Nonreducing Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes β-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

Background:Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry.Aim:The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA.Results:The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples.Conclusion:FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Major cause of antibody artifact bands on non-reducing SDS-PAGE and methods for minimizing artifacts

When monoclonal antibodies (mAbs) are analysed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), method-induced artifacts are a frequent phenomenon. Previous studies suggested that incomplete denaturation and disulfide-bond scrambling are two main causes of artifact bands. Thus, in practice samples are normally heated and treated with alkylating agent iodoacetamide (IAM) before loading to promote denaturation and block free sulfhydryl groups, respectively. In this work, we further studied the major cause of artifact bands on non-reducing SDS-PAGE and ways of eliminating artifacts with two purified mAbs. In both cases, it was found that artifact bands on non-gradient Tris-glycine gels are mainly caused by incomplete denaturation under typical gel conditions. In general, heating minimizes artifact bands due to incomplete denaturation but it also generates some extra bands. Combining heating with IAM treatment achieved slightly better results than heating alone. As an alternative to heating, treating the samples with 8 M urea also allows close to complete denaturation of samples and thus minimizes artifact bands. In addition, we learned that untreated samples (samples that are not heated or treated with urea) may look different on Bis-Tris gel depending on gel composition (non-gradient vs. gradient) and the buffer used (MES vs. MOPS). In certain case, the apparent lack of artifact bands on gradient Bis-Tris gel may be in fact due to insufficient resolution. In conclusion, this study further confirmed that full-denaturation of sample is critical for minimizing/avoiding artifact bands on non-reducing SDS-PAGE.

Trastuzumab-monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2-positive human breast cancer.

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody-drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV-vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.

Characteristics and Protein Subunit Composition of Flour Mill Streams from Different Commercial Wheat Classes and Their Relationship to White Salted Noodle Quality

Proximate characteristics and protein compositions of selected commercial flour streams of three Australian and two U.S. wheats were investigated to evaluate their effects on the quality of white salted noodles. Wheat proteins of flour mill streams were fractionated into salt‐soluble proteins, sodium dodecyl sulfate (SDS)‐soluble proteins, and SDS‐insoluble proteins with a sequential extraction procedure. SDS‐soluble proteins treated by sonication were subsequently separated by nonreducing SDS polyacrylamide gel electrophoresis (SDS‐PAGE). There was a substantial amount of variation in distributions of protein content and protein composition between break and reduction mill streams. SDS‐insoluble proteins related strongly to differences in protein quantity and quality of flour mill streams. The soluble protein extracted by SDS buffer included smaller glutenin aggregates (SDS‐soluble glutenin) and monomeric proteins, mainly gliadin (α‐, β‐, γ‐, and ω‐types) and albumin and globulin. SDS‐soluble proteins of different flour mill streams had similar protein subunit composition but different proportions of the protein subunit groups. Noodle brightness (L) decreased and redness (a) increased with increased SDS‐insoluble protein and decreased monomeric gliadin. Noodle cooking loss and cooking weight gain decreased with increased glutenin aggregate (SDS‐soluble glutenin and SDS‐insoluble glutenin) and decreased monomeric gliadin. Noodle hardness, springiness, cohesiveness, gumminess, chewiness, tensile strength, breaking length, and area under the tensile strength versus breaking length curve increased with increased glutenin aggregate. Monomeric gliadin contributed differently to texture qualities of cooked noodles from glutenin aggregate. Monomeric albumin and globulin were not related to noodle color attributes (except redness), noodle cooking quality, and texture qualities of cooked noodles. The results suggested that variation in protein composition of flour mill streams was strongly associated with noodle qualities.

Biochemical and biological analysis of Philodryas baroni (Baron’s Green Racer; Dipsadidae) venom

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bβ- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 μg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Isolation and characterization of immunoglobulin M of Asian sea bass, Lates calcarifer and its level in serum

Asian sea bass immunoglobulin M (IgM) was purified from the sera of Lates calcarifer by affinity chromatography. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions revealed that the sea bass IgM was a tetrameric protein with a molecular weight of 896 kDa; it contained an equimolar heavy chain and light chain with molecular weight of 83 kDa and 27 kDa respectively. However, besides the covalently linked tetrameric IgM, noncovalently linked tetramer dissociated into dimeric and monomeric forms also demonstrated by non-reducing SDS-PAGE. Carbohydrate moieties were found to be linked with both heavy and light chains. A polyclonal rabbit anti-Asian sea bass IgM was prepared which showed a specific reaction of anti-fish IgM antibody with IgM of sea bass. Sea bass IgM concentration was determined in the serum by indirect ELISA. The average IgM concentration in the sera of the healthy sea bass was 5.4±1.8 mg ml−1; it amounted to 16.7% of the total serum protein.

Isolation and characterization of immunoglobulin M of Asian sea bass, Lates calcarifer and its level in serum

Abstract Asian sea bass immunoglobulin M (IgM) was purified from the sera of Lates calcarifer by affinity chromatography. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions revealed that the sea bass IgM was a tetrameric protein with a molecular weight of 896 kDa; it contained an equimolar heavy chain and light chain with molecular weight of 83 kDa and 27 kDa respectively. However, besides the covalently linked tetrameric IgM, noncovalently linked tetramer dissociated into dimeric and monomeric forms also demonstrated by non-reducing SDS-PAGE. Carbohydrate moieties were found to be linked with both heavy and light chains. A polyclonal rabbit anti-Asian sea bass IgM was prepared which showed a specific reaction of anti-fish IgM antibody with IgM of sea bass. Sea bass IgM concentration was determined in the serum by indirect ELISA. The average IgM concentration in the sera of the healthy sea bass was 5.4±1.8 mg ml−1; it amounted to 16.7% of the total serum protein.

Delivery of bioactive, gel-isolated proteins into live cells.

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.

Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gbetagamma subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gbetagamma complex from cells cultured in the presence of [(3)H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Ggamma and Gbetagamma. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gbetagamma to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gbetagamma depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

Neutrophil Elastase Cleavage of Human Factor IX Generates an Activated Factor IX-Like Product Devoid of Coagulant Function

In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa -(Arg145) and β-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).© 1998 by The American Society of Hematology.

Neutrophil Elastase Cleavage of Human Factor IX Generates an Activated Factor IX-Like Product Devoid of Coagulant Function

AbstractIn preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa ’-(Arg145) and β-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).© 1998 by The American Society of Hematology.

Bovine herpesvirus 1 glycoprotein M forms a disulfide-linked heterodimer with the U(L)49.5 protein.

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U(L)10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U(L)10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U(L)49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448-1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U(L)49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.

Characterization of the allergens of the house dust mite Euroglyphus maynei

Background: The house dust mite Euroglyphus maynei inhabits homes in many parts of the world and is the source of many allergens. Objective: The purpose of this study was to biochemically and immunologically characterize the major allergens of E. maynei. Methods: Proteins in an extract of E. maynei were separated into 20 fractions by using preparative isoelectric focusing. Most proteins and allergens were contained in fractions 7 to 17 with pH of 4.8 to 8.0. The fractions were further characterized by nonreducing sodium dodecylsulfate–polyacrylamide gel electrophoresis and immunoblotting with the serum of 16 individuals sensitive to E. maynei. Results: Molecular weights and isoelectric points were assigned to 47 IgE-binding proteins in an E. maynei extract, and the frequency of IgE binding to each allergen was determined. Twenty-two of the allergens were recognized by more than 50% of the 16 sera, and all but one of the subjects had IgE that bound to more than 10 allergens (range, 0 to 32). One of the proteins was identified as the allergen Eur m 2. Conclusion: E. maynei is the source of at least 47 individual allergens that have been characterized by molecular weight, isoelectric point, and IgE-binding properties. (J Allergy Clin Immunol 1997;100:222-8.)

Identification of a component of the sea urchin hyaline layer, HLC-175, which undergoes proteolytic processing during development

To define the role(s) played by the sea urchin extraembryonic matrix, the hyaline layer, we have previously purified and characterized a number of the protein components of this structure. We are currently studying the timing and significance of the proteolytic processing of these species. The localization of HLC-175 in the egg and 1-hr-old embryo was determined by indirect immunofluorescence analysis. The relationship between HLC-175 and the 109- and 81 kDa species was determined by a combination of native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and protein gel blot analyses using the anti-175, -109 and 81 kDa antisera. Using gel exclusion chromatography we have fractionated a mixture of proteins extracted from the surface of 1-hr-old sea urchin embryos. A set of fractions eluting from the column contained three species of apparent molecular masses 175-, 109- and 81 K. These species comigrated on analysis by either non-reducing SDS-PAGE or native gel electrophoresis. Inclusion of the reducing agent, dithiothreitol, in the solubilizing solutions abolished comigration of these polypeptides. When polyclonal antisera were prepared against each of these antigens cross-reactivity between the 175- and 109 kDa species and between the 175- and 81 kDa species was detected. Developmental protein gel blot analyses revealed a precursor-product relationship between the 175- and the 109- and 81 kDa polypeptides. Indirect immunofluorescence analysis confirmed the localization of HLC-175 to the hyaline layer. The results reported here clearly identify HLC-175 as a component of the hyaline layer. In addition, we have shown that between the early- and mid-blastula stages of development HLC-175 is processed into two smaller species. This reaction will serve as a useful probe to identify the putative cell surface remodeling activity(ies).

Covalent Dimerization of Recombinant Human Interferon-γ

An apparently nonreducible covalent dirner has been consistently observed as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These studies endeavor to understand the nature of this covalent dimerization. Amino acid sequence analysis of a recombinant human interferon-γ stored for 2 years showed a major sequence starting with the intact N-terminal methionine and a minor sequence corresponding to the C-terminal seven amino acids of the intact protein. Moreover, when the same material was analyzed by gel filtration in the presence of 8 M urea, a minor peak corresponding to the dimer was observed prior to the monomer peak. Reducing and nonreducing SDS-PAGE also showed a minor band corresponding to a dimer. These results suggest that the reactions of C-terminal processing and dimerization have occurred during storage. Reverse-phase chromatography of stored, unfractionated material showed three peaks. Mass spectral analysis of the first, second, and third peaks gave molecular weights of 16,900, 16,100, and 33,000. Since no major cleavage was observed in the N-terminal region of the protein, the observed masses suggest that the first peak corresponds to residues 1 to 144 (a full-length molecule), the second peak to residues 1 to 137 (des 7 interferon-γ), and the third peak to a dimer. Calculation of theoretical molecular weight from the amino acid sequence suggests that this dimer corresponds to some combination of the intact protein and des 7 protein. Tryptic peptide maps in conjunction with sequence and mass analyses identified a new tryptic peptide in the map of the dimer corresponding to residue 133 to 137 followed by residues 1 to 7. The conclusion is recombinant methionyl human interferon-γ undergoes a specific cleavage at the C-terminal side of residue 137 phenylalanine, and a conventional peptide bond was formed between residues 137 and 1 methionine.

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.

Extracellular vesicle-associated and soluble trypsin-like enzyme fractions of Porphyromonas gingivalis W50.

Soluble vesicle-associated trypsin-like enzyme fractions (VSF) were prepared by sonication from extracellular vesicles (ECV) from strains W50 and W50/BE1. High-(H), intermediate-(I) and low-(L) molecular-weight VSF enzyme subfractions were identified by non-dissociative gel filtration chromatography with Mr 160, 95 and 60 kDa respectively. The chromatographic profiles of W50 VSF from 48-h and 72-h cultures were identical. W50/BE1 VSF displayed a higher ratio of the 160 to 60 kDa components. This ratio was reduced in VSF from 72-h cultures. Extracellular soluble protein (EP) trypsin profiles were similar to their respective VSF, but the 60 kDa component predominated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a loss of soluble extracellular polypeptides with culture age. A polyclonal antiserum to EP subfraction L reacted in immunoblots with a 50 kDa peptide of subfraction L of W50. Whole EP and its subfraction H displayed a 50 kDa immunoreactive peptide but no peptides of higher molecular weight. This antiserum reacted with a similar sized peptide, and with lower-molecular-weight components in whole ECV. Gelatin substrate zymography of whole EP following non-reducing SDS-PAGE revealed a major 80 kDa protease that increased with culture age. Minor protease bands of 70 and 50 kDa were also detected.

Heterogeneity of intestinal receptors for Escherichia coli heat-stable enterotoxin

The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis. Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa. Exhaustive reduction of these samples resulted in a similar pattern of labeling. Affinity-labeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of beta-mercaptoethanol. Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit. Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit. The 78-, 71-, and 56-kDa subunits were not further reducible. These data suggest heterogeneity of the ST receptor subunit structure and organization in rat intestinal epithelia.