Subsequent Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

The overlooked difference between human endogenous and recombinant erythropoietins and its implication for sports drug testing and pharmaceutical drug design

Sequential deglycosylation by exoglycosidase treatment (Reagent Array Analysis Method, RAAM) and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a profound structural difference between human endogenous and recombinant erythropoietins. While both proteins behaved similarly upon digestion with Arthrobacter ureafaciens α-sialidase and Steptococcus pneumoniae β-D-galactosidase, the action of N-acetyl-β-D-glucosaminidase from Steptococcus pneumoniae was partly blocked by endogenous but not recombinant erythropoietins. Consequently, further treatment with Jack bean α-D-mannosidase and Helix pomatia β-D-mannosidase led to only very limited additional deglycosylation of endogenous EPO, while rhEPO glycans continued to be degraded. The behaviour was visualized by SDS-PAGE combined with Western blotting. While the apparent molecular masses of most endogenous glycoforms did not further decrease after treatment with the first three enzymes, masses of most rhEPO glycoforms continued to drop after digestion with the two mannosidases. Both, human urinary and serum EPO showed this blocking effect, and all of the tested 28 recombinant epoetins were accessible to further degradation by exo-mannosidases. The majority of EPO pharmaceuticals is produced in Chinese hamster ovary (CHO) cell lines, few in other ones (i.e. baby hamster kidney (BHK) or human fibrosarcoma (HT-1080) cells). Since human endogenous EPO is primarily produced by the kidneys, tissue specific glycosylation might explain the altered deglycosylation behaviour. This difference was overlooked since EPO was first isolated from human urine in 1977. The results might prove useful for anti-doping testing and future EPO drug development.

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.

Purification and Characterization of Human Serum N-Acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase

Many lysosomal enzymes are recognized and selected by a unique marker in the form of mannose 6-phosphate groups which are present exclusively on their N-linked oligosaccharides. Two enzymes act sequentially to catalyze the addition of mannose g-phosphate groups to the proteins: N-acetylglucosamine phosphotransferase (GlcNAc phosphotransferase) and N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (phosphodiester α-GlcNAcase). We report here the purification and partial characterization of phosphodiester α-GlcNAcase from human serum. The enzyme was purified over 600,000-fold by utilizing ammonium sulfate precipitation, fractionation on wheat germ agglutinin-Sepharose, Fe3+-chelating Sepharose, and Cu2+-chelating Sepharose, and renaturation from gel slices after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein observed after renaturation and subsequent SDS-PAGE and silver staining had an apparent molecular mass of 118 kDa, which is slightly smaller than bovine liver phosphodiester α-GlcNAcase (Mullis et al. (1994) J. Biol. Chem. 269, 1718-1726). Serum enzyme activity does not require Triton X-100 and is not stimulated by its addition. These results suggest that the enzyme found in serum represents a form secreted after proteolysis in the Golgi of the membrane-bound enzyme. The serum enzyme hydrolyzed UDP-GlcNAc to UDP and GlcNAc and hydrolyzed GlcNAc-P-ManαMe into αMeMan-P and GlcNAc. The enzyme had no hydrolyzing activity toward UDP-GalNAc, UDP-Glc, [6-3H] GlcNAcβ1-3Galβ1-4Glc, p-nitrophenyl-α-N-acetylglucosaminide, p-nitrophenyl-β-N-acetylglucosaminide, p-nitrophenyl-α-N-galactopyranoside, or p-nitrophenyl-β-N-galactopyranoside. The enzyme activity was strongly inhibited by UDP-GlcNAc and GlcNAc-1-phosphate, had a pH optimum between pH 6.0 and 7.0, and was inhibited by FeCl3, FeSO4, and CuSO4. The Km values for UDP-GlcNAc and GlcNAc-P-ManαMe were 0.94 and 0.45 mM, respectively. Over 77% of enzyme activity remained after incubation for 10 min at 70°C, demonstrating an unusual thermostability of the serum enzyme.

Stimulation of rat parietal cell function by histamine and GLP-1-(7-36) amide is mediated by Gs alpha

It was the aim of the present study to determine in rat parietal cells whether Gs alpha, the stimulatory subunit of adenylate cyclase, mediates adenosine 3',5'-cyclic monophosphate (cAMP)-dependent parietal cell function in response to histamine and glucagon-like peptide 1 (GLP-1)-(7-36) amide. Cytoplasmic membrane from enriched (83 +/- 5%) rat parietal cells were incubated for 30 min with 30 microCi/ml [32P]NAD+ and 40 micrograms/ml preactivated cholera toxin (CT), a pharmacological tool for activation of Gs alpha. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed [32P]ADP ribosylation of Gs alpha represented by three proteins with molecular masses ranging from 42 to 45 kDa. In intact parietal cells, CT (10(-12)-10(-8) M) caused marked stimulation of [14C]aminopyrine accumulation and cAMP production confirming the functional importance of Gs alpha in regulation of H+ production. Identical membrane preparations were preincubated (2 h, 4 degrees C) in parallel with and without RM/1, a rabbit polyclonal anti-Gs alpha-antibody. Subsequently, adenylate cyclase was stimulated by histamine, GLP-1-(7-36) amide, CT, or forskolin. At a 1:10 dilution, the antiserum completely abolished adenylate cyclase activity in response to maximal concentrations of histamine, GLP-1-(7-36) amide, and CT while reducing forskolin stimulation by only 22.0 +/- 4.9%. At 1:50, RM/1 reduced responses to histamine, GLP-1-(7-36) amide and CT by 20-30% but failed to inhibit forskolin-stimulated enzyme activity. At 1:100, the antiserum was ineffective versus all stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of a glycoprotein, GP80, in the outermost layer of the developing endosperm of immature seeds of carrot

A glycoprotein (GP80) from the immature fruits of carrot (Daucus carota L.) plants was purified through the sequential application of salt extraction, (NH4)2SO4 precipitation, chromatography on columns of concanavalin A-Sepharose, hydroxylapatite, and boronate, and electroelution, with subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its apparent relative molecular mass (Mr) was estimated to be 80 k by both SDS-PAGE and gel filtration at high ionic strength. The Mr of the polypeptide portion was estimated to be 74 k after deglycosylation by trifluoromethanesulfonic acid (TFMS). Antibodies were produced by immunizing rabbits with purified and deglycosylated GP80. Using immunoblotting of total protein extracts from each organ and tissue GP80 was detected only in immature seeds. It appeared at 4 d after flowering (DAF), accumulated until 15 DAF, then its level suddenly decreased and it disappeared completely at 23 DAF. Another glycoprotein, with an Mr of 78 k (GP78), cross-reacted with GP80-specific antibodies. GP78 appeared at 17 DAF and disappeared at 37 DAF. Histochemical examination of fruits using Sudan Black B and periodic acid-Schiff reagent (PAS) showed that the appearance of GP78 coincided with the rapid filling of the central cavity with endosperm cells, which have cell walls and contain lipid storage granules. In immunohistochemical studies, both GP80 and GP78 were found at the outermost layer of the developing endosperm.

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [ 32P] ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with 32P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term 32P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity. When fat body was pulse-labeled with 32P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro 32P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify 32P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.

Binding of the adhesive protein complex (LFA-1/Mac-1/p150,95) to concanavalin A.

At least 30 proteins from human PMNL plasma membranes capable of binding concanavalin A (Con A), can be identified after surface labeling with 125I and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation of the labeled proteins after solubilization in non-ionic detergents, with monoclonal antibodies (MAbs) directed against a family of leukocyte membrane proteins (LFA-1, Mac-1, p150,95, and the beta subunit these glycoproteins share), indicated that Mac-1 and p150,95 were bound by Con A. Dissociation of the alpha and beta subunits with sodium dodecyl sulfate, electrophoresis, transfer to nitrocellulose paper, and subsequent binding of these proteins by Con A demonstrated Con A retention by Mac-1-alpha, p150,95-alpha, and the common beta subunit. Affinity of Con A for LFA-1-alpha from human peripheral blood PMNL could not be confirmed by direct binding or electroblotting. Similar experiments in a patient deficient in LFA-1, Mac-1, p150,95, and the beta subunit confirmed that Mac-1-alpha and the beta subunit were important Con A-binding proteins.

Isolation and molecular weight determination of two immunoglobulin heavy chains in the channel catfish, Ictalurus punctatus

1. 1. Channel catfish ( Ictalurus punctatus), a teleost fish, were immunized over a 4 month period with 4 intraperitoneal injections of bovine serum albumin (BSA) in Freund's adjuvant. 2. 2. The catfish anti-BSA antibody was purified by affinity chromatography and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 3. 3. By elution of catfish anti-BSA antibody from BSA-afiinity columns with 3.0MKSCN and subsequent SDS-PAGE, two immunoglobulin heavy chains were demonstrated in the channel catfish. The molecular weights and the relative percentages found of the two immtmoglobulin heavy chains were 72,000 (94%) and 56,000 (6%). The molecular weight of the single light chain found was 23,000. 4. 4. Using the 72,000 mol. wt heavy chain and 23,000 mol. wt light chain and including a molecular weight of 15,000 for the J-chain, the molecular weight of the predominant channel catfish tetrameric IgM immunoglobulin molecule was calculated to be 775,000. 5. 5. Using the 56,000 low mol. wt heavy chain, the molecular weight of a second subclass of the channel catfish tetrameric IgM molecule was calculated to be 647,000. 6. 6. After Sephadex G-200 gel filtration, anti-BSA antibody activity was found only in the 14S globulin fraction by indirect hemagglutination testing.

Monoclonal antibodies to rabbit sperm autoantigens. II. Inhibition of human sperm penetration of zona-free hamster eggs.

Monoclonal antibodies (mAbs) to the rabbit sperm autoantigens RSA-1, 2 and 3 were found to cross-react with human ejaculated spermatozoa. The mAbs show by indirect immunofluorescent staining that the antigen is concentrated in the middle-piece region of the tail, with lesser amounts on the head region. Binding of mAbs to human sperm extract was 75% of the maximum binding to rabbit spermatozoa. Immunoadsorbent chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identified three sperm antigens of 87,000, 15,000 and 13,000 daltons (kd). The mAbs significantly inhibited human spermatozoa from penetrating zona-free hamster oocytes. It is concluded that human sperm, like rabbit sperm, may have a low molecular weight family of membrane glycoproteins with a higher molecular weight extrinsic protein bound together in a complex on their surfaces.

Interaction of human polymorphonuclear leukocyte elastase with human IgM. In vitro production of an Fabμ-like fragment

Human granulocyte elatase has ample opportunity to interact with immunoglobulins at both the intracellular and extracellular level. Thus, the susceptibility of human IgM to proteolysis by this enzyme was investigated. Treatment of monoclonal IgM with elastase in the presence of 2 m M cysteine for 18 hr at 37°C resulted in the formation of a major fragment (Frag. A) and small peptides. Fragment A had an approximate mol. wt of 50,000 as determined by gel filtration. Reduction and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Frag. A revealed 2 bands having mol. wt of 23,000 and 30,000, respectively. Ouchterlony analysis showed Frag. A to contain light (K) chain and a portion of the heavy (μ) chain. Fragment A appeared to be antigenically identical with Fab produced by trypsin or papain digestion of IgM but completely different from Fc5μ produced by trypsin digestion of IgM. Fc5μ was digested to small peptides by the enzyme. These findings show that human PMN elastase can specifically degrade IgM in vitro, producing an Fab-like fragment and small peptides.

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.

Investigation of the symmetry of oligomeric enzymes with bifunctional reagents.

The symmetry of proteins composed of identical polypeptide chains has been investigated by means of cross-linking with bifunctional reagents and subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis. The majority of the investigations were performed with diimidates of different chain lengths (C3-C12), which react exclusively with amino groups. Aldolase, catalase, fumarase, pyruvate kinase, tetrameric proteins with identical polypeptide chains, reveal a D2 symmetry, i.e. they appear to be composed of two pairs of polypeptide chains. The validity of this conclusion is demonstrated with lactate dehydrogenase. This enzyme, shown by X-ray analysis to have a D2 symmetry, yields after cross-linking and subsequent polyacrylamide electrophoresis the band pattern expected for a protein with this quaternary structure and similar to the pattern obtained with the above enzymes. 2. The influence of the experimental conditions on the cross-linking reaction has been investigated. The selectivity of the bifunctional reagent for the different contact domains within the quaternary structure of a protein depends on the reaction time, the chain length and on the concentration of the reagent. In general the D2 symmetry becomes more obvious with increasing chain length and with increasing concentration of the diimidate. Diethylpyrocarbonate showed very little selectivity.