Rapid Electrophoretic Mobility Research Articles

Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24

Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis-GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl-prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.

Molecular and cytological characterization of SspI-family repetitive sequence on the chicken W chromosome.

A genomic clone, pWS44, isolated from the chicken W chromosome-specific genomic library contained a partial (226-bp) sequence of a novel SspI-family repetitive sequence. A genomic clone, pWPRS09, containing a 508-bp SspI fragment (a repeating unit of the family) was subsequently obtained and sequenced. This 0.5-kb unit is tandemly repeated about 11,300 times. FISH to mitotic and lampbrush W chromosomes indicates that the SspI-family is located on the chromomere 6 between heterochromatic and distal non-heterochromatic regions on the short arm. The SspI-family sequence was proved to be a good positional marker in FISH mapping of active genes in the non-heterochromatic region on the lampbrush W chromosome. The presence of SspI-family repetitive sequence is limited to the genus Gallus (chickens and jungle fowls). The 0.5-kb repeating unit contains a 120-bp stretch of polypurine/polypyrimidine sequence (GGAGA repeats), shows no DNA curvature, and rapid electrophoretic mobility in 4% polyacrylamide gel at 4 degrees C. The SspI-family forms a relatively diffused chromatin structure in nuclei. These features are distinctly different from those of XhoI- and EcoRI-family sequences on the W chromosome. The total amount of non-repetitive DNA in the chicken W chromosome is estimated to be about 10 Mb.

CTG repeats associated with human genetic disease are inherently flexible

The lengthening of tracts of CTG, CGG and GAA triplet repeats during progression of a pedigree has been associated with more than 12 human genetic diseases, including fragile X syndrome, myotonic dystrophy and Friedreich’s ataxia. These repetitive sequence elements have the potential to form alternative DNA secondary structures that may contribute to their instability. The alternative DNA secondary structures may mediate errors during DNA replication, repair or recombination of the triplet repeat, leading to expansion. Here we show that DNA composed of pure CTG or CGG repeats exhibits anomalously fast mobility on polyacrylamide gels, confirming a previous observation for DNA containing CTG and CGG triplet repeats flanked by mixed sequence DNA. Moreover, we show that even short tracts of duplex CTG repeats have an unusual helix structure. CTG repeats reduce overall curvature associated with phased A-tract or GGCC curves, but alone they do not introduce curvature into DNA. The reduction in curvature of phased A-tracts by CTG repeats is similar to that afforded by an interspersed flexible region associated with a (TT)·(TT) mispair. CTG-containing DNAs exhibit a rapid rate of cyclization, consistent with a flexible helix. These results suggest that tracts of (CTG)·(CAG) repeats are inherently flexible. In addition, our results suggest that the unusual rapid electrophoretic mobility of CTG- or CGG-containing DNA may be a consequence of an extended flexible DNA chain.

Common G6PD Variant from Saudi Population and its Prevalence

Biochemical characterization of erythrocyte glucose-6-phosphate-dehydrogenase from 18 unrelated deficient Saudi Subjects from Al-Hassa and Al-Qatif areas of the Eastern Province was carried out according to WHO procedures. This had led to the identification of one genetically determined common variant, "G6PD Mediterranean". The overall prevalence rate of this deficiency in these areas was determined to be in excess of 42%. None of the subjects studied displayed any sign of favism, a condition usually associated with G6PD Mediterranean, which is characterized by a very low intracellular enzyme activity.

Anomalous Rapid Electrophoretic Mobility of DNA Containing Triplet Repeats Associated with Human Disease Genes

Eight human genetic diseases have been associated with the expansion of CTG or CGG triplet repeats. The molecular etiology behind expansion is unknown but may involve participation of an unusual DNA structure in replication, repair, or recombination. We show that DNA fragments containing CTG triplet repeats derived from the human myotonic dystrophy gene migrate up to 20% faster than expected in nondenaturing polyacrylamide gels, suggesting the presence of an unusual DNA helix structure within the CTG triplet repeats. The anomalous migration is dependent upon the number of triplet repeats, the length of the flanking DNA, and the percentage and temperature of the polyacrylamide. The effect could be reduced by the addition of actinomycin D. Applying a reptation model for electrophoresis, the results are consistent with a 20% increase in persistence length of the DNA. PCR products containing CTG or CGG repeats from the spinocerebellar ataxia type I gene (SCA1) or the fragile X FMR1 gene, respectively, also showed higher electrophoretic mobility. These are the first sequences of defined length for which a dramatic increase in mobility can be attributed to sequence-dependent structural elements in DNA.

Actobindin binds with high affinity to a covalently cross-linked actin dimer.

Actobindin, a 9.8-kDa protein purified from Acanthamoeba castellanii, contains two actin-binding sites that can simultaneously bind two actin monomers. However, actobindin inhibits actin polymerization to a greater extent than can be explained by its affinity for actin monomers (site-specific KD = 3.3 microM). This paradox would be resolved if actobindin could interfere with the nucleation phase of polymerization by using both binding sites to bind simultaneously to an actin oligomer because the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-linked actin dimer prepared from cross-linked F-actin binds to actobindin with high affinity (apparent KD = 11 nM) in accordance with theoretical predictions for simultaneous binding of two actin subunits per single actobindin and consistent with the hypothesis that actobindin might bind to native actin oligomers and prevent them from nucleating polymerization. Furthermore, the interaction with cross-linked dimer exhibits specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, only this isomeric dimer is produced when cross-linking reagents are added to actin undergoing polymerization in the presence of actobindin. Therefore, if actobindin inhibits polymerization by interacting with a native dimer whose conformation is similar to that of the cross-linked dimer with slower electrophoretic mobility, then actobindin must either block the cross-linking sites or convert the dimer to a different conformation.

Reactivity of cloned, expressed human FcγRIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of FcγRIII

We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.

Immunochemical behaviour of a tumour-associated antigen obtained from an AKR mouse lymphoma

A soluble tumour-associated antigen was prepared by homogenizing AKR lymphoma cells (L15) followed by treatment with acetone and glycine-HCl buffer pH 3. It was analyzed by immunoelectrophoresis, rocket electrophoresis in the presence of Concanavalin A and SDS-PAGE. It showed rapid electrophoretic mobility, and the major component had an estimated molecular weight of 70,000 daltons. The molecular composition of the antigen included a glycoprotein. Specific antibodies against this antigen were demonstrated in BALB/c mice in which the AKR lymphoma was conditioned to grow by introducing the L15 cells into a subcutaneously implanted glass cylinder. In this model antibodies were found in both tumour-bearing (progressor) and tumour-rejecting (regressor) animals.In vivo experiments showed that pretreatment of BALB/c mice with L15 acellular extracts, 10 days before tumour challenge, led to a significant increase in allogeneic tumour growth.A rabbit anti-tumour extract serum was prepared and was rendered tumour -specific by exhaustive absorption with normal AKR serum and tissues. It shared the same epitopic specificity with tumour progressor or regressor mouse sera since indirect immunofluorescence using L15 cells could be reciprocally inhibited.

Biochemical characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase).

Lysophospholipase from human eosinophils is a protein previously considered based upon antigenic, enzymatic, and electrophoretic similarities to be the single component of Charcot-Leyden crystals, which are formed in vivo in association with eosinophilic diseases. The identity of eosinophil lysophospholipase and solubilized Charcot-Leyden crystal protein is now established by biochemical criteria, and a basis for the ease of aggregation and crystallization of the protein is identified in its prominent hydrophobicity. Chromatographically purified enzyme and Charcot-Leyden crystal protein formed in vitro functioned as lysophospholipases with identical Michaelis constants (Km approximately equal to 22 microM) for the substrate lysopalmitoylphosphatidylcholine and had blocked amino-terminal residues and almost identical amino acid compositions. The propensity of lysophospholipase to aggregate was not due to extensive intermolecular disulfide bonding because it contained a single cysteine residue as assessed by amino acid analyses and incorporated 0.986 mol of p-chloromercuribenzoic acid/mol of native enzyme or 0.958 mol of iodoacetic acid/mol of reduced and denatured enzyme. By equilibrium dialysis, lysophospholipase bound 3.820 g of detergent/g of protein in 1% sodium dodecyl sulfate and 0.506 g of detergent/g of protein in 10 mM sodium deoxycholate. In addition, monomeric protein demonstrated enhanced binding of detergent as evidenced by its aberrantly rapid electrophoretic mobility in 1%, but not 0.1%, sodium dodecyl sulfate. The hydrophobic nature of this protein, which accounts for 10% of the protein of the eosinophil, may contribute to its unique propensity for crystallization in vivo.

Chemical and biological properties of lipopolysaccharide from a marine bacterium, Photobacterium phosphoreum PJ-1.

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ-1, were studied. This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2-keto-3-deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium. This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection: the early lethal action appeared within 30 min after injection of 250 micrograms or less, and the late lethal action occurred gradually after 16 hr at doses of 500 microgrms or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 micrograms per mouse and in only the late phase it was 500 micrograms. These results show that in spite of structural differences in regard to KDO content, LPS from P. phosphoreum PJ-1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross-reaction with other LPS.

New mechanism for post-translational processing during assembly of a cytoplasmic membrane protein?

Insertion of nitrate reductase into the Escherichia coli cytoplasmic membrane was examined by following the fate of pulse-labeled enzyme in both the membrane and cytoplasm during various times after the addition of an unlabeled chase. The polypeptide composition of this labeled enzyme was determined by autoradiography of immunoprecipitated material after separation on sodium dodecyl sulfate-polyacrylamide gels. The data presented here indicate that immediately after appropriate insertion of the enzyme into the membrane, a post-translational event occurs which converts the cytoplasmically synthesized form of subunit B (B') to the form found in the completely assembled enzyme (B). B' is distinguished from B by its more rapid electrophoretic mobility. B' was found in the cytoplasm of all strains tested, in the membrane of strains with defects in enzyme insertion (hemA and chlE), and as a transient component in the membrane of wild-type cells.

Proteoglycan of rapid electrophoretic mobility

Proteoglycan of rapid electrophoretic mobility

Nucleotide sequences associated with differences in electrophoretic mobility of envelope glycoprotein gp70 and with GIX antigen phenotype of certain murine leukemia viruses.

Previous genetic and biochemical studies led to the identification of two large RNase T1-resistant oligonucleotides, designated the G(IX) (+) and G(IX) (-) oligonucleotides, whose presence in the genomes of closely related murine leukemia viruses is mutually exclusive and predictive of two properties of the viral envelope glycoprotein gp70. Viruses harboring the G(IX) (+) oligonucleotide induce expression of the gp70-associated antigen G(IX) and possess gp70s with more rapid electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide gels than viruses that possess the G(IX) (-) oligonucleotide. The latter viruses fail to induce G(IX) on infected fibroblasts. The G(IX) (+) and G(IX) (-) oligonucleotides lie in corresponding positions in the 3' third of the oligonucleotide maps of their respective viruses. We have determined the nucleotide sequences of the G(IX) (+) and G(IX) (-) oligonucleotides. The sequence of the G(IX) (-) oligonucleotide is U-A-U-C-U-C-A-A-C-C-A-C-C-A-U-A-C-U-U-A-A-C-C-U-C-A-C-C-A-C-[unk]-G, and the sequence of the G(IX) (+) oligonucleotide is U-A-U-C-U-C-A-A-C-C-A-C-C-A-U-A-C-U-U-G. Thus, a single base change could result in the interconversion of the two oligonucleotides. Consideration of the amino acids specified by the two oligonucleotides suggests that this single base difference may result in the presence of an additional oligosaccharide chain in the gp70s of the G(IX) (-) viruses. Evidence supporting this prediction has been obtained by M. R. Rosner, J.-S. Tung, E. Fleissner, and P. W. Robbins (personal communication). It is entirely possible that the single nucleotide change that apparently results in a different electrophoretic mobility of the gp70s of the G(IX) (+) and G(IX) (-) viruses is also responsible for the presence or absence of the G(IX) antigenic determinant; however, the validity of this possibility awaits further investigation.

Isolation and properties of a 7 S rat immunoglobulin different from IgG.

A 7 S rat immunoglobulin, of rapid electrophoretic mobility, was characterized and isolated from normal or hyperimmune rat serum and was designated "IgR". This immunoglobulin belongs to a class different from IgG and was originally described as IgA. IgR has a molecular weight of 160 000, is present in the serum and milk as a monomer and is readily absorbed by the intestine of suckling rats. In hyperimmunized animals, about 20% of the antibodies were of the IgR class. IgR is devoid of anaphylactic capacity and no homologies could be established between it and immunoglobulins of other animal species.

Electrophoretic polymorphism of glutathione peroxidase.

SUMMARYA method for the detection of glutathione peroxidase after electrophoresis on starch gel has been devised. Blood samples from 780 persons of Caucasian or Negro origin were studied. An electrophoretically fast enzyme, designated as the Thomas variant, was found in 6·4 % of 392 Negro donors and 0·8 % of 388 Caucasian donors. This variant was inherited in an autosomal dominant fashion. A slightly rapid electrophoretic mobility was noted in some cord blood samples and in a father and daughter with a refractory anaemia. Because this variant, designated ‘Musi’ differed so little from the normal variant, it was difficult to further characterize it, but it did not appear that this variant was genetically determined.Glutathione peroxidase from most other tissues had the same electrophoretic mobility as the enzyme from erythrocytes, but the enzyme from cultured human fibroblasts moves more rapidly. Various laboratory animals were found to have electrophoretically distinguishable glutathione peroxidase, but efforts to hybridize hamster enzyme to human enzyme were unsuccessful.

Properties of a Cold Hemagglutin in Associated with Leptospiral Hemolytic Anemia of Sheep

The cold hemagglutinin which appears after leptospiral infection of sheep was isolated consistently from the plasma by absorption to autologous erythrocyte stroma at 4 C and subsequent elution at 25 C. Immunoelectrophoretic tests disclosed that the purified preparations consisted primarily of a single detectable component with the electrophoretic, positional, and antigenic characteristics of ovine immunoglobulin M. The properties of the cold hemagglutinin included a rapid sedimentation rate, elution in the leading peak from G-200 Sephadex, high anionic-binding capacity, and rapid electrophoretic mobility. Thus, these methods also supported the identification of the cold hemagglutinin as a homogeneous protein with properties like immunoglobulin M. The occurrence of the cold hemagglutinin after the phase of leptospiremia and concurrent to the onset of hemolytic anemia suggests that this antibody may play a significant role in the genesis of leptospiral acquired hemolytic anemia.

Hemoglobin-Seattle (α2Aβ276Glu): An Unstable Hemoglobin Causing Chronic Hemolytic Anemia

Abstract Mild to moderate chronic hemolytic anemia in a mother and her two sons was found to be associated with the presence of about 40 per cent abnormal hemoglobin of rapid electrophoretic mobility: Hb-Seattle. Hb-Seattle was found to be unstable in vitro. It was shown to be due to a replacement of alanine by glutamic acid at position 76 of the β hemoglobin chain. The substitution occurs at the surface of the β hemoglobin chain. The hemoglobin instability was attributed to an abnormal interaction between the substituted glutamic acid and histidine at position β77.

Rapid Detection of Australia Antigen by Counterimmunoelectrophoresis

Abstract This report describes the detection of Australia antigen by counterelectrophoresis in agar gel. With this technique small amounts of antigen with relatively rapid electrophoretic mobility can be detected by specific precipitation with antibody in 1 hr (1, 2). In addition to its speed the technique is at least 10 times as sensitive as the Ouchterlony double diffusion method. Materials and Methods. Sera containing Australia antigen were obtained from patients with acute viral hepatitis. Specific antisera were from hemophiliacs who had received numerous blood transfusions and were thus repeatedly exposed to hepatitis virus. The standard sera containing Australia antigen used in this laboratory, as well as the homologous antisera, have been shown to have the same specificities as those employed by Blumberg (3) and Prince (4). Counterelectrophoresis is carried out on Kodak projection slides (3.25 × 4 in) covered with 10 ml of 0.85% Agarose in Veronal buffer, 0.05 M, pH 8.2.

Atypical Antigenic Properties of a γA Myeloma Protein

Summary and Conclusions An unusual paraprotein of very rapid electrophoretic mobility and γA antigenicity has been found to have its L chain antigenicity obscured or hidden, although associated Bence Jones protein in the urine exhibited easily demonstrable λ specificity. The intact protein was not typable with anti-κ or anti λ sera and when injected into rabbits failed to provoke either anti-κ or anti λ response. Mercaptoethanol reduction of the protein revealed a normal proportion of L chains, which were readily shown to have λ specificity and which had an acid urea starch gel electrophoretic mobility identical to that of the reduced Bence Jones protein. The isolated L chains provoked a λ-specific response when injected into rabbits. The reduced and alkylated paraprotein (its L and H chains not yet separated by acid sephadex filtration) was readily typable as λ-specific when the same typing sera was employed which failed entirely to react with the unreduced whole protein. Five other γA myeloma proteins showed enhanced precipitation with κ and λ typing sera after mercaptoethanol reduction of the paraproteins. It is concluded that the atypical antigenic properties displayed by the paraprotein described in this report probably reflect structural features producing steric hindrance against reaction with homologous antibodies.

Étude d'une β 2a-globuline cryo-précipitable

From pathologie serum a cryo-precipitable globulin with the electrophoretic properties of β 2A-globulin has been isolated, which in the ultracentrifuge shows 4 components of S o 20, w of 6.75, 9.4, 11.2 and 13.4, respectively. From this globulin fraction were isolated, by preparative ultracentrifugation, 2 distinct groups of differing molecular weights both of which retain the antigenic specificity of β 2A-globulin; one is a homogeneous component of S o 20, w 6.75 and molecular weight 180,000 and rapid electrophoretic mobility; the other is heterogeneous containing heavy components with slower electrophoretic mobility. Treatment with cysteine causes the 2 fractions to lose their property of cryo-precipitation and indicates that the S o 20, w 11.7 and 13.4 fractions are polymeric forms of the other components. These results are in agreement with previous reports in the literature concerning the β 2A-globulin of normal and myeloma sera. It is concluded that normal β 2A-globulin, defined immunoelectrophoretically is heterogeneous containing components of differing molecular weight.