PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE FROM THE AMEBA NAEGLERIA-FOWLERI, AN AMP-SENSITIVE ENZYME

被引:37
作者
MERTENS, E [1 ]
DEJONCKHEERE, J [1 ]
VANSCHAFTINGEN, E [1 ]
机构
[1] INST HYG & EPIDEMIOL, B-1050 BRUSSELS, BELGIUM
关键词
D O I
10.1042/bj2920797
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
PP(i)-dependent phosphofructokinase (PP(i)-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PP(i)-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30-degrees-C in the presence of AMP. N. fowleri PP(i)-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25 %. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30-degrees-C in the presence of 0.5 mM AMP. Reactivation was also observed at 30-degrees-C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed K(m) values of 10 and 15 muM for fructose 6-phosphate and PP(i), respectively, in the forward reaction, and of 35 and 590 muM for fructose 1,6-bisphosphate and P(i) in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased V(max.) about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 muM.
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页码:797 / 803
页数:7
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