Gradient sievorptive chromatography. Focusing system for the separation of cellular components

Ornithine decarboxylase (ODC) was purified 25000-fold from calf liver to apparent homogeneity by methods developed to circumvent the lability of the enzyme. Appropriate ratios of sample protein applied to column size and/or gradient size were derived for each purification procedure (ion-exchange, gel filtration ahd hydroxylapatite chromatography, electrophoresis, and thiol affinity chromatography) to maintain enzymatic activity. The enzyme was labile to dilution at all steps of the purification; the inclusion of poly(ethylene glycol) or additional protein decreased but did not eliminate the activity loss. The purified enzyme had a Stokes radius of 3.14 and a molecular weight of 54000. The Km for ornithine was 0.12 mM, and pyridoxal phosphate was 2.0 microM; the pH optimum for the decarboxylation reaction was 7.0. Analysis by sievorptive ion-exchange chromatography indicated the presence of three ionic forms. In the presence of Tris-barbital buffer containing thioglycolic acid, the ODC preparation assumed an apparent molecular weight of 100000 and a Stokes radius of 4.5 and retained full catalytic activity.

Section: Resultsmentioning

confidence: 99%

Ornithine decarboxylase from calf liver. Purification and properties

Haddox¹,

Russell²

1981

“…This procedure is the ion filtration technique as described by Kirkegaard et al (1972). The effluent (10 mL) containing RNA polymerase activity was concentrated to 3.4 mL by application at 0.35 ( 4)2$04 to a 30-mL DEAE-Sephadex column equilibrated at 0.1 M (NH4)2S04 in TGMED and eluted with 0.45 M (NH4)2S04 in TGMED (although not previously described, this concentration method is a direct consequence of the ion filtration and gradient sievorptive elution phenomena; for full discussion of these phenomena, see Kirkegaard et al, 1972, andKirkegaard, 1973). The effluent (3.4 mL) from the concentration column was resolved into RNA polymerases I, II, and III by gradient sievorptive elution on a 30-mL DEAE-Sephadex column as previously described Morris & Rutter, 1976).…”

Section: Methodsmentioning

confidence: 99%

Biochemistry of the amatoxins: preparation and characterization of a stably iodinated α-amanitin

Morris¹,

Venton²,

Kelley³

1978

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.

“…The resulting supernatant fraction was adjusted to 70% saturation by the addition of 0.21 g/mL of ammonium sulfate and centrifuged as before. The 40-70% ammonium sulfate pellet, which contains all the DNA ligase activity, was dissolved in 0.75 M KC1 and purified by gradient sievorptive chromatography (Kirkegaard, 1973). The sample was applied to a 350-mL DEAE-Sephadex A-25 column containing a linear gradient of from 0 to 125 mM KC1 in 0.1 M Tris (pH 7.5)-1 mM 2-mercaptoethanol-0.1 mM EDTA and was eluted with 1 M KC1.…”

Section: Methodsmentioning

confidence: 99%

L cell DNA ligase joins RNA to DNA on a DNA template

Bedows¹,

Wachsman²,

Gumport³

1977

L cell DNA ligase catalyzes a covalent linkage between 5'-hosphoryl oligodeoxyribonucleotides and 3'-hydroxyl oligoribonucleotides on a complementary polydeoxyribonucleotide template. This reaction occurs to a substantially lesser extent than does the sealing of DNA to DNA. The joining of [5'32P]d(pA)12-18 to (Ap)11A on poly[d(T)] or of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) ON POLY[D(C)] was demonstrated by the formation of alkaline phosphatase resistant radioactivity. The 32P of the hybrid reaction products became sensitive to the action of alkaline phosphatase after treatment with alkali. Furthermore, hydrolysis of the products of the linkage of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) on poly[d(C)] with micrococcal nuclease and spleen phosphodiesterase resulted in the formation of [3'-32P]IMP. Attempts to seal [5'-32p[-(pA)12 to d(Ap)11-17A on poly[d(T) or [5'-32P]oligo(pI) to d(Gp)11-17G on poly[d(C)] were unsuccessful.