes the arythrocytes by two-dimensional gel electrophoresis. Three different electrophoretic phenotypes (type 1-1, 2-1, and 2-2) of the polypeptide have been identified in a Japanese population. Family studies indicate that the phenotypes are determined by two common alleles at a single autosomal locus. The polypeptide is present in the cytosol of various kinds of cells and is abundant in erythrocytes. The data on a gel filtration of the erythrocyte cytosol proteins on a Sephadex G-100 column suggest that the polypeptide exists as a dimer in cells. In nine out of 79 individuals, the phenotypes of the polypeptide were different from those of glyoxalase 1 (GLO1) which has similar properties in subunit size, cell distribution, and allele frequencies. These date indicate that the polypeptide with mol. wt. of 20,000 is a new polymorphic cellular polypeptide. We propose that the polypeptide be temporarily designated as cytosol polypeptide with mol. wt. of 20,000 (CP20) and that the gene for CP20 be designated as CP20. The gene frequencies of two common alleles (CP20 ¹ and CP20 ²) are 0.955 and 0.045, respectively, in a Japanese population.     

Study of the structural polypeptides of Chilo suppressalis iridescent virus (Iridovirus type 6)]

We report a procedure for the purification of Chilo iridescent virus (Iridovirus type 6), an evaluation of the purification procedure, and the results of analyses of the virion proteins by acrylamide gel electrophoresis. Purity was evaluated in three ways, i.e., by analysis of purified virions from artificial mixtures of infected and labeled uninfected larvae, electrophoresis at neutral pH, and electron-microscopic examination. Analysis of the polypeptides of purified CIV gave the following results: (i) after solubilization with SDS-B-mercaptoethanol, 16 polypeptides could be resolved in Coomassie brillant blue-stained electrophoretograms with molecular weights ranging from 18,000 to 115,000; (ii) after solubilization with SDS-urea, 26 polypeptides could be resolved with molecular weights ranging from 10,000 to 230,000 daltons.     

Genetic polymorphism of complement factor H in cattle

Bovine factor H was found to be polymorphic by the combined techniques of SDS-polyacrylamide electrophoresis of bovine plasma and immunoblotting. Three phenotypes (S, SF, F) were identified in a sample population of 149 cattle. Variant S and F differed by an apparent molecular weight of 5000 daltons. Family studies demonstrated Mendelian segregation of variants S and F. The data indicate that these genetic variants of bovine factor H are encoded by two codominant alleles at a single autosomal locus.     

Structural Proteins of Simian Virus 40: Phosphoproteins

All five structural polypeptides of infectious simian virus 40 grown in African green monkey kidney cells were found to be phosphorylated. The polypeptides with the largest and smallest molecular weights are phosphorylated to a somewhat lower extent than the other polypeptides. The protein moiety of "empty" virus, which is essentially devoid of deoxyribonucleic acid, exhibited a degree of phosphorylation similar to that of infectious virus. In the major polypeptide (molecular weight: 49,000), the phosphate appears to be bound to the seryl or threonyl residues, or both. The nature of the phosphate-polypeptide bond in the other viral polypeptides remains obscure.     

Characterization of chloroplast thylakoid polypeptides in the 32-kDa region: polypeptide extraction and protein phosphorylation affect binding of photosystem II-directed herbicides

In order to distinguish between two photosystem II proteins with apparent molecular weights of about 32 kDa, mild extraction procedures were used to remove several thylakoid membrane components. A 32-kDa protein that stained intensely with Coomassie brilliant blue could be extracted from the thylakoid membranes without removing the 32-kDa herbicide receptor protein, which stained poorly with Coomassie brilliant blue. The nonextracted protein was readily detectable after in vivo polypeptide labeling with [35S]methionine or after in vitro covalent tagging with [14C]azidoatrazine. The procedures used to extract the intensely stained, 32-kDa polypeptide resulted in changes in herbicide-binding characteristics, presumably due to conformational changes in the herbicide-binding environment. Alterations of membrane surface charge by protein phosphorylation also influenced herbicide binding.     

Isolation and characterization of surface antigens from Schistosoma mansoni. I. Evaluation of techniques for radioisotope labeling of surface proteins from adult worms

Radiolabeled surface proteins of adult Schistosoma mansoni were prepared by in vitro labeling of whole worms, and by labeling freeze-thaw surface membrane extracts. Incorporation of 125I into surface proteins was attempted using the lactoperoxidase, chloramine-T, iodosulfanilic acid, and Bolton-Hunter methods. Radiolabeling of whole worms with lactoperoxidase, chloramine-T and iodosulfanilic acid yielded a single protein peak (mol wt greater than 100,000) on SDS-PAGE, and showed considerable incorporation of label in the lipid fraction. Bolton-Hunter labeling of whole worms yielded four major peaks with molecular weights of 100,000, 60,000, 30,000 and 21,000, and minor peaks with molecular weights of 26,000, 36,000, 43,000, 68,000 and 78,000; three of the four major peaks corresponded to prominent bands in Coomassie blue-stained gels. Although carbohydrate-labeling techniques were not successful, a single carbohydrate band, molecular weight greater than 100,000, was detected was PAS staining. Radiolabeling of freeze-thaw extracts yielded results similar to those obtained with whole worms. Electron microscopy revealed the tegument to be left intact and undamaged after labeling with the Bolton-Hunter reagent.     

Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis

A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Sch?gger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.     

Evidence for transferrin polymorphism in Spanish wild rabbits

Serum samples from 412 Spanish wild rabbits were analysed by starch and polyacrylamide gel electrophoresis. Three different transferrin (Tf) phenotypes (A, AB and B) were observed by both methods. The occurrence of two codominant alleles (TfA and TfB with frequencies of 0.89 and 0.11 respectively) at an autosomal locus (Tf) was supported by the population data on genetic equilibrium. Electrophoretic mobility differences between the Tf variants A and B could not be explained by differences in sialic acid or iron contents. Each of the two Tf variants were shown to have two sialic acid residues by neuraminidase treatment. These variants had similar affinities for iron, and iron binding did not lead to the conversion of one variant into the other.     

Crystallization of amino-terminal domains and domain fragments of variant surface glycoproteins from Trypanosoma brucei brucei

Crystals were produced from variant surface glycoproteins (VSG) of Trypanosoma brucei brucei antigenic variants MITat 1.2, 1.6, and ILTat 1.22, 1.23, 1.24, 1.25, and 1.26. Purified VSGs had molecular weights from 60,000 to 68,000 on sodium dodecyl sulfate-polyacrylamide gels, whereas the crystals obtained were composed of polypeptides of approximate Mr 40,000-50,000. Amino-terminal amino acid sequences determined from the crystallized VSGs were identical to sequences obtained from the respective intact proteins, indicating that the crystals contained VSG amino-terminal fragments. Crystallization conditions and lattice dimensions of the crystals are given.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Characterization of a 28-kD polymorphic polypeptide detected by two-dimensional electrophoresis of human platelets.

A genetic polymorphism of a human platelet polypeptide with a molecular weight of 28 kD detected by two-dimensional electrophoresis was investigated in family and population studies, and cell distribution. The 28-kD polypeptide showed autosomal codominant inheritance of two alleles. The gene frequencies of the two alleles were 0.925 and 0.075, respectively. The 28-kD polypeptide was observed in lymphocytes, neutrophils, eosinophils and monocytes, in addition to platelets. This polypeptide showed good reproducibility in electrophoresis, and appears to be useful as a genetic marker of the human genome in gene mapping and pedigree analysis.     

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphorylation state, glycosylation state, copy number, isoelectric point, and molecular weight.     

Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2^v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome 11. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the S-2^v variant polypeptide is lower by approximately 62,500 than that of the S-2 polypeptide. Amino acid analysis indicates that the two sericin polypeptides have similar compositions. These results are consistent with the idea that the variant allele arose by deletion within the S-2 coding sequence in the Src-2 gene locus as the result of unequal recombination.     

Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Human chromosome 11 carries at least four genes controlling expression of cell-surface antigens

We have mapped two new genes to chromosome 11 which control the cell-surface expression of two distinct antigens defined by monoclonal antibodies. One of the antigens has a general tissue distribution and is associated with a molecular complex of two polypeptides of 80,000 dalton and 40,000 dalton molecular weight. The second antigen has a restricted tissue distribution and is carried on a polypeptide of 100,000 daltons. We have used a combination of genetic and biochemical techniques to demonstrate that these new markers are distinct from the antigens defined by the monoclonal antibodies F10.44.2 and W6/34 which are also encoded by genes on chromosome 11. It is concluded that human chromosome 11 carries at least four distinct genes controlling cell-surface antigen expression.