A new procedure is described for extracting, purifying, and crystallizing alkaline phosphatase orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from human intestinal mucosa. Active enzyme, extracted with trichiorotrifluoroethane, is subsequently purified by chromatography on Sephadex G-200 and DEAE Sephadex. The acetone and ammonium sulfate precipitation steps that are part of the conven...

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cystein...

Homogeneous myo-inositol oxygenase (EC 1.13.99.1) has been obtained in 45% yield by a 550-fold purification from a centrifuged homogenate of hog kidney. Of particular importance in developing the purification procedure was the finding that although the enzyme becomes less catalytically active during purification, it could be reactivated by incubating with 1 m~ Fe(II) and 2 m~ cysteine. The mole...

Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the ...

The purification of the Escherichia coli dnaB protein by affinity chromatography on nucleotides bound to agarose is described. The dnaB protein, which contains an associated ribonucleoside triphosphatase activity (Wickner, S., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787) binds to immobilized ATP, ADP, and UDP, but not to AMP. The type of linkage of ATP to agar...

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simpl...

A procedure is described for the purification of the alpha-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH(4))(2)SO(4), chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradi...

There are a number of nuclear enzymes that bind nicotinamide, thymidine or nucleotides. We use this fact to prepare affinity columns for the purification of nuclear enzymes. In particular the nuclei of eukaryotic cells have an enzyme that catalyses the synthesis of the homopolymer poly(ADP-ribose) from NAD. Isolation and partial purification has been achieved (Yamada et al., 1971 ; Ueda et al.,...

An enzyme located in the particles of bovine adrenal medulla catalyzes the conversion of 3,4-dihydroxyphenylethylamine to the hormone norepinephrine (1). The extraction and partial purification of this enzyme have recently been described (2). With the purified enzyme, it was shown that the hydroxylation of 3,4-dihydroxyphenylethylamine is coupled to a stoichiometrically equivalent oxidation of ...

It is well known that thiaminase which destroys thiamine is distributed in the viscera of fresh-water fish, shellfish and ferns, and produced by some microorganisms. Recently, thiaminase ‡U, which catalyzes the hydrolysis of thiamine, was purified and crystallized by Kimura et al. (1) and Ikehata (2). The purification of thiaminase ‡T, which catalyzes a transfer reaction in the presence of a ba...