540th MEETING, OXFORD
1179
first begin to eat solid food. It is possible that the high circulating glycosidase activities in
the sera of pregnant and young rabbits are associated with the metabolism of oligosaccharides derived from maternal milk.
Each tissue had its own specific pattern of development for N-acetylglucosaminidase
during the first 35 days after birth. In liver N-acetylglucosaminidase activity was higher
in newborn animals than in adults (Pt0.005)and continued to rise until day 14 after
birth; thereafter activities fell and adult values were seen in 28-day-old animals.
In spleen and kidney (Figs. 2c and 2d) N-acetylglucosaminidase activity in newborn
animals was lower than in adults (P= 0.0005). In kidney the pattern of development was
biphasic, whereas in spleen N-acetylglucosaminidase activity rose steadily until adult
activities were attained by 35 days. Electrophoresis showed that in kidney and liver all
the adult forms were present in newborn animals. In the spleens of these rabbits no forms
of the enzyme were detectable, but as enzyme activity increased with age the various
forms appeared.
In neonatal heart and brain N-acetylglucosaminidase activity was similar to that in
the adult tissues (Figs. 2e and 2f). Brain enzyme activity increased until 14 days
(0.005 >P>0.0025), after which time the mean activity did not vary statistically. Examination of cerebral tissue by electrophoresis showed that these increases were accompanied
by the appearance of an electrophoretic form.
Liver and hearts of neonatal rabbits had higher acid a-mannosidase activity than did
the same tissues in mature animals (0.01 >P>0.005; 0.005 >P>0.0025 respectively).
In spleen both acid and neutral forms had lower activities thanin adults (0.01 >P>0.005),
although in no tissue were the changes in activity as marked as for N-acetylglucosaminidase. For young rabbits both enzyme forms in kidney and heart had similar activities, in
contrast with the adult animals, where the neutral form had higher activity than the acid
(P= 0.01 and 0.05 >P>0.025 respectively).
These results show that in early life the changes in rabbit tissue N-acetylglucosaminidase activity are different from a-mannosidase and it may be that each glycosidase
has a distinct pattern of development. By 5 weeks after birth, the earliest stage at which
young laboratory rabbits are capable of existence independent from their mothers, adult
activities and electrophoretic forms of the enzymes were established in all tissues studied
except brain. In both spleen and brain changes in N-acetylglucosaminidase activity were
accompanied by alterations in electrophoretic patterns. Perhaps these changes are associated with developmental processes in early life; in brain this may be myelination and in
spleen it may be the change from a haemopoietic to haemolytic function.
Carroll, M., Dance, N., Masson, P. K., Robinson, D. &Winchester,B. G. (1972) Biochem. Biophys. Res. Commun. 49, 579-583
Price, R. G. & Dance, N. (1972) Biochim. Biophys. Actu 271, 145-153
Robinson, D., Price, R. G. & Dance, N. (1967) Biochem. J . 102, 525-561
Stirling, J. (1972) Biochim. Biophys. Actu 271, 154-162
Suzuki, I., Kushida, H. & Shida, H. (1969) Seikuguku 41, 334341
-
The Effect of Insulin Deficiency on the Glutathione Hnsulin
Transhydrogenase Activity of Rat Liver
J. HYWEL THOMAS and SUSAN M. WAKEFIELD
Department of Biochemistry, St. Thomas’s Hospital Medical School,
London, SEl IEH, U.K.
and RICHARD H. JONES
Department of Medicine, St. Thomas’sHospital Medical School, London, SE1 IEH, U.K.
Glutathione-insulin transhydrogenase (glutathione- protein disulphide oxidoreductase,
EC 1.8.4.2) inactivates insulin by reducing the disulphide bonds in the presence of a
Vol. 1
1180
BIOCHEMICAL SOCIETY TRANSACTIONS
thiol suchas GSH(Katzen &Tietze, 1966;Varandani, 1966). Recentlyit has beendemonstrated that the inactivation and degradation of insulin by rat liver takes place in a sequential manner (Varandani et al., 1972). Initially, the interchain disulphide bonds of
insulin are cleaved by GSH-insulin transhydrogenase and subsequently, the A and B
chains undergo proteolysis to small-molecular-weight components. This would suggest
that GSH- insulin transhydrogenase is
in the degradation of
insulin by the liver.
Morgan & Wiesman (1968) found a correlation between decreased liver ‘insulinase’
activity and decreased plasma insulin concentrations in starved and alloxan-diabetic
rats; they suggested that insulin is an inducer for liver ‘insulinase’ activity. By using a
specific assay method we have now investigated the effect of starvation and experimentally induced diabetes on the activity of GSH-insulin transhydrogenase in rat liver.
GSH - insulin transhydrogenase activity was determined by recording the glutathione
reductase-catalysed oxidation of NADPH by GSSG generated in the reduction of insulin
by GSH (Katzen & Stetten, 1962). The values of NADPH oxidized were corrected for
the non-enzymic reduction of insulin and for the oxidation of NADPH in the absence of
insulin. Enzyme activity is expressed both in terms of nmol of NADPH oxidized/min per
mg of protein and pmol of NADPH oxidized/min per liver.
Male albino Wistar rats of 180-200g were used in these experiments. Rats were made
diabetic either by an intraperitoneal injection of alloxan at a dose of 120mg/kg body wt.
or by means of an intravenous injection of streptozotocin (Upjohn Co., Kalamazoo,
Mich., U.S.A.) at a dose of 65mg/kg body wt. At the same time, a similar group of rats
were weighed and set aside as controls. All animals had access to food and water at all
times. Onset of diabetes was indicated by the development of glycosuria, detected by
Clinistix (Ames Co., Slough, Bucks., U.K.), and confirmed by high blood glucose concentrations when the animals were killed, measured by the glucose oxidase method of
Trinder (1969). After 8 days both treated and control animals were weighed and killed
by decapitation. Blood was taken for measurements of blood glucose and serum insulin
concentrations. Serum insulin was determined by a double-antibody immunoassay procedure (Morgan & Lazarow, 1963).The livers were quickly removed, weighed and homogenized in 0.25 M-sucrose containing 1mM-EDTA. The homogenates were centrifuged
at 12000g for lOmin at 4°C and the supernatant was assayed for GSH-insulin transhydrogenase activity (see Table 1).
With both alloxan- and streptozotocin-treated rats there was a decrease of GSHinsulin transhydrogenase activity to about half that of the normal untreated animals.
Treatment with either alloxan or streptozotocin resulted in blood glucose concentration
three- to four-fold higher than in the control group. The serum insulin concentrations in
the diabetic animals were less than 5 % of those of normal, fed animals.
In another series of experiments twelve rats were starved for 2 days. Four were killed
at the end of this period, four were re-fed for 3 days before being killed and the final group
of four animals were injected intraperitoneally with actinomycin D (Merck, Sharp and
Dohme Ltd., Hoddesdon, Herts., U.K.) at a dose of 40pglkg body wt. per day throughout the period of 3 days’ re-feeding. A control group of four animals was allowed free
access to food. GSH - insulin transhydrogenase activity, blood glucose and serum
insulin concentrations of the untreated, starved and re-fed animals are shown in Table 2.
Starvation for 2 days resulted in a 40 % decrease in liver GSH - insulin transhydrogenase
activity when expressed in activity per unit of protein, and a 60% decrease when the
results are expressed in terms of activity per liver. In the re-fed group the activity was
normal. In the actinomycin D-treated animals, however, the GSH -insulin transhydrogenase activity, expressed in terms of activity per unit of protein, was 68 % of that in the
untreated animals. Serum insulin and blood glucose concentrations decreased on starvation and returned to normal values on re-feeding.
The results indicate that the activity of GSH-insulin transhydrogenase is lowered in
alloxan- and streptozotocin-diabetes and in starvation and that this correlates with the
low insulin concentrations in these conditions. A possible interpretation of these
findings is that blood insulin serves as an inducer for the synthesis of liver GSH-insulin
1973
0
P
c
2
+
8.9 0.9
8.4 rt 0.7
7.8 f 0.9
(g)
Liver wt.
+
(nmol of NADPH
oxidized/min per mg of protein)
6.35 f 0.80
2.64f0.30
3.45 0.64
,
(pmol of NADPH
Blood glucose Serum insulin
oxidized/min per liver) (mg/100 ml)
(punits/ml)
3.10f0.32
107f 10
110+ 11
4
333 f83
1.55 f 0.42
(5
452 If:60
1.47 f0.19
Treat men t
None
Starved 2 days
Starved, then re-fed
Starved, then re-fed
factinomycin D
Body wt.
(g)
199f 3
143f 3
18822
166+3
Liver wt.
(g)
10.8+ 1.0
5.1 f0.2
10.6 f0.5
9.2+ 0.5
+
(nmol of NADPH oxidized/
min per mg of protein)
4.53 0.2
2.74f 0.37
4.21 f0.6
3.10+ 1.78
+
(pmol of NADPH
Blood glucose Serum insulin
oxidized/min per liver) (mg/lOOml)
(punitslml)
3.09 f 0.14
96+4
105+ 9
61 f 7
23+ 7
1.28 f 0.15
3.42 0.54
97f5
96f 11
2.61 f0.48
109i~8
98 f20
Each result is given as a m e a n f s .D. for four animals. One group of four animals was starved for 2 days. Two other groups of animals were starved for
2 days and then re-fed for 3 days before being killed. Animals in one of these groups were injected intraperitoneally with actinomycin D (40pg/kg
body wt. per day) during the period of re-feeding.
GSH - insulin transhydrogenase activity
Table 2. Effect of starvation and re-feeding on the liver GSH- insulin transhydrogenase activity, blood glucose concentration and serum insulin
concentration of rats
Treatment
None
Alloxan
Streptozotocin
Body wt.
(d
182f 7
1645 11
167+ 12
GSH - insulin transhydrogenase activity
Each result is given as a m e a n i s m for eight animals. Each of eight rats in a group was given a single intraperitoneal injection of alloxan (120mg/kg
body wt.) or a single intravenous injection of streptozotocin (65 mg/kg body wt.)
Table 1 . Effect of alloxan- and streptozotocin-induced diabetes on the liver GSH- insulin transhydrugenase activity, blood glucose concentration and
serum insulin concentration of rats
t!
co
+
-
I?
F
0
0
"0
2
zm
3
8
v,
1182
BIOCHEMICAL SOCIETY TRANSACTIONS
transhydrogenase, as has been suggested by Morgan & Wiesman (1968) and by
Varandani et al. (1971). The fact that GSH-insulin transhydrogenase activity is
restored completely on re-feeding, but only partially in the presence of actinomycin D,
would seem to support this hypothesis. Such control of the activity of this hepatic enzyme
could serve as a feedback mechanism to regulate the amount of insulin available to the
systemic circulation.
The authors gratefully acknowledge the excellent technical assistance of Mrs. Marian
Johnson and Miss Carol Mason. They also thank Weddel Pharmaceuticals Ltd. for a gift of ox
insulin, and Dr. W. E. Dulin, Upjohn Co., Kalamazoo, Mich., USA., for a gift of streptozotocin.
Katzen, H. M. & Stetten, D., Jr. (1962) Diabetes 11, 271-280
Katzen, H.M.& Tietze, F. (1966)J . Biol. Chew. 241, 3561-3570
Morgan, C. R.& Lazarow, A. (1963) Diabetes 12, 115-126
Morgan, C. R.& Wiesman, H. J. (1968)Proc. SOC.Exp. Biol. Med. 127, 763-765
Trinder, P. (1969)J . Clin. Pathol. 22, 246
Varandani, P. T. (1966)Biochirn. Biophys. Acta 118, 198-201
Varandani, P.T.,Nafz, M. A. & Shroyer, L. A. (1971) Diabetes 20, Suppl. 1, 342
Varandani,P.T.,Shroyer,L.A. &Nafz, M.A.(1972)Proc. Nut. Acad. Sci.U.S.69,1681-1684
Pteroylpoly-y-L-glutamatesin the Liver and Kidney of the Monkey
(Cjmarnolgus sp.)
GILLIAN E. DAVIDSON, JOSEPH P. BROWN and JOHN M. SCOTT
Department of Biochemistry, Trinity College, Dublin 2, Irish Republic
Present knowledge of mammalian pteroylglutamate metabolism is based largely on
work carried out on the rat. This animal, however, being coprophagous, obtains some of
its dietary pteroylglutamates via microbial action in the intestine and may not be a valid
model for the human situation, where the ingested pteroylglutamates are principally of
higher-plant origin.
The synthesis of pteroylpolyglutamates from exogenous [3H]PteGlu* by the liver and
kidney of the monkey (Cynamolgzis sp.) has been studied. The animals were injected
intramuscularly with 200pCi of 3‘,5’,9-[3H]PteGlu (15 Ci/mmol; The Radiochemical
Centre, Amersham, Bucks., U.K.) and killed after 24h or 72h. The tissues were removed
and the pteroylglutamates extracted as previously described (Houlihan et al., 1973).
The y-linked polyglutamates of p-aminobenzoate (H2NPhCO-Glul_,) formed by
alkaline oxidation of pteroylpolyglutamates with K M n 0 4 were separated on a 20 cm
x 0.5 cm column of DEAE-cellulose (Whatman DE52) eluted with a non-linear gradient
composed of 550ml of 5mM-potassium phosphate buffer, pH7.0, in the mixing chamber
and 235m1 of the same buffer, 1.OM with respect to KCI, in the reservoir. The radioactive
peaks were identified by their elution position compared with standards prepared and
identified as described previously (Houlihan et al., 1972, 1973).
Endogenous p-aminobenzoylpolyglutamateswere detected in column fractions by a
microbiological assay for p-aminobenzoic acid with a mutant of Escherichia coli
(N.C.I.B. 8109) after alkaline hydrolysis of samples to remove the glutamate residues
(Lampen et al., 1949; Kozloff et al., 1970).
Pteroylmonoglutamates were liberated from the folates in tissue homogenates by the
action of ‘conjugase’ (y-glutamyl carboxypeptidase), prepared from guinea-pig intestinal mucosa (Bernstein et al., 1969), after precipitation of the existing liver or kidney
proteins with trichloroacetic acid. ‘Conjugase’-treated extracts were kept at pH 1 for 2 h
to isomerize any 5CHO-H4PteGlu present to 5,10CH=H4PteGlu, which on subsequent
adjustment to pH7.5 is converted into 10CHO-H4PteGlu. This last-mentioned com-
* Abbreviations: PteGlu etc., pteroylglutamateetc. [Biochem.J . (1967)102, 19-20].
1973