Ornithine decarboxylase from calf liver. Purification and properties

Haddox, Mari K.; Russell, Diane Haddock

doi:10.1021/bi00526a030

Cited by 38 publications

(17 citation statements)

References 20 publications

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“…In addition, spermidine is the source of the 4-aminobutyl moiety that is used in the posttranslational conversion of lysine to hypusine; hypusine serves a vital role in the function of the protein synthesis initiation factor elF-5A (Park et al, 1997). Be cause of the importance of polyamines, ODC has been characterized from a variety of organisms including Escherichia coli (Applebaum et al, 1977), Trypanosoma brucei (Phillips et al, 1987), Neurospora crassa (DiGangi et al, 1987), Saccharomyces cerevisiae (Tyagi et al, 1981), and tobacco (Heimer and Mizsrahi, 1982), as well as many mammals (e.g., Haddox and Russell, 1981;Seely et al, 1982;Gupta and Coffi no, 1985).…”

Section: Introductionmentioning

confidence: 99%

Ornithine Decarboxylase Encoded by Chlorella Virus PBCV-1

et al. 2002

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Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A207R, which encodes a protein with 37-41% amino acid identity to ornithine decarboxylase (ODC) from many eukaryotic organisms. The a207r gene was cloned and the protein was expressed as a His-A207R fusion protein in Escherichia coli. The recombinant protein catalyzes pyridoxal 5'-phosphate-dependent decarboxylation of ornithine to putrescine, the first step in the polyamine biosynthetic pathway. The enzyme has a pH optimum of 9.0 and a temperature optimum of 42 degrees C, and it requires dithiothreitol for maximal activity. The enzyme has a K(m) for ornithine of 0.78 mM and a specific activity of 100 micromol/min/mg protein. PBCV-1 ODC is quite sensitive to the competitive inhibitor L-arginine and the irreversible inhibitor difluoromethylarginine but it is less sensitive to the irreversible inhibitor difluoromethylornithine. The a207r gene is expressed both early and late in PBCV-1 infection and is highly conserved among the chlorella viruses. The 42-kDa PBCV-1 ODC (372 amino acids) is the smallest ODC in the databases and, to our knowledge, is the first virus-encoded ODC.

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Section: Introductionmentioning

confidence: 99%

Ornithine Decarboxylase Encoded by Chlorella Virus PBCV-1

et al. 2002

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“…With more purified enzyme, inactivated by dialysis, which presumably provokes thiol/disulphide interchanges, natural reducing agents of low Mr proved ineffective or scarcely effective in restoring ODC activity ( [12,16]; the present work).…”

Section: Discussionmentioning

confidence: 67%

“…It is well known that ODC is a thiol-dependent enzyme [12][13][14][15][16][17][18], and exogenous, non-physiological thiols, usually DTT, are added to purification and assay buffers to avoid Actually, the activity of purified ODC depends on DTT concentration in the assay buffer, although it should be noted that high concentrations of DTT are inhibitory, particularly at low ornithine concentrations, resulting in an apparent increase in Km [23][24][25]. On the other hand, the physiological factor(s) and reducing system(s) involved in maintaining ODC in the active status are not known.…”

Section: Discussionmentioning

confidence: 99%

“…ODC activity can be inhibited in vitro by compounds able to react with thiol groups, including GSSG [13,14], and by microsomal oxidases [15]. Synthetic dithiols, usually DTT, have to be added to buffers during ODC purification, whereas GSH or other physiological reductants are ineffective or, at best, poor substitutes for DTT [12,16]. Removal of DTT by dialysis results in inactivation of ODC, accompanied by polymerization [12,17], presumably owing to formation of protein disulphides.…”

Section: Introductionmentioning

confidence: 99%

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Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

Flamigni

Guarnieri

Caldarera

1988

Biochemical Journal

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Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated 'A', eluted near the void volume (Mr greater than or equal to 60,000), and the other designated 'B', eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

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“…Posttranslational modification also may explain the widely varied specific activity of the purified OrnDCase molecule (26). …”

Section: Discussionmentioning

confidence: 99%

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

Russell¹

1983

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

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This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [a-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, adifluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [2P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 ,uM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32p]_ guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.Several investigators have indicated that the control of RNA polymerase I (EC 2.7.7.6) activity is not through increased synthesis of the enzyme but rather through modification of the enzyme structure that facilitates the attachment of the enzyme complex to rDNA gene sites (1-5). RNA polymerase I activity appears to be dependent upon the presence of an extremely labile half-life protein that is sensitive to amino acid pool sizes (6, 7). Ornithine decarboxylase (OrnDCase, EC 4.1.1.17), the initial enzyme in the polyamine biosynthetic pathway, has the properties of such a protein (8)(9)(10)(11)

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Ornithine decarboxylase from calf liver. Purification and properties

Cited by 38 publications

References 20 publications

Ornithine Decarboxylase Encoded by Chlorella Virus PBCV-1

Ornithine Decarboxylase Encoded by Chlorella Virus PBCV-1

Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

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