We have studied the effect of protein phosphokinase (EC 22.214.171.124; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 126.96.36.199; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity...
We have observed low-molecular weight carboxyterminal fragments of the human choriogonadotropin (hCG) beta-subunit in the urines of several women with choriocarcinoma, and we have characterized one fragment in detail. Its apparent molecular weight by gel chromatography on Sephadex G-100 was 14,200. The fragment was not adsorbed to concanavalin A-Sepharose, indicating that it lacked the asparagine-linked carbohydrate groups of intact hCG beta. It was active in radioimmunoassays (RIA) using antisera either to the hCG beta carboxyterminal peptide (CTP) or to the desialylated hCG beta CTP (hCG beta as-CTP), indicating the presence of not only the hCG beta carboxyterminus but also desialylated O-serine-linked carbohydrate side chains on the fragment. It lacked luteinizing hormone/choriogonadotropin radioreceptor activity and hCG beta conformational immunoreactivity (SB6 RIA). On Sephadex G-100 gel chromatography, the elution profiles of this fragment and the hCG beta as-CTP(115-145) prepared by trypsin digestion of as-hCG were essentially indistinguishable (apparent molecular weights 14,200 and 14,000, respectively). The immunological characteristics of the fragment in both hCG beta CTP and hCG beta as-CTP RIA were indistinguishable from those of the hCG beta as-CTP(115-145) glycopeptide. Carboxyterminal fragments of hCG beta were evident in urine specimens obtained from 10 of 11 patients with choriocarcinoma but not in those obtained from normal subjects who were given an intravenous infusion of highly purified hCG. Of six pregnant women...
Synthesis of somatostatin-14 (S-14) could occur through direct enzymatic processing of precursor somatostatin (prosomatostatin) or via sequential breakdown of prosomatostatin leads to somatostatin-28 (S-28) leads to S-14. If direct processing is important, it should theoretically generate S-14 and a molecule equivalent to prosomatostatin without the S-14 sequence. In an attempt to identify such a molecule, I characterized the molecular forms of S-28(1-12)-like immunoreactivity (S-28(1-12) LI) in the rat pancreas and compared the relative amounts of these forms with those of S-14-like immunoreactivity (S-14 LI). Pancreatic extracts were chromatographed on Sephadex G-50 and Sephadex G-75 columns (Pharmacia Fine Chemicals Inc., Piscataway, NJ) under denaturing conditions and immunoreactivity in the eluting fractions was analyzed by region-specific radioimmunoassays (RIAs). For RIA of S-28(1-12) LI we used a newly developed rabbit antibody R 21 B, 125I-Tyr12 S-28(1-14), and S-28(1-12) standards. This system detects S-28, S-28(1-12), high molecular weight forms of S-28(1-12), but not S-14. S-14 LI was measured using antibody R149, which detects S-14, S-28, and higher molecular weight S-14-like substances, but not S-28(1-12). Three forms of S-28(1-12) LI were identified: Mr 9...
Nitrate reductase (NR)-inactivating proteins from corn roots (Wf-9 × 38-11) and rice cell suspension cultures were tested against a partially purified NR obtained from corn leaves (W64A × W182E). The corn protein was purified 921-fold and the rice protein, 1,660-fold using standard purification procedures. Approximate molecular weight values were 75,000 for the corn protein, and 150,000 for the rice protein as determined by Sephadex G-100 gel filtration. The Sephadex-treated proteins were characterized by electrophoresis on polyacrylamide gels. With a running pH of 9.4 the corn protein remained at the origin whereas the rice protein migrated with an RF value of 0.49. With a running pH of 4.0 the corn protein migrated with an RF value of 0.25. With the corn protein the activities of NR inactivation and hydrolysis of azocasein were detected in the same protein band. The rice protein, however, had no associated protease activity. From sodium dodecyl sulfate gel electrophoresis, there was one major protein band with an estimated molecular weight of 66,000 in corn protein. In rice protein four bands were observed with estimated molecular weights of 73,000, 66,000, 62,500, and 58,500, respectively.
Two forms of α-galactosidase (α-d-galactoside galactohydrolase, E.C. 188.8.131.52) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total α-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total α-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.
The elution pattern of serum proteins and the distribution of rubella HAI activity in 95 sera from 65 cases were determined after gel filtration with (a) Sephadex G-200 and (b) Bio-Gel A-5M. Rubella HAI antibody in peak 1 after Sephadex G-200 fractionation of early convalescent sera consists of both IgM and high molecular weight IgA. However, these two classes of antibody can be distinguished by gel filtration with Bio-Gel A-5M. Bearing these differences in mind, the results of fractionation with the two gels correlate very well although the use of Bio-Gel A-5M gives a slightly less sensitive technique for the diagnosis of recent infection.
To investigate a possible function of plasma albumin in the vectorial transport of organic anions by the liver, the plasma disappearance of sulfobromophthalein (BSP) and its interaction with plasma and liver cytosolic proteins were studied in normal rats and mutant Nagase analbuminemic rats (NAR). After intravenous administration of BSP, plasma BSP decreased rapidly in both NAR and control animals: plasma clearance values of BSP in NAR and controls were 12.45 and 7.40 ml/min per kg, respectively. Gel exclusion Sephadex G-100 chromatography of BSP with control rat serum revealed a protein peak in the void volume and another in the albumin fraction. BSP chromatographed exclusively with the albumin fraction; binding of BSP to plasma albumin occurred stoichiometrically. Similar studies with NAR serum revealed a single protein peak, in the void volume; a small amount of BSP chromatographed with this protein peak. The amount of BSP that chromatographed with NAR serum protein(s) was 8% of that with control rat serum albumin. Sephadex G-100 chromatography of BSP with control rat liver cytosol revealed four peaks of protein-bound BSP in fractions corresponding to the void volume (fraction X), albumin, glutathione S-transferases (fraction Y...
A cytokinin-binding protein has been isolated from wheat germ via ammonium sulfate precipitation, carboxymethyl Sephadex chromatography, and affinity chromatography on a column substituted with a derivative of kinetin riboside. On Sephadex G-200, the protein migrated with an apparent molecular weight of 122,000 daltons. The dissociation constant for kinetin was determined by equilibrium dialysis to be 1.2 micromolar; N6-benzylaminopurine and N6-(Δ2-isopentenyl)adenine were also strongly bound. Little affinity was exhibited toward either cis-zeatin or trans-zeatin.
DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.
Various types of rubella complement-fixing (CF) antigen preparations derived from infected BHK-21 cell cultures were examined for stability to certain chemical and physical agents. Antigens studied included: infected culture fluids concentrated 100-fold, ether-treated fluid concentrates, alkaline buffer extracts of infected cells, and large- and small-particle CF antigens separated by Sephadex gel filtration. All preparations were stable at -20 C for months, and were unaffected by three cycles of freezing and thawing. Ether treatment rendered antigens highly susceptible to thermal inactivation at 56 C; untreated antigens were inactivated slowly and showed a persistent fraction of activity even after 2 hr. Large-particle antigen was found to be slightly more susceptible than small-particle antigen to thermal inactivation and ether treatment. CF activity in alkaline buffer extracts was more labile than that in untreated or ether-treated fluid concentrates upon dialysis, Sephadex gel filtration, and sucrose density gradient centrifugation.
A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg2+, Ag+, Zn2+, and KMnO4.
The serotype antigens of Streptococcus mutans have been described as cell wall-associated polysaccharides. In this study, an additional wall polysaccharide antigen was purified and characterized from S. mutans strain 6715-T2, a mutant of 6715 (serotype g). Strain 6715-T2 lost the serotype antigen during animal passage. Rhamnose-containing carbohydrate fractions were solubilized from bacterial cells by extraction with 5% trichloroacetic acid at 4 degrees C for 18 h and with 0.01 N HCl at 100 degrees C for 20 min. Extracts were combined and purified on columns of diethylaminoethyl-Sephadex A-25 and Sephadex G-100. The purified sample contained 59% rhamnose, 31% glucose, 2.2% protein, and 0.24% phosphorus. The purified rhamnose-glucose polysaccharide (RGP/6715-T2) reacted strongly with antisera to whole cells of 6715-T2. Agar gel diffusion and comparative immunoelectrophoresis studies revealed that RGP/6715-T2 was serologically distinct from the serotype g and d polysaccharide antigens. These techniques also indicated immunological identity between RGP/6715-T2 and RGP/B13, a rhamnose-glucose polymer previously isolated from S. mutans B13, a serotype d strain. Antigen immunologically identical to RGP/6715-T2 was detected both in Rantz-Randall extracts from whole cells of S. mutans strains 6715...
The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [3H]gibberellin A1 (GA1) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the 3H-activity bound to this protein was largely [3H]GA1 and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA1. Both biologically active and inactive GAs and non-GAs were able to inhibit GA1 binding. [3H]GA1 binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.
NADP+-isocitrate dehydrogenase (threo-DS-isocitrate: NADP+ oxidoreductase [decarboxylating]; EC 184.108.40.206) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg2+ or Mn2+, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP+, dl-isocitrate, Mn2+, and Mg2+. Their Km values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP+. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP+ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP+ and isocitrate...
Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF.
An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The Km values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP+ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP+ oxidoreductase. The effect of NaCl and MgCl2 concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP+ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.
The amination of α-ketoglutarate (α-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca2+. The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca2+ in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca2+ had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca2+.
Intercellular fluids of compatible race-cultivar interactions of Cladosporium fulvum and tomato contain specific elicitors of necrosis. These elicitors which are of fungal origin induce chlorosis and necrosis in resistant but not in susceptible plants. With the tomato cultivar Sonatine (carrying resistance gene Cf9, resistant to the fungal races 0, 4, 5, 2, 2.4, and 2.4.5 but susceptible to race 220.127.116.11) as the test plant for assaying necrosis-inducing activity, we isolated and partially characterized an elicitor of necrosis on this cultivar. The elicitor bound to CM-Sephadex but not to DEAE-Sephadex; it was stable to heat (10 minutes at 100°C), HCl (0.01 normal), NaOH (0.01 normal), and NaIO4 (0.02 molar), sensitive to pronase and protease (from Bacillus polymyxa) but not to other proteases such as α-chymotrypsin and trypsin. After electrophoresis of partially purified elicitor preparations under low pH conditions, the necrosis-inducing activity was association with a peptide with an apparent molecular weight of 5500. Races 0, 4, 5, 2.4, and 2.4.5 but not race 18.104.22.168 produced this elicitor in high yields. The elicitor is probably a product of avirulence gene A9 which is present in all races except in race 22.214.171.124 and induces necrosis in cultivars carrying resistance gene Cf9.
Tomatoes (Lycopersicon esculentum L.) contained a high level of β-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of β-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, β-galactosidases I and III levels decreased but the β-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, Km value with p-nitrophenyl-β-galactoside, and stability with respect to pH and temperature. β-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of β-1, 4-linked galactose. The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.
A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE-Sephadex A-25. The major cadmium fraction was purified further by gel filtration on Sephadex G-75 in 1 molar KCl buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was 1:2.7. Spectroscopic measurements indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days.