Activity of fructose-1,6-bisphosphatase (EC 3.1.3.11), one of the key gluconeogenic enzymes, was measured in human fetal brain and liver during development. Fructose-1,6-bisphosphatase was distributed throughout the different regions of the brain. In contrast to the partially purified enzyme from the brain, the liver enzyme was dependent on Mg2+ for maximal activity, EDTA, citrate, oleate and linoleate were stimulatory, whereas 5'-AMP inhibited the activity of the liver enzyme

Biswas, Tapas

Majumder, Arun Lahiri

Mukherjee, K. L.

Thakurta, Gitanjali Guha

English

Publications of the IAS Fellows

J. Biosci., Vol. 4, Number 2, June 1982, pp. 167-173.     © Printed in India. 
 
 
 
 
 
 
Fructose-1, 6-bisphosphatase in human fetal brain and 
liver during development 
 
 
TAPAS BISWAS, ARUN LAHIRI MAJUMDER†, GITANJALI GUHA 
THAKURTA* and K. L. MUKHERJEE* 
Biochemistry Laboratory, Department of Botany, School of Life Sciences, Visva-Bharati, 
Santiniketan 731 235 
 
* Department of Biochemistry, Institute of Post Graduate Medical Education and Research, 
Calcutta 700 020 
 
MS received 24 August 1981; revised 22 March 1982 
 
Abstract.  Activity of fructose-1,6-bisphosphatase (EC 3.1.3.11), one of the key gluconeogenic 
enzymes, was measured in human fetal brain and liver during development. Fructose-1,6- 
bisphosphatase was distributed throughout the different regions of the brain. In contrast to 
the partially purified enzyme from the brain, the liver enzyme was dependent on Mg2+ for 
maximal activity, EDTA, citrate, oleate and linoleate were stimulatory, whereas 5'-AMP 
inhibited the activity of the liver enzyme.
 
Keywords. Human fetus; brain and liver fructose bisphosphatase; fructose-phosphate inter- 
conversion; myo—inositol synthesis.
 
 
 
Introduction 
 
During development, the human fetus depends on the mother for the supply of 
glucose. The glucose homeostasis of the fetus not only aids development but also 
contributes to extrauterine adaptation of the fetus, in which neonatal energy 
metabolism supported by gluconeogenesis plays a  decisive function.
 
In order to understand the possible role of gluconeogenesis during the 
development of human fetus, the present investigation on the nature and 
properties of fructose-1,6-bisphosphatase (EC 3.1.3.11), a key gluconeogenic 
enzyme was undertaken. The two organs considered for this study were the fetal 
liver and brain. Although the brain is not ordinarily considered to be a gluconeo- 
genic organ, it was taken into consideration because of the unequivocal demonstra- 
tion of the enzyme therein (Majumder and Eisenberg, Jr., 1977). The present 
communication deals with the preliminary studies on fructose bisphosphatase 
during development of human fetal liver and brain.
 
 
† To whom correspondence should be addressed. 
 
167 
168 Biswas et al. 
 
Materials and methods
 
Human fetuses of different gestation periods (determined by considering the last 
menstrual period) were obtained from- normal mothers undergoing hysterotomy 
and ligation in the SSKM hospital attached to the Institute of Post Graduate 
Medical Education and Research, Calcutta, as a part of the Medical Termination 
of Pregnancy Clinic. The project was cleared by the Ethical Sub-Committee of the 
Institute of Post Graduate Medical Education and Research. The fetuses were put 
on ice immediately after the operation and the liver and brain were dissected
within 30 min,after the operation.
 
 
Fructose- 6-bisphosphate, sedoheptulose-l,7-bisphosphate, glucose-6-
phosphate, fructose-6-phosphate, and sodium β-glycerophosphate were purchased 
from Sigma Chemical Company, St. Louis,Missouri, USA. Sodium salts of oleate 
and linoleate (Applied Science, USA) were kind gifts of Dr. Eisenberg, Jr. of the 
National Institutes of Health, Bethesda, USA. Partially purified fructose 
bisphosphate was isolated from human fetal brain of different stages of gestation 
following essentially the procedure of Majumder and Eisenberg, Jr. (1977) 
described for the enzyme from rat brain. A 25% homogenate of a portion of tissue 
from different parts of brain in 0.15 Μ KCl containing 0.01 Μ NaHCO 3 was
centrifuged at 10,000 g for 30 min in a refrigerated Remi K-24 centrifuge. The 
supernatant was heated for 3 min at 60°C and again centrifuged at 15,000 g as 
before. The heated supernatant was adjusted to pH 4.8 with 5 Μ acetic acid and 
immediately centrifuged at 10,000 g. The supernatant thus obtained was adjusted 
to pH 7.5 (termed acid fraction), dialysed against 0.005 Μ Tris-HCl, pH 7.5 and 
used as the enzyme source.
 
A somewhat different procedure was adopted for the isolation and partial 
purification of the enzyme from human fetal liver. A 10,000 g supernatant was 
obtained after centrifuging a 25% 0.15 Μ KCl/0.01 Μ NaHC03 homogenate of 
fetal liver. This was centrifuged at 100,000 g for 1 h in a MOM (Hungary) Ultra- 
centrifuge. The supernatant obtained was heated for 3 minutes at 60°C and the
coagulated protein centrifuged off at 20,000 g in a Remi K-24 (India) refrigerated 
centrifuge. The supernatant thus obtained (termed heat fraction) was dialysed 
against 0.005 Μ Tris-HCl, pH 7.5 and used as the enzyme in this study.
 
Assay for fructose bisphosphatase activity was carried out by the procedure of 
Majumder and Eisenberg, Jr. (1977) by measuring the released inorganic phosphate 
(Ames 1966). The standard incubation mixture contained the following (all in 
µmol ml); Tris-HCl buffer (pH 7.5/9.5) 100; MgSO4, 5; fructose-1, 6-bisphosphate, 
0.1-0.5; and enzyme, appropriate amount. It was incubated at 37°C for an hour, 
deproteinized by the addition of 5% trichloroacetic acid and the supernatants 
assayed for inorganic phosphate. Parallel sets were run using fructose-6- 
phosphate as substrate for determination of any non-specific phosphatase activity.
 
Protein was measured by the method of Lowry et al. (1951) using bovine serum
albumin as standard.
Fructose-bisphosphatase from developing human fetal tissues.    169
 
Results and discussion 
 
A comparison of the fructose bi-phosphatase activity at different stages of
gestation revealed appreciable change in the activity in both fetal brain and liver
during development (table 1). The relative enzyme activity at the neutral and
 
Table 1. Fructose-l,6-bisphosphatase activity of human fetal brain & liver at different gestation
periods. 
 
 
The enzyme activity was determined in the 10,000 g supernatant as described in Methods and 
expressed as µmol Pi liberated per mg protein per hour after correction for the Fructose-6- 
Phosphatase activity, if any. Other details are in Methods.
 
— Not detected
 
alkaline range varied at different stages of development, although there was a 
tendency towards even distribution at the two pH values. The fructose-6- 
phosphatase activity of the different samples, if at all, was about 20% that of 
fructose bisophosphatase activity. Variation was noticed among the specific 
activity values for the brain and liver enzymes. However, in case of brain, the 
activity of the enzyme during fetal life was much lower than that of the adult organ 
but higher than the adult rat brain (Majumder and Eisenberg, Jr., 1977).
170                                          Biswas et al.
 
Table 2 describes fructose bisphosphatase activity isolated from the fetal brain
of different gestation periods at different stages of purification as outlined under
Methods. It was evident that, in all the specimens, except for that of 21-24 weeks 
 
 
Table 2. Fructose-l,6-bisphosphatase and fructose-6-phosphatase activity during purification 
of human fetal brain obtained at different stages of development.
 
 
 
Fru-P2ase and F-6-Pase activities were determined as described in Methods and expressed as 
µmol Pi liberated per mg protein per hour. Procedures for partial purification are described in 
Methods. 
 
Fru-P2ase — fructose-l,6-bisphosphatase
F-6-pase — fructose-6-phosphatase.
 
 
 
 
of gestation, there was a progressive increase in the specific activity during purifi- 
cation. Better purification was achieved with specimens between 8-12 weeks of
gestation, a stage also characterized by no detectable fructose-6-phosphatase
activity. The ratio of the activities at pH 7.5 and pH 9.5 was close to 1. It may be 
worth mentioning here that although mammalian liver fructose bisphosphatase is
reported to exhibit two activities at pH 7.5 and pH 9.5 as a result of proteolytic
modification (Horecker et al., 1975) the rat brain enzyme failed to show two peaks 
of activity over a pH range of 6 to 10 (Majumder and Eisenberg, Jr., 1977). Human 
fetal brain fructose bisphosphatase exhibited a similar property. Scanning of
Fructose-bisphosphatase from developing human fetal tissues.                171
 
different parts of the fetal brain e.g. cerebrum, midbrain and cerebellum showed 
that the enzyme was distributed in all these regions. Little variation in the 
distribution was noticed in samples between 8 to 24 weeks of gestation (table 3). 
 
Table 3. Distribution of fructose-l,6-bisphosphatase activity in different regions of human fetal 
brain, from different stages of development.
 
 
 
10,000×g supernatant from different regions of fetal brain, of different stages of gestation was 
used as enzyme source. Fructose-bisphosphatase activity was corrected for Fructose-6- 
phosphatase activity, if any.
 
 
Some properties of the fructose bisphosphatase partially purified from fetal 
brain (acid-fraction) and liver (heat-fraction) were studied. Substrate specificity 
of the enzymes from the two sources, using various sugar phosphates, such as 
fructose-l,6-bisphosphate,sedoheptulose-1,7-bisphosphate,glucose-6-phosphate,
fructose-6-phosphate and β-glycerophosphate (each 0.5 mM) revealed that both 
the enzymes were unable to hydrolyze glucose-6-P, fructose-6-phosphate and ß- 
glycerophosphate. The brain enzyme hydrolyzed sedoheptulose-1, 7-bisphosphate 
to 20% of the fructose-1, 6-bisphosphate activity whereas 37% of the activity was 
recorded for liver enzyme with the same substrate.
 
Further studies revealed that while the liver enzyme was dependent on divalent 
cation for maximal activity, the brain enzyme had no requirement for it. Mn2+ at 
5 mM was optimally effective and could be partially replaced by Mn2+ (2mM). 
The liver enzyme was stimulated by EDTA (0.1 mM) and inhibited by 5'-AMP (0.5 
mM). Citrate (0.5 mM), oleate (0.5 mg/ml) and linoleate (0.5 mg/ml), increased 
the activity of the liver enzyme approximately by 2,1.5 and 2.3 fold respectively. 
This property was also shared by the rabbit liver enzyme (Baxter et al., 1972, 
Carlson et al., 1973). All these compounds had little effect on the brain enzyme 
activity. 
 
Studies on the gluconeogenic enzymes during fetal life were mainly undertaken 
in human, sheep, guinea-pig and baboon (Aurricchio and Rigillo, 1960; Ballard and 
 
172 Biswas et al. 
 
Oliver, 1965; Jones and Ashton, 1972; Levitsky et al., 1976). For obvious reasons, a 
detailed study of these enzymes in the human fetus, in particular, was lacking and 
subhuman primates were often chosen for this purpose (Robinson et al., 1980; 
Sherwood et al., 1980). Most of these studies dealt with determination of activities 
of different gluconeogenic enzymes in cell extracts from liver or kidney. In the 
present study we have restricted ourselves to the demonstration of the fructose 
bisphosphatase activity and the partial purification of the enzyme from both 
the organs upto a stage where it exhibits absolute specificity towards hydro-
lysis of fructose bisphosphate. The preliminary characterization of the liver 
enzyme reported here, showed that with respect to the divalent cation requirement, 
EDTA stimulation and 5'-AMP inhibition, it was similar to the enzyme from other 
mammalian sources and recently reported adult human liver enzyme (Dzugaj and 
Kochman, 1980).
 
Though not considered gluconeogenic (Scrutton and Utter, 1968), existence of 
fructose bisphosphatase in brain was suggested by [14C]-pyruvate and [14C]-
glutamate incorporation studies in guinea-pig brain slices (Phillips and Coxon, 
1975) and subsequently its presence in rat brain was demonstrated (Majumder and 
Eisenberg, Jr., 1977; Hevor and Gayet, 1978). Majumder and Eisenberg, Jr. (1976- 
1977) suggested that in brain, this enzyme along with the phosphofructokinase, 
may be regulated by fatty acids and phospholipids for controlled utilization of 
glucose-6-phosphate for synthesis of myo-inositol by the myo-inositol synthase 
reaction. Keeping this in view, a probable coupling between the two enzymes has 
recently been suggested (Majumder, 1981). Since during its development, the 
human fetal brain requires a high pool of myo-inositol for synthesis of polyphos- 
phoinositides of the myelin (Eichberg and Dawson, 1965; Eichberg and Hauser, 
1973),this system was thought to be ideal for this study. As preliminary to this we have 
demonstrated the presence of a fructose bisphosphatase in human fetal brain 
during development and studied some of their properties. Partial purification of 
myo-inositol synthase from rat brain was reported (Maeda and Eisenberg Jr 1980) 
and human fetal brain has been found to have appreciable synthase activity 
(Adhikari and Majumder, unpublished observation). Further studies would 
require a detailed characterization of the purified enzyme from both liver and 
brain as well as that of myo-inositol synthase. Work in this direction is now in 
progress. 
 
Acknowledgement 
 
This work is supported by the Council of Scientific & Industrial Research, Govt. of 
India. ALM is a Career Awardee of the University Grants Commission, New Delhi. 
 
 
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Fructose-1, 6-bisphosphatase in human fetal brain and liver during development

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