scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Abstract Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Motility and the Polar Flagellum Are Required for Aeromonas caviae Adherence to HEp-2 Cells

2001 ◽  
Vol 69 (7) ◽  
pp. 4257-4267 ◽  
Author(s):  
Ali A. Rabaan ◽  
Ioannis Gryllos ◽  
Juan M. Tomás ◽  
Jonathan G. Shaw
Keyword(s):  
Epithelial Cells ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kanamycin Resistance ◽  
Polar Flagellum ◽  
Bacterial Cells ◽  
Aeromonas Caviae ◽  
Human Epithelial Cells

ABSTRACT Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of A. caviae to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the A. caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH, and flaJ. Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing, mutant analysis, and Western blotting demonstrated that A. caviae has a complex flagellum filament composed of two flagellin subunits encoded by flaAand flaB. The predicted molecular mass of both flagellins was ∼31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was ∼35,500 Da. This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays. Single mutations in either flaA orflaB did not result in loss of flagella but did result in decreased motility and adherence by approximately 50%. Mutation offlaH, flaJ, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence. However, mutation of flaG did not affect motility but did significantly reduce the level of adherence. Centrifugation of the flagellate mutants (flaA, flaB, and flaG) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (flaH, flaJ, and flaA flaB) increased adherence slightly. We conclude that maximum adherence of A. caviae to human epithelial cells in vitro requires motility and optimal flagellar function.

scholarly journals Defensin-rich dense granules of human neutrophils

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 757-765 ◽  
Author(s):  
WG Rice ◽  
T Ganz ◽  
JM Jr Kinkade ◽  
ME Selsted ◽  
RI Lehrer ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Human Neutrophils ◽  
Total Acid ◽  
Density Fractions ◽  
Dense Granules ◽  
Blood Neutrophils

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

Characterization of a rat adrenocortical inner zone-specific antigen and identification of its putative precursor

1992 ◽  
Vol 9 (2) ◽  
pp. 95-102 ◽  
Author(s):  
S. Barker ◽  
S. M. Laird ◽  
M. M. Ho ◽  
G. P. Vinson ◽  
J. P. Hinson
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Antigen ◽  
Kidney Tissue ◽  
Immunoblot Analysis ◽  
Fresh Tissue ◽  
Mitochondrial Fractions ◽  
Doc Production

ABSTRACT We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization. Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30 000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. Invivo treatment with ACTH (100 μg/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro. In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60 000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 μmol/l) or dibutyryl-cAMP (100 μmol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2. As in the in-vivo treatments, the increase in IZAg1 was associated with a corresponding increase in corticosterone and 18-OH-DOC production. These findings suggest that the IZAb recognizes a protein (IZAg2) which occurs in unstimulated adrenal cells. On stimulation by steroidogenic agents, this protein becomes processed to yield a smaller protein (IZAg1) which is associated with enhanced adrenal steroidogenesis.

scholarly journals Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide

1982 ◽  
Vol 204 (1) ◽  
pp. 323-327 ◽  
Author(s):  
E M Danielsen ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aminopeptidase N ◽  
Single Chain ◽  
Polyadenylated Rna ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Translation Mixture

A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide.

scholarly journals Purification and Characterization of Turnip Mosaic Virus Helper Component Protein

1999 ◽  
Vol 89 (7) ◽  
pp. 564-567 ◽  
Author(s):  
R. Y. Wang ◽  
T. P. Pirone
Keyword(s):  
Molecular Mass ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Turnip Mosaic Virus ◽  
Biologically Active ◽  
Helper Component ◽  
Pass Through

The helper component (HC) protein of turnip mosaic virus (TuMV) was concentrated by differential centrifugation followed by ammonium sulfate precipitation. The partially purified HC was then loaded onto a Ni2+-resin column that bound the HC; a histidine tag was not required for binding. The HC eluted from the column migrated as a band of about 50 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In its native state, the HC did not pass through an ultrafiltration membrane with a molecular mass cutoff of 100 kDa, which suggested that the HC is in a multimeric form when it is biologically active. The molecular mass of the multimeric form was determined by gel filtration to be approximately 145 kDa. Purified HC retained its activity for several months at -20°C. Using a protein blotting-overlay protocol, purified HC interacted in vitro with an aphid-transmissible TuMV isolate, but not with a non-aphid-transmissible isolate.

Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum

1991 ◽  
Vol 260 (6) ◽  
pp. R1168-R1175
Author(s):  
L. Bosca ◽  
K. B. Storey
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Busycon Canaliculatum ◽  
Native Molecular Mass ◽  
Dependent Protein

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

scholarly journals Glycoprotein IIb Leu214Pro Mutation Produces Glanzmann Thrombasthenia With Both Quantitative and Qualitative Abnormalities in GPIIb/IIIa

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1562-1571 ◽  
Author(s):  
Christine M. Grimaldi ◽  
Fangping Chen ◽  
Changhong Wu ◽  
Harvey J. Weiss ◽  
Barry S. Coller ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Homologous Region ◽  
Glanzmann Thrombasthenia ◽  
Binding Surface ◽  
Mutant Receptors

Abstract Glanzmann thrombasthenia is an inherited bleeding disorder due to a functional reduction or absence of platelet GPIIb/IIIa (αIIbβ3) integrin receptors. Based on a prolonged bleeding time and absence of platelet aggregation in response to physiologic agonists, a 55-year-old white man was diagnosed as having Glanzmann thrombasthenia. The patient's platelet fibrinogen level was ≈5% of normal. As judged by complex-dependent monoclonal antibody (MoAb) binding, surface expression of platelet GPIIb/IIIa receptors was less than 5.5% of normal, whereas the binding of an anti-GPIIIa specific MoAb (7H2) was ≈12% of normal. Immunoblot analysis of the patient's platelet lysates showed ≈35% of normal levels of GPIIIa, ≈30% of normal levels of GPIIb, and an abnormally migrating fragment of GPIIb. Biotinylation of the surface proteins on the patient's platelets followed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed only GPIIb and GPIIIa subunits of normal size. Surface expression of platelet αvβ3 receptors was 192% of normal, suggesting that the patient's' defect was in GPIIb. Sequence analysis of the patient's GPIIb cDNA identified a T to C transition at nucleotide 643, predicting a Leu214Pro substitution. Direct sequencing of GPIIb exon 6 indicated that the patient is homozygous for the mutation. The nature of the Leu214Pro mutation was analyzed by expression in Chinese hamster ovary (CHO) cells. As judged by subunit-specific MoAb binding, surface expression of mutant receptors was ≈60% of normal, but these receptors were not recognized by the complex-dependent monoclonal antibodies, 10E5 and 7E3. In addition, mutant receptors pretreated with the ligand-induced binding site MoAb AP5 were not recognized by the activation-dependent MoAb PAC-1 and mutant expressing CHO cells did not adhere to immobilized fibrinogen. These data suggest that the Leu214Pro mutation in GPIIb disrupts the structural conformation, and either directly or indirectly, the ligand binding properties of the heterodimeric complex. This is in accord with studies from other integrins that have implicated a β-turn in a homologous region as important in ligand binding. Thus, the Leu214Pro mutation appears to produce the Glanzmann thrombasthenia phenotype by both qualitative and quantitative abnormalities. In addition, the mutation appears to confer susceptibility of the GPIIb subunit to proteolysis.

scholarly journals Rh(D) antigen expression and isolation of a new Rh(D) cDNA isoform in human erythroleukemic K562 cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1975-1981 ◽  
Author(s):  
K Suyama ◽  
R Lunn ◽  
S Haller ◽  
J Goldstein
Keyword(s):  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Premature Stop Codon ◽  
K562 Cells ◽  
Antigen Expression ◽  
Reading Frame ◽  
Rabbit Reticulocyte Lysate System ◽  
D Antigen

Abstract Human erythroleukemic K562 cells are known to have several erythroid properties. K562 cells possess Rh mRNAs, but expression of Rh proteins has not previously been reported. We immunoprecipitated Rh protein from K562 cell lysate using rabbit anti-Rh and detected Rh(D) antigens on K562 cells using fluorescence-activated cell sorting (FACS). These results suggest that K562 cells will be useful as an expression model for most Rh antigens. We also cloned a new Rh(D) cDNA isoform (RhK562- II), from a K562 cDNA library using polymerase chain reaction (PCR) with 5′ and 3′ end oligonucleotides of the published Rh(e/E) antigen encoding cDNA sequence as primers. Sequence analysis showed that RhK562- II is composed of 951 nucleotides (316 amino acids), identical to the first 939 nucleotides (exons 1 to 6) of one of the Rh(D) cDNAs (RhXIII), except for nucleotide 654 (C-->XG exchange). However, this exchange is the same as that of another published Rh(D) cDNA (RhPII cDNA). RhK562-II is deprived of exons 7 and 8 (nucleotides 940 to 1,153), followed by an identical sequence up to the 3′ end of the open- reading frame of the RhXIII cDNA, which causes a frame-shift mutation and produces a premature stop codon. In vitro expression of RhK562-II using the transcription and translation rabbit reticulocyte lysate system produced two major Rh-related proteins (30 kD and 25 kD), which were immunoprecipitated by rabbit polyclonal anti-Rh and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Sign in / Sign up

Export Citation Format

Share Document