Details

Autor(en) / Beteiligte

• DIGANGI, JOSEPH JOHN

Titel

THE PURIFICATION, CHARACTERIZATION, AND REGULATION OF ORNITHINE DECARBOXYLASE IN NEUROSPORA CRASSA

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1986

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Ornithine decarboxylase (ODC), a key enzyme of polyamine biosynthesis, was purified about 670 fold to homogeneity from derepressed crude extracts of the fungus, Neurospora crassa. Antisera were raised against pure preparations of native and denatured ODC protein. The enzyme purification procedure included ammonium sulfate precipitation and molecular sieving, followed by ion exchange and HPLC-ion exchange chromatography. The M(,r) = 110,000 enzyme consisted of two M(,r) = 53,000 subunits and had a specific activity of 2,600 umole/hr/mg. The final preparation contained less prominent polypeptides of lower molecular weight. All the polypeptides were derived from ODC. This was shown by the specific binding of a radioactive suicide inhibitor, difluoromethylornithine (DFMO), and by their similar peptide digest patterns. DFMO binding and immunotitrations of crude and purified ODC showed that the catalytic activity of ODC protein remained constant during its purification. Pure Neurospora ODC had a K(,m) for ornithine of 0.35 mM, a K(,m) for pyridoxal 5'-phosphate of 0.16 uM, and a K(,i) for the competitive inhibitor alpha-methylornithine of 0.28 mM. The pH optimum was about 7, and the enzyme was stabilized by dithiothreitol and the non-ionic detergent, Brij 35. ODC was not significantly inhibited by putrescine or spermidine in vitro. Previous work showed that ODC is regulated by the polyamines in vivo. Spermidine prevents ODC formation, and putrescine appears to promote ODC inactivation. In this work, immunoblots showed loss of ODC protein during inactivation. Immunotitration showed that ODC protein was lost proportionately with ODC activity during inactivation. Steady-state cultures starving for spermidine but accumulating large amounts of putrescine had lower OCD activity than cultures starved for both putrescine and spermidine. However, both cultures displayed the same ratio of ODC protein to ODC activity. The data do not exclude the appearance of small amounts (<25%) of inactive ODC molecules during inactivation, but degradation of the protein appears to be the most prominent feature of ODC inactivation in Neurospora.