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.     

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: 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 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.     

Gene frequencies of S-adenosylhomocysteine hydrolase (SAHH) in a Japanese population

Summary Genetic polymorphism of S-adenosylhomocysteine hydrolase (SAHH) was investigated in a total of 214 red blood cell samples from unrelated Japanese using the starch gel electrophoresis and the enzyme-specific staining procedures. Three common phenotypes were observed which corresponded to SAHH 1, SAHH 2-1, and SAHH 2, controlled by two alleles, SAHH^*1 and SAHH^*2. The estimated gene frequencies of SAHH^*1 and SAHH^*2 in Japanese were 0.953 and 0.047, respectively. This result was not different from European samples reported by Bissbort et al. (1983).     

Genetic polymorphism of human urine deoxyribonuclease I

Summary A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes —DNASE1 1, 1–2, and 2 — and the other four rare phenotypes — DNASE1 1–3, 2–3, 2–4, and 3–4 — represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 ^* 1, ^* 2, ^* 3, and ^* 4. The frequencies of the DNASE1 ^* 1, DNASE1 ^* 2, DNASE1 ^* 3, and DNASE1 ^* 4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.     

Isolation of a novel rat testicular metalloprotein binding cadmium and zinc

Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration of¹⁰⁹Cd- or⁶⁵Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of¹⁰⁹Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/ mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10±4 kD/pI 6.3) Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (<60 kD) protein in rat testic cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of¹⁰⁹Cd- and⁶⁵Zn-labeled cytosol demonstrated that¹⁰⁹Cd and⁶⁵Zn cochromatography with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.     

A new method for FXIIIA genetic variants determination using isoelectric focusing in 1 M urea

Summary A new method for separating genetic variants of the A subunit of human coagulation factor XIII using ultrathin layer polyacrylamide gel isoelectric focusing in 1M urea followed by immunoblotting is described. The pattern obtained by this method differs from that reported previously: Three sets of unrelated band patterns are observed and can be explained by the existence of two additional gene loci, designated FXIIIA2 and FXIIIA3, besides the previously reported FXIIIA locus, now renamed FXIIIA1. The FXIIIA2 locus is polymorphic and shows three commonly occurring phenotypes, FXIIIA2 1, FXIIIA2 2-I, and FXIIIA2 2. These are determined by two common alleles, FXIIIA2^*1 and FXIIIA2^*2, with respective frequencies of 0.7965 and 0.2035 in the Japanese population. The studied population conforms to a Hardy-Weinberg equilibrium, and family data confirmed autosomal codominant transmission. The FXIIIA3 locus is monomorphic.     

Genetic polymorphism of the seventh component of complement in a Japanese population

Summary Genetic polymorphism of C7 in a Japanese population has been described, using polyacrylamide gel isoelectric focusing electrophoresis followed by an electrophoretic blotting technique. Phenotypes of C7 were classified into six common patterns, and observed phenotypes were produced by autosomal codominant at a single locus with three alleles. Three common alleles, designated C7^*B, C7^*M and C7^*A, were found, and gene frequencies calculated from 494 individuals showed C7^*B=0.858, C7^*M=0.096 and C7^*A=0.046, respectively. It is noteworthy that both C7^*M and C7^*A have polymorphic frequencies in the Japanese population. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. Results indicate that the electrophoretic blotting technique, which has high specificity and sensitivity, is applicable in the study of heterogeneity of protein antigens.     

Patterns of proteins synthesized by non-proliferating murine L cells

When L cells were plated at high density (2 × 10⁵/cm²), proliferation ceased within 3 days, but the cells remained viable and capable of reinitiating DNA synthesis for at least 16 days. SDS-polyacrylamide gel electrophoresis of [³⁵S]methionine labeled polypeptides at various intervals after plating revealed distinct changes in the relative abundance of major cellular proteins beginning 9 days after DNA synthesis ceased. An 84 000 mol. wt polypeptide increased markedly in amount while a polypeptide of 94 000 mol. wt disappeared. Autoradiograms following pulse-labeling showed that these changes were due to increased synthesis of the 84 000 mol. wt polypeptide and decreased synthesis of the 94 000 mol. wt polypeptide. Increased synthesis of a 109 000 mol. wt polypeptide occurred without a concomitant change in its relative abundance. These alterations in the pattern of proteins synthesized revert to normal after feeding with serum-free medium even though DNA synthesis does not resume. Therefore, it appears that even though no abrupt changes in the synthesis of major cellular proteins occurred upon cessation of proliferation, eventually a distinct adaptive pattern of protein synthesis develops in response to the changing culture conditions.     

Evidence for two additional common alleles at the PGM1 locus (Phosphoglucomutase-E.C.:2.7.5.1)

Summary Lysates of erythrocytes, leukocytes, lymphocytes, and extracts of sperms were investigated for the PGM₁ isozymes by three techniques: starch gel electrophoresis, high voltage thin-layer agarose gel electrophoresis, and thinlayer isoelectric focusing on polyacrylamide gel. On starch, only the well known common phenotypes 1, 2-1, and 2 were demonstrable. On agarose, different distances of the two main cathodal bands (a, b) among the phenotypes 2-1 were noted. Furthermore, on agarose, some types considered as homozygous on starch gel had a single, sharp banded pattern, while others were broad and blurred. Optimal separation was achieved by isoelectric focusing on polyacrylamide gel. In 291 leukolysates, 10 different phenotypes were identified. These are considered as gene products of 4 different common alleles at the PGM₁ locus as suggested by preliminary family investigations. In a random population from Hessen these four alleles, had the following frequencies: PGM ₁ ^a1 0.6186, PGM ₁ ^a2 0.1718, PGM ₁ ^a3 0.1426, and PGM ₁ ^a4 0.067. The preliminary designation a1, a2, a3 and a4 was chosen as the assumed polymorphism was demonstrated on acrylamide and agarose. The sum of the frequencies PGM ₁ ^a1 and PGM ₁ ^a3 (the gene products of which have apparently the same electrophoretic mobility on starch) is similar to the frequency of the old PGM ₁ ¹ allele (0.757) in Caucasoids, PGM ₁ ^a2 and PGM ₁ ^a4 have a frequency of 0.2388 corresponding with the frequency of the old allele PGM ₁ ² .     

Erythroid 5-aminolevulinate synthetase from trout (Salmo gairdneri R.)

A mitochondrial and cytosolic erythroid ALA-synthetase have been found in trout. The polypeptide existent in the cytosol is probably a precursor of the 90,000 mol. wt mitochondrial ALA-synthetase. The erythroid ALA-synthetase is about 20,000 mol. wt larger than the hepatic enzyme. The differences in mol. wt and catalytic properties between erythroid and hepatic enzyme support the existence of two forms of the ALA-synthetase in teleostei.     

Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting,immunological and biochemical characterization

Summary The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6–7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1–2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI ^*1 and PCI ^*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI ^*1 and PCI ^*2 alleles were 0.988 and 0.012, respectively.     

Genetic studies on free-ranging macaques of Cay Santiago. II. 6-phosphogluconate dehydrogenase and NADH-methemoglobin reductase (NADH-diaphorase).

For the population of 395 semi-free-ranging rhesus macaques (Macaca mulatta) that inhabited Cayo Santiago in 1976, 6-phosphogluconate dehydrogenase phenotypes of 378 animals were determined. Three phenotypes, controlled by two autosomal codominant alleles,PGD^A andPGD^B, were found by electrophoretic methods. The frequencies of the alleles are 0.898 and 0.102, respectively. The population, composed of five troops and peripheral males, is in Hardy-Weinberg equilibrium at this locus. The allele frequencies at the 6-phosphogluconate dehydrogenase locus in the population in 1976 were compared with frequencies in 1973; a statistically significant difference was found in one troop. The phenotypes of NADH-methemoglobin reductase (NADH-diaphorase) were determined electrophoretically for 372 animals. These phenotypes are probably the products of two autosomal codominant alleles,Dia¹ andDia², with frequencies of 0.786 and 0.214, respectively. The population is in equilibrium at this locus also. Tests of homogeneity at the dehydrogenase and reductase loci indicate that the allele frequencies are significantly different among the five troops in the population. Observed and expected phenotypic ratios in progeny were compared at the dehydrogenase and the reductase loci. The only significant deviation from expectation occurs among offspring of mothers heterozygous at the reductase locus. The observed distributions of alleles at the 6-phosphogluconate dehydrogenase locus and the NADH-methemoglobin reductase locus are probably the results of stochastic processes.     

Phenotype and gene frequencies of acid phosphatase (s-AcP) in the human parotid saliva

Summary Genetic polymorphism of the human parotid salivary acid phosphatase (s-AcP) in the Japanese population is described. The use of polyacrylamide gel isoelectric focusing electrophoresis with the pH range of 4.0–6.5 enabled us to discern three variant patterns controlled by two codominant alleles at the single autosomal locus. The two alleles were designated s-AcP: A and s-AcP:a, and the gene frequencies calculated from 183 Japanese subjects were s-AcP:A=0.2268±0.022, s-AcP:a=0.7732±0.022, respectively. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for gene duplication of ORM1 and genetic polymorphism of ORM2

Summary It has been demonstrated that the genetic polymorphism of human serum orosomucoid (ORM) is controlled by polymorphic ORM1 and monomorphic ORM2 loci. In this study a Japanese family was encountered in which several members had puzzling electrophoretic patterns consisting of four bands. The ORM patterns were due to the products of a duplicated ORM1 locus haplotype (ORM1 ^* 2·1) or the products of new variant alleles at the ORM2 locus. The ORM1 ^* 2·1 haplotype is very common in the Japanese population, occurring at an allele frequency of 0.16. The increased occurrence of ORM1 2-1 and the heterogeneity in band intensity among ORM1 2-1 phenotypes could be explained in terms of a duplicated gene ORM1 ^* 2·1. The ORM2 locus proved to be polymorphic, with six alleles in the Japanese population. Dedicated to Professor Dr. K. Nishigami on the occasion of his 60th birthday     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Genetic polymorphism of the complement C2 in Japanese

Summary Genetic polymorphism of the second component of human complement (C2) was investigated in 521 unrelated healthy adult Japanese using isoelectric focusing in polyacrylamide gel followed by a specific hemolytic overlay method. Besides the phenotypes reported previously (C, AC and BC), a relatively infrequent double-banded phenotype (tentatively named A'C) was observed. Moreover, a homozygous variant (A) and a heterozygous double variant (AB) were observed. The estimated frequencies for the common allele. C2 ² (=C2 ¹ ), and the variant alleles, C2 ^A , C2 ^B (=C2 ² ) and C2 ^A were 0.939, 0.034, 0.022, and 0.006, respectively.The results of further typing for HLA-A,-B,-C specificities indicated the presence of significant associations of C2 ^A with HLA-B15 and with A26, and of C2 ^B with HLA-Bw61. These findings support our previous observation that in Japanese there are allelic combinations showing linkage disequilibrium between C2 and HLA loci which are different from those in Caucasians, and that the C2 structural locus is more closely linked to HLA-B than to HLA-A.C2 hemolytic activities of each phenotypes were assayed. The mean activity of type AC sera was significantly higher than that of type C or type BC, while there were no differences in the activities among the types C, BC or A'C.Also presented are two pedigrees demonstrating the segregation of C2 with HLA alleles in which a homozygous C2A or C2B individual was observed.