Chromatography of a halophilic enzyme on hydroxylapatite in 3.4 M sodium chloride

Norberg, Per; B, von Hofsten

doi:10.1016/0005-2744(70)90239-1

Cited by 9 publications

(5 citation statements)

References 6 publications

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“…Initial attempts to purify GCR from extracts of H. halobium by techniques that have proven useful in the purification of other halobacterial enzymes were unsuccessful. Ion-exchange chromatography on DEAE-cellulose and hydrophobic chromatography on several alkyl-agarose resins (n-butyl-, n-octyl-and n-dodecylagarose) in buffer containing (NH4)2SO4 (14,18) and hydroxylapatite chromatography in buffer containing NaCl (24) each gave less than fourfold purification (data not shown). Also, several affinity resins were tested for their ability to bind GCR in buffers containing molar levels of either NaCl or (NH4)2SO4.…”

Section: Resultsmentioning

confidence: 99%

“…Only a small number of methods have been described that are suited to the purification of halobacterial enzymes (14,18,22,24), and of these methods most have strict salt requirements and result in satisfactory but low purification factors. The present use of immobilized-metal-ion resins in the isolation of two enzymes from H. halobium demonstrates that immobilized-metal-ion affinity chromatography in high-salt buffers can be a valuable chromatographic method for purifying halobacterial enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…oped that function well in buffers containing molar levels of NaCl or (NH4) 2SO4 (14,18,22,24), and a small number of halobacterial enzymes have been purified to homogeneity. Our limited success in purifying the GCR from H. halobium by established methods led us to examine additional approaches to purification in high-salt buffers.…”

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confidence: 99%

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The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Sundquist

Fahey

1988

J Bacteriol

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An NADPH-specific disulfide reductase that is active with bis-'y-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-y-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2" resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2O resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native Disulfide-specific reductases play a central role in the maintenance of aerobic cell cytoplasms in a chemically reduced state. Glutathione reductase (EC 1.6.4.2) is an NADPH-dependent disulfide reductase that is present in aerobic eubacteria and eucaryotic cells and maintains the peptide glutathione (y-Glu-Cys-Gly) predominantly in its reduced or thiol form (13, 39). Reduced glutathione is found at millimolar levels in cells and is a coenzyme for several enzymes that reduce intracellular disulfides and detoxify potent oxidants, such as peroxides (8,15,21). These processes yield the disulfide form of glutathione, which is cycled back to the thiol by glutathione reductase.Glutathione may not be the only thiol antioxidant utilized in aerobic cells; several aerobic bacteria that lack glutathione have been shown to produce other low-molecularweight thiols and disulfide reductase activities (23,32,35). One such group of bacteria are the halophilic archaebacteria, which have been shown to lack reduced glutathione but contain millimolar levels of -y-glutamylcysteine (,y-Glu-Cys) (23). This finding suggested that the halobacteria may employ a redox metabolism similar to that established for glutathione but based on -y-Glu-Cys. Central to this metabolism would be a disulfide reductase that is active with -y-Glu-Cys disulfide (bis-y-glutamylcystine), and the detection of an NADPH-dependent reductase activity in crude extracts of Halobacterium halobium indicated that this enzyme was present (23). In this paper we report the complete purification of an NADPH-specific bis-y-glutamylcystine reductase (GCR) from extracts of H. halobium. The characteristics of GCR and the possible functions of -y-Glu-Cys in the halobacteria are also discussed.Purification of most halobacterial enzymes requires the use of high-ionic-strength buffers (,u > 2 M) to retain enzyme activity (17). Chromatographic methods have been devel-* Corresponding author.oped that function well in buffers containing molar levels of NaCl or (NH4) 2SO4 (14,18,22,24), and a small number of halobacterial enzymes have been purified to homogeneity. Our limited success in purifying the GCR from H. halobium by established methods led us to examine additional approaches to purification in high-salt buffers. Immobilizedmetal-ion affinity chromatography (also known as metal chelate affinity chromatography) (26, 27), a technique used...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Sundquist

Fahey

1988

J Bacteriol

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“…1975 0.25 0.50 1.00 1.50 2.00 2.50 3.00 DEAE-cellulose chromatography seems not to have been applied before to the purification of halophilic enzymes, although other ion exchangers have been used (Aitken & Brown, 1972;Hochstein & Dalton, 1973). Hydroxyapatite chromatography (Norberg & von Hofsten, 1970;Dundas, 1970;Hochstein & Dalton, 1973) and gel filtration (Aitken & Brown, 1972) have been used before for the purification of halophilic enzymes. The elution pattern of the citrate synthase from Sephadex G-200 in buffer 1 was the same as that previously obtained in buffer 2 (Cazzulo, 1973).…”

Section: Discussionmentioning

confidence: 99%

Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum

Higa¹,

Cazzulo²

1975

Biochemical Journal

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1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.

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“…Not only is it inconvenient for largescale purification, but it is also a fire hazard. The present paper demonstrated that the nuclease could be conveniently purified by adsorption to hydroxyapatite, which is recommended for purifying proteins in the presence of high salt concentration (6,19).…”

Section: Discussionmentioning

confidence: 82%

Properties of the halophilic nuclease of a moderate halophile, Micrococcus varians subsp. halophilus

Kamekura¹,

Ōnishi²

1978

J Bacteriol

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The halophilic nuclease of Micrococcus varians ATCC 21971 hydrolyzed thymidine 5'-monophospho-p-nitrophenyl ester at a rate that increased with the NaCl concentration up to saturation. The nuclease attacked RNA and DNA exonucleolytically and processively, producing 5'-mononucleotides. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 99,000, approximately the same as that previously determined for the native enzyme. Examination of amino acid composition showed that acidic amino acids were in high excess over basic amino acids.

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Chromatography of a halophilic enzyme on hydroxylapatite in 3.4 M sodium chloride

Cited by 9 publications

References 6 publications

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum

Properties of the halophilic nuclease of a moderate halophile, Micrococcus varians subsp. halophilus

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