A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Glycoprotein (gp-51) antigen not associated with the bovine leukemia virus in FLK culture supernatant liquid]

After elimination of the virus fraction non-associated with bovine leukemia virus (free) glycoprotein gp-51 antigen has been preparatively isolated from the supernatant of the FLK cell culture. For this purpose ultrafiltration through PM-30 membrane, thrice-repeated isoelectric focusing with different (pH 3.0-10.0, pH 4.0-6.0) ampholine intervals and affinity chromatography with ConA-sepharose are used. The SDS-polyacrylamide gel electrophoresis has determined it to be a homogeneous protein with molecular weight 51,000 daltons. The results of the enzyme-linked immunosorbent assay, immunoelectrophoresis and agar-gel immunodiffusion test confirm the specific activity of isolated gp-51 antigen. The gp-51 antigen may be used for identification of antibodies in the blood serum of leukemia virus of infected cattle and for monoclonal screening of antibodies.     

Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.     

Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa.

The isoelectric points of three proteases (I, II and III), separated from culture supernatants of Pseudomonas aeruginosa strain PAKS-I by isoelectric focusing, were 8.5, 6.6 and 4.5 respectively. Collagenase activity was not detected. More than 75% of the extracellular protease activity of this strain was due to protease II. This enzyme also possessed elastase activity. When purified by ammonium sulphate precipitation, isoelectric focusing and gel chromatography, protease II showed one band on disc electrophoresis and one band on conventional immunoelectrophoresis. The pH optimum, stability and effect of inhibitors and substrate concentration were examined. The molecular weight was 23000 +/- 5000. Protease II was lethal for mice when injected intraperitoneally at a high dose (minimum lethal dose 0.1 mg). Dermonecrosis and subcutaneous haemorrhages were produced in new-born mice upon subcutaneous injection of 10 microgram protease II. A sensitive test for cytotoxicity showed no evidence of cytoplasmic membrane damage to HeLa cells or human diploid embryonic lung fibroblasts by protease II. Morphological changes similar to those produced by trypsin were found.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Heterogeneity of human alpha-fetoprotein as revealed by isoelectric focusing in urea-containing gels.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.     

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.     

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.     

Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs

Dardas, T. J. (Michigan State University, East Lansing), and V. H. Mallmann. Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs. J. Bacteriol. 92:76-81. 1966.-Normal guinea pig serum was separated into seven fractions by electrophoresis on cellulose acetate membranes. Thirty antigens were found by immunoelectrophoresis: albumin, 6 alpha(1) globulins, 11 alpha(2) globulins, 6 beta(1) globulins, 5 beta(2) globulins, and gamma globulin. Hyper-alpha(2)-globulinemia was detected in sera from guinea pigs 14 days after inoculation with viable virulent Mycobacterium bovis. An additional alpha(2) globulin, not demonstrable prior to infection, was detected concomitantly with the hyper-alpha(2)-globulinemia by immunoelectrophoresis. The additional alpha(2) globulin was tentatively named alpha(2)-T. It persisted until the death of the guinea pigs. Neither hyper-alpha-globulinemia nor the alpha(2)-T antigen was detected by cellulose acetate electrophoresis and immunoelectrophoresis of sera from guinea pigs sensitized with heat-killed M. bovis. Both changes were due to the disease, not to delayed sensitivity alone.     

Studies of rhesus monkey (Macaca mulatta) hemoglobin.

One hemaglobin was found in the adult rhesus monkey (Macaca mulatta) by the technique of isoelectric focusing. In addition to the adult hemoglobin, one fetal hemaglobin was observed in neonates of the same species. The alpha and non-alpha globins of rhesus monkey hemoglobins showed similar electrophoretic mobilities as those of human hemoglobin A by cellulose acetate membrane electrophoresis. It appeared that the rhesus monkey hemoglobin system is a good model system for the study of human hemoglobin development.     

Two-dimensional electrophoresis on the layers of cellulose acetate membrane

A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.     

A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiff's reagent identified sulfated glycopeptides on cellulose acetate membrane.     

Purification and characterization of Aleutian disease virus

Virus was isolated from infected mink organs by a combination of tissue homogenization, fluorocarbon extraction and ultracentrifugation. The final preparation was analysed by crossed immunoelectrophoresis and electronmicroscopy. Virions had a capsid diameter of 22 nm. Preparative agarose electrophoresis separated virions from contaminating ferritin. Crossed immunoelectrophoresis of virus gave a single precipitate with sera from infected mink. Crossed immunoelectrophoretic analysis with intermediate gels showed that a part of the virus preparation was complexed with antibody. Serum from a certain mink was found to contain precipitating antibody to (poly)nucleotid. Virus and virus-antibody complexes were found to focus at pH 4.0-4.4 in isoelectric focusing. In SDS-polyacrylamide gel-electrophoresis the main virus protein was found to have a molecular weight of 69000. This study gives further support to the classification of aleutian disease virus as a parvovirus.     

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.     

Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Evidence against the occurrence of tissue-specific variants and isoenzymes of phosphoglucose isomerase

Phosphoglucose isomerase (EC 5.3.1.9) from a variety of rat and human tissues has been studied by several electrophoretic techniques. When heart, liver, kidney, brain, erythrocyte, or skeletal muscle extracts were subjected to electrophoresis on cellulose acetate, a broad electrophoretic band was observed. When tissues were subjected to isoelectric focusing in sucrose density gradients or polyacrylamide gels, multiple peaks were observed unless the extracts were prepared and focused in the presence of reducing agents. In the presence of 2-mercaptoethanol or dithiothreitol phosphoglucose isomerase from different tissues co-focused as a single peak (apparent isoelectric pH = 9.23 for human and 8.40 for rat), suggesting that the enzyme from the various tissues is identical, and that tissue-specific variants do not exist. The electrophoretic multiplicity previously reported by other investigators may be due to artifacts arising from oxidation of the enzyme.     

Purification and Characterization of the L Component of Streptococcus zymogenes Lysin

By utilizing conventional techniques of pressure ultrafiltration, gel filtration chromatography, diethylaminoethyl cellulose chromatography, and preparative polyacrylamide electrophoresis, the L component of the group D lysin produced by Streptococcus zymogenes strains has been purified to a state of homogeneity as determined by the techniques of disc-gel electrophoresis at pH 9.3 and 4.3 and isoelectric focusing. The L component was found to be a protein possessing a molecular weight of 11,000 with a slight net negative charge at physiological pH.     

alpha 2-Macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts

alpha 2-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F2B2 . Similar amounts of alpha 2-macroglobulin complexes (between 40 and 90 micrograms/ml) were generated in cystic fibrosis and control sera. Endocytosis of the complexes by normal human fibroblasts was compared to the amount of complexes detected by the F2B2 -radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F2B2 failed to inhibit binding of purified alpha 2-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low uptake complexes could not be distinguished from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.