Purification by affinity chromatography of thermostable glyceraldehyde 3‐phosphate dehydrogenase from <i>Thermus aquaticus</i>

Hocking, Jennifer; Harris, Ieuan

doi:10.1016/0014-5793(73)80812-9

Cited by 50 publications

(26 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The removal of NAD was checked by measuring the absorbance at 280 nm and 260 nm (A2so/Az60 > 1.4). The dialyzed enzyme was applied onto a column of NAD-Sepharose (1 ml bed volume; prepared according to Hocking and Harris [7]), and subsequently rinsed batchwise with 3 x 1 ml 0.1 M NaCl, 3 x 1 ml 0.2 M NaC1, 3 x 1 ml 0.3 M NaCl and 3 x 1 ml 0.4 M NaCl (all in buffer A), after which the bound GAPDH was eluted with buffer A containing 1 mM NAD. The peak fractions were pooled and ammonium sulphate was added (1 M final concentration) after which the enzyme was stored at 4°C.…”

Section: Purification Of Glyceraldehyde-phosphate Dehydrogenasementioning

confidence: 99%

“…5 (1) is the glycosomal GAPDH from T. brucei. For the calculation of percentage homology the areas containing insertions or deletions have not been taken into account (la) T. brucei cytosolic (2) E. coli (3) B. stearothermophihs (4) T. aquaticus (5) Yeast (6) Drosophila melanogaster (7) Lobster (8) Chicken (9) pig (10) Rat (11) brucei cytosolic GAPDH, therefore, must await the elucidation of its complete sequence. The large difference in sequence of the two T. brucei GAPDH isoenzymes is at complete variance with what we have previously found in the case of the glycosomal and cytosolic isoenzymes of phosphoglycerate kinase.…”

Section: Y D S V H G K F K H S V S T T K S K P S V a K D D T L V~' N mentioning

confidence: 99%

See 1 more Smart Citation

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Misset

Beeumen²,

Lambeir

et al. 1987

European Journal of Biochemistry

View full text Add to dashboard Cite

Trypanosoma bruceicontains two glyceraldehyde-phosphate (GAPDH; EC 1.2.1.12) isoenzymes; one is located in glycosomes and represents 80% of the total activity, whereas the other is present in the cytosol. The purification of the cytosolic GAPDH, which is identical in both bloodstream-form and insect-stage trypanosomes, is described, and the enzyme compared with its glycosomal counterpart. Cytosolic GAPDH is specific for NAD. It is a tetrameric enzyme with subunits of 33.5 kDa, 5 kDa smaller than those of the glycosomal GAPDH. The native enzyme has a pl of 7.9, which is 1.5 pH units less basic than the glycosobal enzyme. Both enzymes display maximal activity at pH 8 but the cytosolic enzyme has a much broader activity profile especially towards lower pH values.Sequence comparison of the first 85 amino acids reveals that the N-terminal parts of both isoenzymes differ by 52%. The N terminus of the cytosolic isoenzyme resembles the corresponding N termini of ten other known GAPDH sequences in that they all lack three amino-acid insertions, which so far only have been found in the glycosomal isoenzyme of T. brucei. This observation explains in part the great difference in subunit size between the two T. brucei isoenzymes and suggests that at least one of these insertions is responsible for import of the glycosomal isoenzyme into the organelle.In Trypanosoma brucei, glyceraldehyde-phosphate dehydrogenase (GAPDH) has been found in two separate cellular compartments: the cytosol and the glycosomes [l]. However, thus far no direct proof has been brought forward for the existence of two separate isoenzymes in this organism.Recently two tandemly linked identical genes coding for GAPDH have been identified in the genome of T. brucei [2]. From sequence data it was inferred that both genes code for the glycosomal enzyme; subunit molecular mass, isoelectric point 131, amino-acid composition and the N-terminal sequence of the isolated glycosomal protein [2] were all in excellent agreement with the properties predicted from the amino acid sequence as deduced from the gene. A gene coding for a possible cytosolic isoenzyme has not yet been identified.In this paper we now present evidence for the existence of a separate cytosolic isoenzyme of GAPDH in cultured procyclic (insect-stage) as well as in bloodstream-form trypanosomes. We describe its purification, characterization and N-terminal amino acid sequence and compare the properties of both the glycosomal and the cytosolic counterparts.

show abstract

Section: Purification Of Glyceraldehyde-phosphate Dehydrogenasementioning

confidence: 99%

Section: Y D S V H G K F K H S V S T T K S K P S V a K D D T L V~' N mentioning

confidence: 99%

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Misset

Beeumen²,

Lambeir

et al. 1987

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Previously several NAD '-Sepharose derivatives have been reported to bind glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle [4,18] and from thermophilic microorganisms [19,20]. Hence, the behavior of the enzyme isolated from sturgeon muscle is similar to that of these enzymes which are known, like the sturgeon enzyme, to bind NAD' strongly with negative cooperativity [9,.…”

Section: Interactions Of Yeast and Sturgeon Glyceraldehyde-3-phosphatmentioning

confidence: 96%

Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase. A Comparative Study of the Enzymes from Yeast and Sturgeon Muscle

1977

View full text Add to dashboard Cite

Sturgeon glyceraldehyde-3-phosphate dehydrogenase has been strongly adsorbed on an 8-azolinked NAD+-Sepharose derivative. Specific elution of the matrix-bound enzyme was obtained with relatively low concentrations of NAD' (20-100 pM) in elution buffer. Recovery of adsorbed enzyme amounted to 90 -95 "/, when an hydrophilic 1,3-diaminopropan-2-01 spacer arm was used instead of the more conventional diaminohexyl derivative. A capacity of 0.8 mg of sturgeon enzyme per g of derivative was estimated either under batchwise conditions or by frontal chromatography. Under similar conditions, purified apoglyceraldehyde-3-phosphate dehydrogenase from yeast did not bind significantly to the same NAD+ derivative. Crude extracts of sturgeon muscle can be purified more than 15-fold upon chromatography on this derivative.Unlike the sturgeon enzyme, yeast glyceraldehyde-3-phosphate dehydrogenase was significantly adsorbed to an N6-(6-aminohexyl)-AMP derivative under chromatographic or batchwise conditions. At least 40 units (i.e. 0.2-0.3 mg) of yeast glyceraldehyde-3-phosphate dehydrogenase can be bound per g of matrix. An 8-fold purification of the enzyme can be obtained upon specific elution with NAD'. Relatively large amounts of yeast enzyme ( i .~. up to 100 mg) can be partially purified by this one-step procedure.The distinct adsorption properties of yeast and sturgeon glyceraldehyde-3-phosphate dehydrogenases are discussed in the light of the known cooperative coenzyme binding properties of these enzymes. The relevance of affinity chromatography to the preparation of sizeable amounts of glyceraldehyde-3-phosphate dehydrogenase is discussed and compared with conventional procedures. Possible interference of glyceraldehyde-3-phosphate dehydrogenase with the 'general ligand' binding of other dehydrogenase and kinases in crude extracts is discussed.Affinity chromatography of NAD+-dependent dehydrogenases has been widely studied over the past five years [I -71. Successful resolution of complex mixtures of several dehydrogenases has been achieved by adsorption of the mixture on affinity matrices containing an immobilized 'general ligand' (derivative of AMP or NAD') [2], followed by a selective elution of the chosen dehydrogenase [2-61. This technique has been found to be very useful for rapid and efficient purification of small amounts (i.e. a few units of activity) of various dehydrogenases [5-81. It is not clear, however, at the present time, if affinity chromatogEnzymes. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2

show abstract

“…In common with other enzymes from T. aquatcs previously studied in this laboratory (3)(4)(5), Taq I is stable and active at temperatures of up to 700. Contaminating nonspecific exo-and endonucleases are inactivated at this temperature and the availability of a thermostable restriction enzyme greatly extends the range of conditions that may be used to study DNA structure and the mode of action of restriction endonucleases.…”

Section: Discussionmentioning

confidence: 99%

“…Restriction-like enzymes of this general type have become indispensable for the physical mapping of genomes and for DNA sequencing (1,2), and consequently enzymes with new specificities are of particular interest. Enzymes from an extreme thermophile, Thermus aquaticus, are exceptionally stable to heat and to protein-denaturing reagents (3)(4)(5), and we have sought to prepare a thermostable restriction endonuclease from this source that would aid in the study of DNA structure and of the mode of action of restriction-like enzymes. We report here on the partial purification of a new restriction endonuclease, Taq I, with a novel specificity, from T. aquaticus (YT-1).…”

mentioning

confidence: 99%

A thermostable sequence-specific endonuclease from Thermus aquaticus.

Sato¹,

Hutchinson²,

Harris³

1977

Proc. Natl. Acad. Sci. U.S.A.

112

View full text Add to dashboard Cite

A sequence-specific endonuclease, Taq I, of novel specificity has been partially purified from an extreme thermophile, Thermus aquaticus. The enzyme cleaves bacteriophage A DNA at many (>30) sites and bacteriophage OX174 RF DNA at 10 sites. The enzyme is active at temperatures u to 700. The cleavage sites on 4X174 RIF DNA have been mr The sequence recognized and cleaved by Taq I has been s own to be the symmetrical tetranucleotide:I ,

show abstract

Purification by affinity chromatography of thermostable glyceraldehyde 3‐phosphate dehydrogenase from Thermus aquaticus

Cited by 50 publications

References 16 publications

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase. A Comparative Study of the Enzymes from Yeast and Sturgeon Muscle

A thermostable sequence-specific endonuclease from Thermus aquaticus.

Contact Info

Product

Resources

About