Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity

Abstract Lipoxygenase was purified from rat liver cytosolic fraction by a method involving two successive chromatographic steps on Sephacryl S-200 and Phenyl Sepharose CL-4B. The enzyme has a molecular weight of 96 Kdal and it seems to be composed of two identical subunits. Chromatofocusing of the enzyme revealed a single band of activity at pi 6.3. The enzyme activity of the purified fraction showed maximum activity at pH 7.0 with a Km for linoleic acid of 1.4 μM and is competitively inhibited by the specific lipoxygenase inhibitor nordihydroguaiaretic acid. The purified enzyme shows absorption and fluorescence spectra similar to those of lipoxygenase from other sources. However, the molecular weight of lipoxygenase purified from liver is found to be different from that of the enzyme from polymorphonuclear leukocytes. It is suggested that there are different isoenzymes of lipoxygenases in mammals.

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Activity Staining and Inhibition Characterization of Dipeptidylpeptidase-III Enzyme from Goat Brain

Enzyme Research ◽

10.4061/2011/897028 ◽

2011 ◽

Vol 2011 ◽

pp. 1-3 ◽

Cited By ~ 2

Author(s):

Pooja Attri ◽

Jasbir Singh ◽

Suman Dhanda ◽

Hari Singh

Keyword(s):

Enzyme Activity ◽

Metal Ions ◽

Potent Inhibitor ◽

Polyacrylamide Gel ◽

Cysteine Residues ◽

Protective Effects ◽

Activity Staining ◽

Chemical Inhibitors ◽

Goat Brain

Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy-β-naphthylamide (Arg-Arg-4mβNA) substrate. This enzyme retained its activity in native 10% polyacrylamide gel when stained using Arg-Arg-4mβNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40–50% inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl2 at 50 μM almost completely restored the enzyme activity. Further ZnCl2 and CoCl2 exerted protective effects on EDTA pretreated enzyme for its susceptibility to DTNB inhibition. Therefore, DPP-III is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation.

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Regulation and Characterization of L-Serine: Pyruvate Aminotransferase in Rat Liver Cytosol and Mitochondria

Zeitschrift für Naturforschung C ◽

10.1515/znc-1977-3-416 ◽

1977 ◽

Vol 32 (3-4) ◽

pp. 249-253 ◽

Cited By ~ 1

Author(s):

Jiro Hoshino ◽

Uta Kühne ◽

Branka Filjak ◽

Hans Kröger

Keyword(s):

Enzyme Activity ◽

Rat Liver ◽

Gel Filtration ◽

Deae Cellulose ◽

High Protein Diet ◽

Mitochondrial Fraction ◽

Protein Diet ◽

Low Protein ◽

Rat Liver Cytosol

Abstract Distribution of rat liver serine: pyruvate aminotransferase between cytosol and mitochondria varies considerably with the dietary and hormonal state of animals. Feeding a high-protein diet or fasting the animals results in an increase in the enzyme activity of both fractions but more marked in the mitochondrial fraction. A low-protein diet exerts the reverse effect. A single administration of dibutyryl cyclic AMP causes a rapid elevation of the enzyme activity in both fractions, which is effectively prevented by cycloheximide, actinomycin D and cortisone. The activity in mitochondria increases with a lag of 2 h following injection of the nucleotide inducer, in contrast to the cytosol enzyme, which increases without any lag. Gel filtration and DEAE cellulose chromatography of the enzyme from both fractions revealed the similar pattern and some kinetic constants of these two types of the enzyme were not significantly different from each other. These results indicate that rat liver serine: pyruvate aminotransferase is synthesized in the extra-mitochondrial site and transfered to mitochondria.

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