Sodium Dodecyl Sulfate-acrylamide Electrophoresis Research Articles

Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)

Reinigung und Charakterisierung von Streptokokken-Hyaluronatlyase

Abstract Hyaluronate lyase of group C strain H 46 A ( Streptococcus equisimilis ) was purified and characterized by isoelectric focusing, sodium-dodecylsulfate-acrylamide electrophoresis, polyacrylamide-gradient-electrophoresis and crossed immunoelectrophoresis. The purification of the hyaluronate lyase was performed successively by adsorption on Florisil, chromatography on DEAE-cellulose, and - for separation of streptokinase - stirring with CPG-pore-glass. The last step was gel filtration on Sepharose 6B. Purified hyaluronate lyase showed a high specific activity. The purified enzyme was found to be antigenically homogeneous. No contaminating streptococcal components could be detected. The molecular size was determined as to be 90000 dalton by gel filtration and 110000 dalton by sodium dodecylsulfate-acrylamide electrophoresis. The amino acid composition was also determined. In the isoelectric focusing, using gels with reducing conditions, one protein band at a pi 4.95 was observed. Under nonreducing conditions two or three diffuse protein bands which showed lower enzymatic activity were found. It might be possible that the hyaluronate lyase exists in two different forms.

Growth response in athymic nude mice to transplanted growth hormone-secreting rat pituitary tumor cells.

Clonal cell lines originally derived from a somatotropic rat pituitary tumor were grafted sc into athymic nude mice, and the growth response induced in the graft carriers was examined. When at least 106 rat tumor cells were injected per mouse, virtually all mice developed slow growing, nonmetastasizing tumors at the injection site. In immature nude mice, the somatotropic effect of the rat GH secreted by the growing tumor was clearly evident, as body weight continued to increase well beyond that of control littermates. Nude mice with a final body weight of over 60 g were regularly produced in this manner. The action of the rat GH in the nude mouse carriers was generalized, as shown by the fact that the gigantic mice resulting from the xenografts maintained apparently normal balanced physical proportions. Exceptionally, however, the islets of Langerhans in the host pancreas showed even greater hypertrophy, presumably due to the known anti-insulin activity of GH. The rat pituitary tumor cells were shown to synthesize and secrete a protein which was precipitated by an antiserum against rat GH and which comigrated with authentic rat GH when analyzed by sodium dodecyl sulfate-acrylamide electrophoresis. Through serial transplantation in nude mice, the tumor cells retained their functional and tumorigenic properties. These results demonstrate that rat GH is active in the mouse, and suggest also that the athymic nude mouse should be useful for the development of other xenogeneic tumor lines that secrete GH.

Partial purification and characterization of prolyl hydroxylase from the free-living nematode Panagrellus silusiae

Prolyl hydroxylase was isolated from the free-living nematode Panagrellus silusiae by ammonium sulfate precipitation and calcium phosphate gel ion exchange from Triton X-100 treated homogenates. The enzyme preparation was highly purified but not to homogeneity as judged by agarose gel filtration and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Gel filtration indicated that the molecular weight of the enzyme approximated 285 000. Analysis by SDS–acrylamide electrophoresis revealed subunits of ~67 000. Complete enzymic activity required α-ketoglutarate, Fe2+, ascorbate, catalase, atmospheric oxygen, and dithiothreitol. This activity was dramatically inhibited by α, α-dipyridyl, phenanthroline, and polyproline II. The purified enzyme preparation synthesized 50 to 100 μg hydroxyproline per milligram chick protocollagen per hour at 30 °C with an estimated Km for the substrate of 80 μg. Thus, a purified prolyl hydroxylase from a simple invertebrate possesses many of the properties of the prolyl hydroxylases of various vertebrates.

Erythrocyte troponin inhibitor‐like protein: Isolation and characterization

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000--24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca2+ and K+-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg2+-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.

Purification and properties of streptococcal hyaluronate lyase.

Hyaluronate lyase (hyaluronidase) has been purified and characterized from a group A type 4 Streptococcus. Production of the enzyme was favored by growth in trypsinized veal infusion in the presence of hyaluronate oligosaccharide and tetrasaccharide. Detectable enzymatic activity was diminished in the presence of N-acetylglucosamine and glucuronic acid. Purification of hyaluronate lyase consisted of 40 to 60% ammonium sulfate precipitation, diethylaminoethyl A-50 Sephadex ion-exchange chromatography, gel filtration with G-200 Sephadex, and adsorption to Sepharose 6B. Purified enzyme was antigenically homogeneous and free of proteinase, deoxyribonuclease, streptolysin 0, and streptokinase. Active hyaluronate lyase was recovered from neutral polyacrylamide gels, and it appeared to be a glycoprotein. A single band was detected by sodium dodecyl sulfate-acrylamide electrophoresis, which had a molecular weight of approximately 50,000. A molecular weight of 70,000 was observed by gel filtration. The purified enzyme had a Km of 3.8 x 10(-4) and a pH optimum of 6.0. Reducing agents increased the activity of crude enzyme at least threefold and were necessary to prevent inactivation of the purified enzyme.

Comparative chemical analyses of the alloantigenic fragments of HL-A antigens.

Papain-solubilized HL-A antigens have been shown to contain two polypeptide fragments: beta2-micro-globulin with a molecular weight of approximately 12,000 and a larger fragment with a molecular weight of about 34,000. The large fragments isolated from two HL-A preparations carrying different specificities appeared homogeneous both by immunoelectrophoresis and sodium dodecyl sulfate-acrylamide electrophoresis. Both HL-A antigen preparations contained the same NH2-terminal (glycine) and the same COOH-terminal residue (serine). The carbohydrate content of the large fragment was 12.9%, making the carbohydrate-free molecular weight approximately 30,000. Small but significant differences have been found in the amino acid compositions and tryptic peptide maps of the two large fragments containing different specificities.

Purification by immunoadsorbtion chromatography of the normal and a mutant form of the B2 subunit of Escherichia coli tryptophan synthase.

Columns containing immobilized immunoglogulin G fractions from normal and immunized animals were used to purify the wild-type beta component of Escherichia coli tryptophan synthase, formula alpha2 beta2, and a mutant form of the beta component. The procedure yielded proteins with no detectable contaminants as measured by analytical acrylamide disc gel electrophoresis and immunodiffusion against proteins subject to sodium dodecylsulfate acrylamide electrophoresis. After elution from the antibody column at pH 11 the normal beta component, by dialysis against pyridoxal phosphate at pH 7.5, could be restored to the enzymatically-active beta2 dimer with a specific activity of 1700 enzyme units/mg protein. This compares with reported values of 2500-3000 enzyme units/mg when the beta2 dimer is purified by conventional means.

Purification and characterization of a staphylococcal epidermolytic toxin.

A staphylococcal exotoxin that causes epidermolysis when injected into the skin of the newborn mouse and man was highly purified by coventional biochemical techniques. With Staphylococcus aureus EV, the epidermolytic toxin was a major protein component of supernatant culture fluids. The initial step in purification was zone electrophoresis in Pevikon carried out at pH 9.0, the isoelectric point of alpha-hemolytic toxin, which remained near the origin. Fractions containing the epidermolytic toxin, but free of alpha-toxin, were then subjected to cation exchange chromatography on carboxymethyl-Sephadex C-50 to remove trace contaminants. A major highly purified epidermolytic toxin migrated as a single band in polyacrylamide gel electrophoresis, sedimented as a single component in the analytical ultracentrifuge, and elicited a single precipitating antibody after injection into rabbits. A smaller amount of a second epidermolytic toxin, identical in molecular weight and antigenicity but differing in electrophoretic behavior from the major molecular species, was also identified. The epidermolytic factor had a molecular weight of 28,600 +/- 400 by sodium dodecyl sulfate-acrylamide electrophoresis and 32,500 +/- 120 by approach to sedimentation equilibrium.

Comparisons of the Molecular Forms of Activated Bovine Factor X: PRODUCTS OF ACTIVATION WITH RUSSELL'S VIPER VENOM, INSOLUBLE TRYPSIN, SODIUM CITRATE, TISSUE FACTOR, AND THE INTRINSIC SYSTEM

Abstract Preparations of bovine Factor X zymogen were converted to enzymatically active forms with Russell's viper venom, with 25% sodium citrate, with a water-insoluble copoly-(maleic acid-ethylene)trypsin, with tissue factor (rabbit brain extract thromboplastin), and with a preparation designed to represent the intrinsic pathway of blood coagulation. The activated Factor X generated in each case was purified by chromatography on diethylaminoethylcellulose. Analytical ultracentrifugation, gel filtration, disc electrophoresis, sodium dodecyl sulfate-acrylamide electrophoresis, amino acid and carbohydrate analyses, carboxyl- and amino-terminal amino acid analyses, and peptide mapping of tryptic digests were used to show the striking molecular similarities of the enzymatically active products. Mixtures containing various combinations of the differently activated Factor X preparations behaved essentially as single species in disc electrophoresis, and after reduction with mercaptoethanol only two bands appeared in sodium dodecyl sulfate-acrylamide electrophoresis, as with each separate product. It was concluded that the diverse activation procedures yield essentially the same molecular form of activated Factor X.

Cocoonase: IV. MECHANISM OF ACTIVATION OF PROCOCOONASE FROM ANTHERAEA POLYPHEMUS

Abstract A glycoprotein obtained from the maxillary galeae of the silkmoth Antheraea polyphemus has been established as the zymogen (prococoonase) of the proteolytic enzyme cocoonase. A study of the in vitro activation of prococoonase and comparison of the properties of the resultant product with those of the native enzyme have confirmed this relationship. This comparison revealed (a) identical electrophoretic mobilities on polyacrylamide gels, (b) identical molecular weights, (c) identical apparent Km values for the hydrolysis of Nα-benzoyl-l-arginine ethyl ester, (d) indistinguishable amino acid compositions, and (e) the same NH2- and COOH-terminal amino acid sequence. The in vitro mechanism of activation of prococoonase by cocoonase was shown to be autocatalytic by kinetic analysis. Cocoonase also resulted from prococoonase activation by bovine trypsin, but the rate was 480 times faster than activation by cocoonase itself. Prococoonase was also activated by porcine enterokinase at about 75% the rate of the autocatalytic activation. Sedimentation equilibrium and sodium dodecyl sulfate-acrylamide electrophoresis of proenzyme and enzyme indicated a molecular weight difference of 2,000 to 3,500. By comparison of the NH2- and COOH-terminal amino acid sequences of prococoonase and cocoonase, it was concluded that activation involves the removal of a 13-residue peptide from the NH2-terminal end of prococoonase. The composition of this peptide is compared to other trypsinogen-trypsin activation peptides and an in vivo mechanism for prococoonase activation is proposed.

Human α-Fetoprotein

Abstract Human α-fetoprotein has been isolated from the sera of hepatoma patients by the combined techniques of preparative electrophoresis, Sephadex gel filtration, and preparative isoelectric focusing. The purity of the isolated α-fetoprotein was demonstrable by (a) a single line on Ouchterlony immunodiffusion versus anti-α-fetoprotein, with no reaction versus a potent polyvalent anti-human serum of anti-albumin; (b) a single band on sodium dodecyl sulfate polyacrylamide electrophoresis; and (c) the production of monospecific antibody upon immunization of rabbits. The molecular weight of α-fetoprotein was 72,000 by sodium dodecyl sulfate acrylamide electrophoresis. Two major molecular forms of α-fetoprotein were found by isoelectric focusing, ion exchange chromatography, and agarose electrophoresis. Both forms were present in most hepatoma and fetal sera, but their relative amounts appeared to have no relationship to gestational age, clinical status, serum concentration, or method of storage. The two forms, with isoelectric points of 4.85 and 5.2, were isolated by preparative isoelectric focusing and were found to convert to a single homogeneous form with slower electrophoretic mobility after treatment with neuraminidase.