Analytical Polyacrylamide Disc Gel Electrophoresis Research Articles

Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis.

Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of 125I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.

Methods for the rapid purification of trehalase from Dictyostelium discoideum.

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.

Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

Embryonic chick duodenum maintained in organ culture responds to 1α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+(3–5 × 10 −5 m) or Zn 2+(10 −5-10 −4 m) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109Cd-binding activity ( 109CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35S-labeled CdBP—the only protein significantly labeled under the conditions employed—migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+- or Zn 2+-stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organcultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3, in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions.

Uroporphyrinogen I synthase from human erythrocytes: separation, purification, and properties of isoenzymes.

Uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from human erythrocytes was separated into two active protein peaks (A and B on DEAE-cellulose, by ammonium sulfate fractionation, on Sephadex G-100, and on DEAE-Sephadex A-50 with a NaCl gradient. The final purification was 613 and 743 times for A and B, respectively. The corresponding yields were 2.2 and 3.4% Fraction A was separated further into two (A1 and A2) active protein bands and fraction B into three (B1, B2, and B3) on analytical polyacrylamide disc gel electrophoresis. Bands A1 and A2 were identical with B1 and B2; B3 represented a third isoenzyme. Molecular weights (mean +/- SEM), measured by gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, were 38,000 +/- 1000 for B1 and 40,000 +/- 1000 for B2 and B3. Isoelectric focusing on 4% polyacrylamide gel separated both fractions A and B into three active protein bands. Maximal activity of the enzyme was found in gel cuts (5-mm) at pH 5.6 for both fractions A and B.

Physical Characterization of the Effect of Heat on Factor B Of the Properdin System

Abstract Purified factor B heated at 50°C for 30 min (ΔB) at physiological concentration (200–400 µg/ml) or higher sedimented as at least 4–5 species in linear sucrose density gradients. The lightest species of ΔB possessed slightly lower sedimentation rate, 5.3S, than native B, 5.8S. Consecutive heavier species differed by ∼ 2.6S. Serum fortified with radiolabeled 131I-B was heated; 131I-ΔB distributed broadely in the gradient. In analytical polyacrylamide disc gel electrophoresis (PAGE), ΔB migrated as multiple bands with the same or slower mobility compared with B, indicative of a polymerization phenomenon. Prior to PAGE, samples of ΔB were treated overnight at room temperature with 1% sodium dodecyl sulphate (SDS) with or without 8 M urea (Ur). This reduced the intensity of the slow bands and, concomitantly, increased the quantity of the fastest band. Upon additional treatment with 1% beta-mercaptoethanol (2-ME), ΔB migrated as a single band with the same mobility as B.

Purification of human factor IX

Factor IX was isolated from human plasma by a six-step procedure employing adsorption to DE-52 cellulose in batches, aluminium hydroxide adsorption, affinity chromatography on heparin-coupled Sepharose, preparative polyacrylamide disc gel electrophoresis, immunosorbent absorption, and DE-52 cellulose chromatography. Determination of factor IX activity and antigen revealed a specific activity of about 290 U per absorbancy unit at 280 nm. Analytical polyacrylamide disc gel electrophoresis showed one major and three to four minor bands with lower electrophoretic mobility. Disc gel electrophoresis in the presence of 10 M urea gave a double major band and two additional minor bands with higher electrophoretic mobility. One major band was observed in SDS polyacrylamide gel electrophoresis, and no change in mobility took place after reduction.

Immunochemical purification and characterization of ovine α-fetoprotein

Ovine α-fetoprotein was successfully isolated from fetal sheep serum by using rabbit anti-ovine α-fetoprotein linked to an agarose immunoadsorbent column. Antibody used in this affinity chromatography column was produced by immunizing a rabbit with highly purified α-fetoprotein·antibody complex to yield a monospecific antiserum to ovine α-fetoprotein. Following affinity chromatography, α-fetoprotein was further purified by preparative polyacrylamide disc gel electrophoresis ultimately yielding a 105-fold purification. The purified α-fetoprotein was homogeneous on analytical polyacrylamide disc gel electrophoresis. Ovine α-fetoprotein was found to be immunochemically related to human α-fetoprotein and to exhibit a molecular weight and amino acid composition similar to other mammalian α-fetoproteins.

Purification and characterization of two human pancreatic elastases.

Two proteases with elastolytic activity (elastases 1 and 2) have been isolated from activated extracts of human pancreatic tissue. The purification procedure for both elastases included ammonium sulfate fractionation followed by ion-exchange chromatography on CM-Sephadex C-50. Elastase 1 was further purified by chromatography on DEAE-Sephadex A-50. The homogeneity of both enzymes was demonstrated by Sephadex G-75 gel filtration, analytical polyacrylamide disc gel electrophoresis at pH 2.3, 4.5, and 8.3, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3. Both enzymes hydrolyzed undyed elastin as well as Remazol brilliant blue elastin and Congo red elastin. Activities and kinetic parameters using several synthetic substrates are also reported. The enzymes were further characterized in terms of molecular weight, amino acid composition, and N-terminal and penultimate amino acid residues. Their inhibition by the human serum protease inhibitors alpha2-macroglobulin and alpha1-antitrypsin was also studied. Elastase 1 appears to be very similar to human protease E (Mallory, P. A., and Travis, J. (1975), Biochemistry 14, 722). Elastase 2 is distinct from all human pancreatic proteases which have been characterized to date.

Human pancreatic carboxypeptidase B. I. Isolation, purification, and characterization of fraction II

Human carboxypeptidase B fraction II has been purified from pancreatic juice by DEAE-‘Sephadex’ chromatography, isoelectric focusing, and ‘Sephadex’ G-100 gel filtration. The enzyme has been characterized by analytical polyacrylamide disc-gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, K m determination, molecular weight determination on ‘Sephadex’ G-100, zinc analysis, and inhibition by metal chelating agents. Human carboxypeptidase B fraction II appeared homogeneous in analytical polyacrylamide disc-gel electrophoresis, but showed two components of 23,500 and 9,200 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Zinc analysis revealed 0.96 gram atoms of zinc per mole of enzyme, and a K m of 65 ± 3 μM was determined for hydrolysis of hippuryl- l-arginine.

Separation of binding proteins for cholecalciferol and 25-hydroxycholecalciferol from chick serum

The serum transport proteins in the chick of vitamin D and 25-hydroxyvitamin D have been studied by means of analytical polyacrylamide disc gel electrophoresis, gel filtration and column chromatography on DEAE-Sephadex. It has been possible to show the presence of, and subsequently separate, two carrier proteins from chick serum. One was found to bind predominantly 25-hydroxycholecalciferol while the other protein binds predominantly cholecalciferol.

Purification of Deoxythymidine Kinase by Preparative Disc Gel Electrophoresis and the Effects of Various Halogenated Nucleoside Triphosphates on Its Enzymatic Activity

Abstract A 530-fold purification of deoxythymidine kinase from extracts of Escherichia coli B cells has been achieved by the use of preparative polyacrylamide disc gel electrophoresis in the final stages of the purification procedure. Purified preparations of the enzyme contained one major protein band when examined by analytical polyacrylamide disc gel electrophoresis. 5-Iodo-2'-deoxycytidine 5'-triphosphate and 5-bromo-2'-deoxycytidine 5'-triphosphate were more potent allosteric activators of deoxythymidine kinase than the naturally occurring effector 2'-deoxycytidine 5'-triphosphate throughout the pH range of 5.2 to 9.3. Although 5-iodo-2'-deoxyuridine 5'-triphosphate (IdUTP) activated the enzymes at basic pH values, it exerted an inhibitory effect in a manner comparable to 2'-deoxythymidine 5'-triphosphate below pH 6.5. Whereas the inhibitory effect produced by IdUTP at pH 5.4 could not be prevented by even a 12-fold excess of dCTP, the activation caused by IdUTP at pH 7.8 is completely prevented by dTTP. Zone sedimentation studies in sucrose gradients have shown that the sedimentation coefficient of deoxythymidine kinase at pH 7.8 is increased by 5-bromo-2'-deoxycytidine 5'-triphosphate and 5-iodo-2'-deoxycytidine 5'-triphosphate to a greater extent than that produced by dCTP. IdUTP caused an even more marked effect on the sedimentation coefficient observed at pH 5.6 and 7.8 and was similar to that produced by dTTP. Linear reciprocal plots were obtained for varying concentrations of deoxythymidine in the presence of each halogenated effector at pH 5.4 and 7.8. At pH 5.4 IdUTP, like dTTP, was a competitive inhibitor of thymidine phosphorylation. The synthesis of 5-iodo-2'-deoxycytidine 5'-triphosphate from dCTP by direct iodination is described.

The Purification and Characterization of O-Acetylserine Sulfhydrylase-A from Salmonella typhimurium

Abstract O-Acetylserine sulfhydrylase-A is one of two highly specific enzymic activities which catalyzes the final step in l-cysteine biosynthesis in Salmonella typhimurium: O-acetyl-l-serine + sulfide → l-cysteine + acetate. The enzyme has been purified to near homogeneity and characterized. It is a pyridoxal phosphate-containing protein of s20,w 4.5 and molecular weight 68,000. Equilibrium sedimentation studies on native and carboxymethylated enzyme, analytical polyacrylamide disc gel electrophoresis in urea, pyridoxal phosphate analysis, tryptic peptide mapping, and amino acid analysis have shown that O-acetylserine sulfhydrylase-A consists of two identical subunits of molecular weight 34,000. The spectrum of native O-acetylserine sulfhydrylase-A has a visible absorption maximum at 412 mµ due to pyridoxal phosphate. When O-acetyl-l-serine is added to the enzyme, the visible absorption maximum shifts to 470 mµ. This shift is dependent upon the concentration of O-acetyl-l-serine, and the equilibrium constant, determined spectroscopically, for this reaction is 6 x 10-7 m. The shift in spectral absorption does not occur in the presence of sulfide. The Km for O-acetyl-l-serine, measured kinetically, in the presence of both substrates is 5 x 10-3 m.