Pyruvate kinase from Aspergillus niger: a regulatory enzyme in glycolysis?

1984 ◽  
Vol 30 (1) ◽  
pp. 16-22 ◽  
Author(s):  
Bibiana Meixner-Monori ◽  
Christian P. Kubicek ◽  
Max Röhr
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Pyruvate Kinase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Acid Accumulation ◽  
Sigmoidal Kinetics ◽  
Atp Inhibition ◽  
Very High

Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in the presence of FDP. However, it was possible to reactivate inactivated pyruvate kinase (after dialysis in the absence of FDP) by adding FDP to the enzyme assay. From these results and because of the very high affinity of pyruvate kinase for FDP (Ka < 0.1 μM), it is concluded that the enzyme probably has FDP bound to the protein in vivo. The significance of this hypothesis to the regulation of glycolysis in A. niger, with special reference to the mechanism of citric acid accumulation, is discussed.

Pyruvate kinase from the earthworm Allolobophora calliginosa: modification of the enzyme during anaerobiosis

1991 ◽  
Vol 69 (1) ◽  
pp. 251-254
Author(s):  
Athanasios I. Papadopoulos ◽  
Basile Michaelidis ◽  
Isidoros Beis
Keyword(s):  
Pyruvate Kinase ◽  
Deae Cellulose ◽  
Relative Activity ◽  
The Body ◽  
E Coli ◽  
In Vitro Incubation ◽  
Sigmoidal Kinetics ◽  
Independent Protein

The relative activity of pyruvate kinase from the body-wall muscle of the earthworm Allolobophora calliginosa was found to drop dramatically within 6 h of exposure to N2, whereas the opposite was observed during recovery. Two forms of pyruvate kinase (designated as peak I and peak II) were separated chromatographically on DEAE-cellulose and eluted at 50 and 150 mM of KCl, respectively. They displayed different kinetic behaviour with respect to substrate phosphoenolpyruvate; peak I exhibited Michaelis–Menten kinetics whereas peak II showed sigmoidal kinetics. The ratio of the enzyme units (peak I/peak II) decreased from 3.38 under normoxic conditions to 0.09 under anoxic conditions. In vitro incubation of the aerobic form of pyruvate kinase in the presence of ATP and Mg++ resulted in a reduction of the enzyme activity by 64%, suggesting the presence of an endogenous cyclic-nucleotide-independent protein kinase capable of phosphorylating pyruvate kinase. After in vitro incubation, alkaline phosphatase from E. coli increased the depressed activity of anaerobic pyruvate kinase, indicating that the enzyme molecule is phosphorylated in vivo during exposure to anoxia.

Regulation by zinc and adenosine 3′,5′-cyclic monophosphate of growth and citric acid accumulation in Aspergillus niger

1976 ◽  
Vol 22 (8) ◽  
pp. 1093-1101 ◽  
Author(s):  
William S. M. Wold ◽  
Isamu Suzuki
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Cyclic Amp ◽  
Zinc Deficiency ◽  
Growth Phase ◽  
Acid Fermentation ◽  
High Zinc ◽  
Acid Accumulation ◽  
Camp Metabolism ◽  
Rates Of Growth

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they excrete citrate but do not proliferate. The phase alternative was controlled by the concentration of zinc: high zinc (2 μM) maintained growth phase, and a zinc 'deficiency apparently signaled the transition to the accumulating phase. Cyclic AMP affected the rates of growth and acidogenesis when added to cultures growing at low but not at high zinc: that is, cAMP did not induce the phase transition, zinc deficiency did. Cyclic AMP enhanced growth early in the fermentation, but at later stages the response of the mycelia to cAMP changed, and then cAMP inhibited growth. When citrate eventually began to accumulate cAMP augmented its synthesis. The growth and acidogenic responses were quite specific to cAMP, and were sensitive to concentrations of about 1 μM. Cyclic AMP also either promoted or retarded the appearance of an unidentified yellow pigment. It is proposed that the growth and accumulating phases are distinct differentiated states, at least with respect to cAMP metabolism.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

A TOXICOLOGICAL AND BIOCHEMICAL STUDY OF ω-FLUORO COMPOUNDS

1957 ◽  
Vol 35 (1) ◽  
pp. 407-417
Author(s):  
J. M. Parker ◽  
I. G. Walker
Keyword(s):  
Respiratory Failure ◽  
Citric Acid ◽  
Biochemical Study ◽  
Toxic Compounds ◽  
Molar Basis ◽  
Acid Accumulation ◽  
Citric Acid Accumulation ◽  
Substituted Urea

A series of ω-fluoro compounds have been examined for their toxicity to mice and their ability to cause accumulation of citric acid in vivo. It was found that the toxic members did cause citric acid accumulation. Since fluoroacetate causes citric acid accumulation, it was concluded that the activity of these compounds and their effect on citric acid levels is in agreement with the hypothesis that these compounds were degraded to a fluoroacetyl derivative. The findings suggested that mice can degrade to fluoroacetate, compounds containing the following terminal radicals: amine, nitro, nitrile, thiocyanate, isothiocyanate, mercaptan, fluoride, chloride, bromide, iodide, tosylate, mesylate, 2-ketone, sulphonyl chloride, 1-alkyne, ether, substituted urea, aldehyde, and carboxyl. When compared on a molar basis several of the toxic compounds containing four or more carbon atoms were more toxic than fluoroacetate. 6-Fluorohexylamiue and 6-fluorohexanoic acid, which were representative of this type of compound, were investigated in this regard. These compounds, like fluoroacetate, were not immediately reactive but appeared to be converted to the toxic end-product more efficiently. In the dog, death from 6-fluorohexylamine was due to respiratory failure.

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