Fast Protein Liquid Chromatography-gel Filtration Research Articles

Identification of two different glyceraldehyde-3-phosphate dehydrogenases (phosphorylating) in the photosynthetic protist Cyanophora paradoxa

Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NAD- and NADP-dependent, have been found in cell extracts of the cyanelle-bearing photosynthetic protist Cyanophora paradoxa. Whereas the two G3P dehydrogenase activities were detected with similar specific activity levels (0.1 to 0.2 U/mg of protein) in extracts of the photosynthetic organelles (cyanelles), only the NAD-dependent activity was found in the cytosol. Thus, a differential intracellular localization occurred. The perfect overlapping of the two G3P dehydrogenase activity peaks of the cyanelle in both hydrophobic interaction chromatography and subsequent FPLC (fast protein liquid chromatography) gel filtration indicated that the two activities were due in fact to a single NAD(P)-dependent G3P dehydrogenase (EC 1.2.1.-) with a molecular mass of 148,000. SDS-PAGE of active fractions from FPLC gel filtration showed that the intensity of the major protein band (molecular mass, 38,000) of the enzyme preparation clearly paralleled the activity elution profile, thus suggesting a tetrameric structure for the cyanelle dehydrogenase. On the other hand, FPLC gel filtration analysis of the cytoplasmic fraction revealed a NAD-dependent G3P dehydrogenase with a native molecular mass of 142,000, being equivalent to the classical glycolytic enzyme (EC 1.2.1.12) present in the cytosol of all the organisms so far studied. The significance of these results is discussed taking into account that the cyanobacteria, photosynthetic prokaryotes which share many structural and biochemical features with cyanelles and are considered as their ancestors, have a similar NAD(P)-dependent G3P dehydrogenase.

Analysis of the immunodominant antigens of Corynebacterium pseudotuberculosis

Antibodies to seven antigens in a whole cell lysate of Corynebacterium pseudotuberculosis ranging in molecular mass from 22 to 120 kilodaltons (kDa) were present in sera of 40 sheep and goats infected with C. pseudotuberculosis. Three antigens of about 120, 68, and 31.5 kDa in size were consistently detected with sera from all animals and twenty-two sera had antibodies to 64, 43, 40, and 22 kDa antigens. None of these antigens were detected by sera from 160 sheep in a C. pseudotuberculosis-free research flock. An NaCl extract of C. pseudotuberculosis cells contained one major protein of about 31.5 kDa and four minor proteins of 68, 64, 43, and 22 kDa in molecular mass as shown by Coomassie Blue staining. Immunoblot analysis demonstrated that the three immunodominant antigens identified in the whole cell extract were contained in the NaCl extract. The 31.5-kDa protein was purified from the NaCl extract by fast-protein liquid chromatography gel filtration to near homogeneity. The purified 31.5-kDa protein showed phospholipase D activity as indicated by synergistic hemolysis with Rhodococcus equi factors and sphingomyelinase activity. The 31.5-kDa protein reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis. This serum also had phospholipase D neutralizing activity. On the basis of its molecular mass, biological activity, N-terminal amino acid sequence analysis, and immunoreactivity, the 31.5-kDa protein was identified as the phospholipase D exotoxin of C. pseudotuberculosis.

Human milk proteins: separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion-exchange chromatography

Human milk proteins are of nutritional and physiological significance to the newborn infant. To further study these proteins, a rapid procedure to separate and analyze human milk whey proteins was developed using fast protein liquid chromatography (FPLC). First, to separate whey proteins from casein, different variables such as low- or high-speed centrifugation at different temperatures with or without adjustment of pH to 4.6 or 4.3 and with or without addition of calcium to whole milk or skim milk were tested. Each variable was evaluated by gel filtration, anion-exchange chromatography, sodium dodecyl sulphate (SDS)-polyacrylamide gradient gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The optimum method for a discrete separation of whey and casein is the adjustment of whole milk to pH 4.3 with addition of 60 mmol calcium/L, followed by ultracentrifugation. Rapid and sensitive separation and analysis of whey proteins was achieved by FPLC gel filtration and anion-exchange chromatography.

Microheterogeneity of the growth-associated neuronal protein B-50 (GAP-43) : Contribution of phosphorylation by protein kinase C

The neuron-specific, growth-associated protein B-50, also known as GAP-43, F1 and neuromodulin, shows a striking heterogeneous behaviour in many chromatographic and electrophoretic systems. A modulatory function has been proposed for the protein in receptor-mediated processes in the presynaptic membrane. Fatty acid acylation, calmodulin binding and phosphorylation appear to be tools in this respect. At least three discrete isoforms were present in separations made by reversed-phase fast protein liquid chromatography (FPLC) of the phosphorylated protein. In anion-exchange FPLC chromatography a conglomerate of eight peaks was eluted, which migrated as eight parallel curves in electrophoretic mobility studies. After dephosphorylation of the protein this number was reduced to two. Under non-reducing conditions, the phosphoprotein was eluted from an FPLC gel filtration column at M r = 270 kDa, i.e. 8–12 times the size of the monomer ( m = 23.6 kDa). In sodium dodecyl sulphate polyacrylamide gel electrophoresis all isoforms showed only B-50 at M r of 48 kDa and its breakdown product ( M r = 40 kDa) in a constant ratio. It was concluded that phosphorylation by protein kinase C of a single serine residue is only one factor in the microheterogeneity of B-50. Multimeric forms may also add to the heterogeneous behaviour of phosphorylated B-50.

Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor.

The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter. Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid. After induction with isopropyl-beta-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments. A scheme to purify the overproduced fragments was developed. Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively. Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca. 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons). However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer. When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons. Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found. Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.

The v-rel oncogene product is complexed to a 40-kDa phosphoprotein in transformed lymphoid cells.

The transforming protein encoded by the v-rel oncogene of avian reticuloendotheliosis virus (REV-T) is a very low copy number molecule in the cytosol of transformed cells. Analysis of cytosolic extracts from a REV-T-transformed lymphoid cell line by gel filtration on Sephacryl S-300 indicated that most of the v-rel oncogene product, pp59v-rel, eluted with an apparent molecular mass of 400 kDa. The size of this complex was confirmed by analysis on a fast-protein liquid chromatography gel filtration column. A 40-kDa cellular protein copurified with pp59v-rel on sequential gel filtration on Sephacryl S-200 and immunoaffinity chromatography with a monoclonal antibody directed against pp59v-rel. The 40-kDa cellular protein could also be immunoprecipitated together with pp59v-rel from cell extracts of [35S]methionine-labeled cells, suggesting that pp59v-rel is complexed with the 40-kDa protein in transformed lymphoid cells. Both the 59- and 40-kDa proteins were phosphorylated when the highly purified preparation containing pp59v-rel was incubated with [gamma-32P]ATP and 10 mM MgCl2 in vitro. The identity of the kinase in the highly purified preparation containing pp59v-rel, however, is unknown. Immune complexes recovered from extracts of REV-T-transformed lymphoid cells labeled with [32P]orthophosphate also contained the 59- and 40-kDa phosphoproteins. These observations suggest that pp59v-rel is complexed with a 40-kDa cellular phosphoprotein to form a 400-kDa heteropolymer in the cytoplasm of transformed lymphoid cells.

Estrogen sulfotransferase of mouse placenta: behaviour and characteristics during partial purification.

Mouse placental estrogen sulfotransferase (ST) was partially purified by fast protein liquid chromatography (FPLC) gel filtration in combination with FPLC anion exchange. Owing to the highly unstable nature of the enzyme, large increases in specific activity were not obtained. Storage of the ST in the presence of thiol groups at -20 degrees C stabilized the enzyme considerably. Forty-three percent of the cytosolic ST was bound to an Affi-Gel blue column and eluted as a broad peak at approximately 0.8 M NaCl. The use of the latter procedure, in combination with FPLC gel filtration, did not increase the specific activity substantially. Larger increases in specific activity were obtained using agarose-hexane-adenosine-3',5'-diphosphate affinity chromatography. The bound ST activity was eluted under a single peak at 1 mM ADP. Increases in specific activity following use of this column averaged 54-fold but could reach 90-fold. Attempts at further purification of this material resulted in low recovery and decreased specific activity. Velocity versus substrate concentration curves show that estrone and particularly estradiol inhibit the partially purified mouse placental sulfotransferase above 0.1-0.25 microM substrate concentrations.

Purification and Species Distribution of Rubisco Activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.

Characterization of a rat salivary sialoglycoprotein complex which agglutinates Streptococcus mutans

Rat saliva agglutinated Streptococcus mutans Ingbritt and NCTC 10449 and Streptococcus sanguis NCTC 7864 but not S. mutans NCTC 10921, GS 5, or LM 7, Streptococcus sobrinus 6715-13 or OMZ 65, or Streptococcus cricetus HS 6, as measured turbidometrically. The specificity of agglutination by rat saliva was the same as that by human saliva. Agglutination was associated with a mucin complex (rat salivary agglutinin complex [rS-A]) of sulfated sialoglycoproteins, with a trace of associated lipid and an apparent Mr of 1.6 X 10(6), isolated by gel-filtration Fast Protein Liquid Chromatography. The complex was dissociated in a high-ionic-strength buffer containing 6 M urea and then fractionated by gel filtration and anion-exchange Fast Protein Liquid Chromatography into four sulfated sialoglycoprotein components, designated rS-A-1Q1, rS-A-1Q2, rS-A-1Q3, and rS-A-2, with rS-A-1Q2 being polydisperse through differential glycosylation of the polypeptide backbone. The dissociation destroyed agglutination activity. The polypeptide backbones contained up to 42% serine plus threonine and up to 40% glycine plus alanine plus proline plus valine. The carbohydrate moiety of the rS-A sialoglycoproteins consisted of N-acetylgalactosamine, sialate, galactose, fucose, N-acetylglucosamine, and small amounts of mannose, with the predominant sugar being N-acetylgalactosamine. Agglutination was inhibited by 1 mM EDTA but was restored by 1.5 mM CaCl2. Agglutination was also inhibited by 5 mM CaCl2; nonimmune sera; cationic polymers; and wheat germ, lentil, soybean, and peanut lectins. However, agglutination was not affected by lipoteichoic acid, various anionic proteins, or various sugars. Neuraminidase treatment of rS-A did not affect activity, but tryptic digestion of S. mutans did prevent agglutination. The results are consistent with calcium bridging the negative groups within the rS-A complex and allowing the approach of rS-A to the bacterial cell surface to effect a specific conformational attachment.