Biomedical subjects

N J Kruger

Publications and source records attributed to N J Kruger.

25 records · Page 2Linked to original sources

PubMed1986

Distribution of pyrophosphate:fructose 6-phosphate phosphotransferase in maize leaves.

In leaves of maize (Zea mays) the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) is much less than that of ATP:fructose 6-phosphate 1-phosphotransferase. A sequential extraction technique was used to study the location of PFP in this tissue. When compared with enzymes known to be restricted to specific locations in maize, the distribution of PFP activity in the sequential extracts indicated that PFP is located predominantly, if not exclusively, in the mesophyll cytoplasm. Although confined to the same site as sucrose synthesis, the level of PFP activity is inadequate to contribute significantly to the gluconeogenic flux from fructose 1,6-bisphosphate to fructose 6-phosphate. The absence of PFP activity from the bundle-sheath demonstrates that this activity is not essential for glycolysis in higher plants.

Journal Article↗

PubMed1985

A source of apparent pyrophosphate:fructose 6-phosphate phosphotransferase activity in rabbit muscle phosphofructokinase.

In the presence of UDPglucose, rabbit muscle phosphofructokinase appeared to use PPi as a phosphoryl donor, as reported previously (Biochem. Biophys. Res. Commun. 121, 842-847). This apparent activity was due to conversion of UDPglucose and PPi to glucose 1-phosphate and UTP, the latter being metabolized by phosphofructokinase. Auxiliary enzymes used in the assays were contaminated by UDPglucose pyrophosphorylase. This contamination was sufficient to account for, and had similar properties to, the apparent PPi-dependent activity. Without auxiliary enzymes phosphofructokinase could not use PPi. These findings indicate that the apparent interconversion of phosphofructokinase and PPi:fructose 6-phosphate phosphotransferase must be re-assessed.

Animals↗

PubMed1985

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings.

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P(2)) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P(2) is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P(2) was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P(2) in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P(2), respectively) were sufficient to account for the highest observed rates of Fru-2,6-P(2) metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P(2) level. Variation in the level of Fru-2,6-P(2) in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.

Journal Article↗

PubMed1985

Reassessment of an apparent hyperactive form of phosphofructokinase from plants.

In the assay of phosphofructokinase (PFK) from endosperm of germinating castor bean (Ricinus communis L.) there is a transient stimulation of initial activity by fructose 2,6-bisphosphate. This activation is due to metabolism of a limited amount of pyrophosphate (a contaminant of commercial ATP) by PPi:fructose 6-phosphate phosphotransferase (PFP), which is present in the extract. Both this activity and the amount of pyrophosphate contamination are sufficient to account for the initial increase in apparent PFK activity. The transient burst of activity is dependent on both of the above factors. Based on studies of a similar hyperactive PFK, others have proposed that PFK and PFP may be interconverted (Balogh et al. 1984 FEBS Lett 169: 287-292). The evidence for such conversions is reinterpreted in the context of the current results.

Journal Article↗

PubMed1984

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm.

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.

Journal Article↗

PubMed1984

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm.

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.

Journal Article↗

PubMed1983

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: effect on PPi:fructose 6-phosphate phosphotransferase.

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PPi:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PPi:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.

Chromatography, Ion Exchange↗