ISSN:
0749-503X
Keywords:
δ-Aminolevulinate dehydratase purification
;
Saccharomyces cerevisiae HEM2 transformants
;
Life and Medical Sciences
;
Genetics
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for δ-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16·2 μmol h-1 per mg protein at pH 9·4 and 37·5°C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr, of 275 000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr ≃ 37 000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1·0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0·359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total δ-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/yea.320060405
