pyruvate kinase isoenzymes in tissues of the developing guinea pig

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 170, 228-241 (1976)

Pyruvate Kinase lsoenzymes in Tissues of the Developing Guinea Pig

ANNE FAULKNER AND COLIN T. JONES

Nufield Institute fbr Medical Research, University of Oxford, Headley Way, Headington, Oxford OX3 9DS

England

Received February 12, 1975

Pyruvate kinase activities and isoenzymes have been followed during the development of the fetal and neonatal guinea pig.

The kinetic properties of the adult isoenzymes were not substantially different from

those previously reported for the rat except pyruvate kinase 1 isolated from liver was far less sensitive to L-alanine inhibition. The kinetic properties of the isoenzymes isolated from the fetal tissues were the same as those of the adult.

Fetal liver contained pyruvate kinase 1 and 4 in comparable activity throughout the period of gestation studied.

Up to five bands of activity in the region of pyruvate kinase 3 and 4 were detected in the brain and muscles of developing guinea pig after electrophoresis. Early in gestation

the bands with mobility close to pyruvate kinase 4 were predominant; during development these disappeared and bands of activity with mobility close to pyruvate kinase 3 were seen.

The properties and distribution of the pyruvate kinase isoenzymes are discussed in

relation to the control of glycolysis in developing tissue. The possible molecular significance of multiple forms between pyruvate kinase 3 and 4 is discussed.

The existence of multiple forms of pyruvate kinase (EC 2.7.1.40) in various animal tissues has been well documented (l- 7). Three major isoenzymes have been identified: PKll found in liver (alternatively named type B or L), PK3l found in muscle and brain (type M or A) and PK4l found in liver, kidney, lung, spleen, and various other tissues (type Mz or C). There is also some evidence for a fourth isoenzyme, PK2,l found in erythrocytes.

On separation these various isoenzymes display distinctive kinetic properties. PKl from rat liver is an allosteric enzyme being activated by FDPl and inhibited by ATP and n-alanine (5, S-11). Because of these properties, a role in the regulation of glycolysis and gluconeogenesis has been at- tributed to this isoenzyme. Recent work has shown that PK4 is also regulated by

1 Abbreviations: PKl, PK2, PK3, and PK4, pyruvate kinase isoenzymes; FDP, fructose 1,8diphos- phate; PEP, phosphoenolpyruvate.

FDP and amino acids (12-14), while PK3 isolated from muscle is not stimulated by FDP and shows normal Michaelis-Menten kinetics except in the presence of L-phenylalanine (15-18).

During the development of the rat, changes in the isoenzyme pattern of various tissues have been observed. Initially PK4 predominates in all tissues but, as development proceeds other isoenzyme types appear in muscle, brain, and liver (19-22). Recently attempts have been made to quantitate these changes in liver (21, 23). The results show that the relatively low activity of PKl in fetal and neonatal life increases sharply on weaning, whereas PK4 drops from a relatively high activity during gestation to a constant low value throughout neonatal and adult life. The much higher ratio of PK4 to PKl activity in the fetal rat liver compared with adult liver and the high ratio found in tumour cells has led to the suggestion that

228

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

PYRUVATE KINASE DURING DEVELOPMENT 229

PK4 is the major isoenzyme in developing livers (19, 21-23). However without infor- mation on developing tissues from other species this view may be misleading.

It is thought that PKl rather than PK4 functions in the control of the interrelation between glycolysis and gluconeogenesis. The absence or low rates of gluconeogenesis in fetal rat liver (24) together with relatively low activites of PKl has supported this view. Study of the changes in PKl, PK4, and gluconeogenesis in the liver of other developing species allows further in- vestigation of such a relationship.

The guinea pig, unlike the rat, is born relatively mature and during late fetal life its liver has the capacity for gluconeogenesis (25, 26). Rates of conversion of glucose into fatty acids suggested that the rate of glycolysis may be low in the fetal guinea pig liver (27). More recent observations have shown relatively high rates of glucose utilization and glycolysis. As part of a study on the factors regulating the control and development of glucose metabolism the properties and isoenzyme distribution of pyruvate kinase have been studied in developing guinea pig tissues.

MATERIALS AND METHODS

Animals

Guinea pigs of the Dunkin-Hartley strain were

maintained on Dixon’s diet 18 (Dixon & Son, Ware, Herts) and mated as described by Elvidge (28). Ges- tational age was determined with an estimated accu-

racy of 2 1.5 days. Animals were stunned by a blow to the back of the neck. Fetuses were removed from the uterus, weighed, bled from the neck and tissues

isolated.

Assay Procedure

Tissues were homogenized in 10 mM Tris-HCl,

pH 7.5, containing 0.1 mM FDP, 1 mM MgCl, and 0.1 mM dithiothreitol, (50% w/v for liver; 33% w/v kidney, lung, spleen, and brain; 20% w/v, cardiac mus-

cle; 10% w/v, skeletal muscle), using a Potter-El- vehjem glass homogenizer fitted with a Teflon pes- tle. Homogenates were centrifuged at 180,OOOg for 45

min at 2”C, and the supernatant used for all experi- ments. Pyruvate kinase was assayed at 25°C by a coupled assay system similar to that described by

Biicher and Pfleiderer (29). Except where stated otherwise, the final reaction mixture contained: 100 mxu Tris-HCl, pH 7.5; 50 mM KCl, 10 mxu MgCl,, 0.5 mM PEP, 0.15 mM NADH, 0.1 mM FDP, 5 U/ml

lactate dehydrogenase and 1 mM ADP. The reaction was started by the addition of ADP to the other

components and followed continuously as the de-

crease in E,,, on an S.P. 1800 recording spectropho- tometer (Pye Unicam Ltd., Cambridge).

DEAE-cellulose Chromatography of Pyru- vate Kinase Isoenzymes

Pyruvate kinase isoenzymes were separated and partially purified by chromatography (5) on DEAE- cellulose (Whatman DE 52). In general, samples (0.4

ml) of the supernatant were applied to a 1.5 x 15 cm DEAE-cellulose column preequilibrated in 20 mM Tris-HCl, pH 7.5, containing 1 mM KCl; 1 mru

MgClz, 0.1 mM EDTA; and 0.1 mM dithiothreitol. PK3 and PK4 were eluted with 20 ml of the equili- brating buffer; PKl was eluted with 30 ml of buffer

containing 0.5 M KCl. A flow rate of 25 ml/h was maintained throughout.

For kinetic work, PKl was eluted with a O-O.5 M

NaCl linear gradient.

Determination of the Recoveries of PKl and PK4

Significant activity was lost during the separation of PKl and PK4 from liver on DEAE-cellulose.

Let the recovery of PKl and PK4 be a and b, respectively, the activity appearing off the column

as PKl be L and as PK4 be M. Then:

Total initial activity (T) = L/u + M/b

MIT = b - bla (LIT)

For extracts with different proportions of PKl and PK4-produced by mixing the supernatants from

liver (PKl and PK4) and lung (PK4, see Results) homogenates-it is possible to determine the isoenzyme recovery by using the above equation, i.e., a and b (Fig. 1). Four independent determinations,

each with five separate isoenzyme mixtures gave a value for PKl recovery of 55.6 ? 6.8% (SD) and for

PK4 recovery of 28.5 c 2.9% (SD). Isoenzyme activities eluted from the DEAE-cellulose were corrected to 100% recovery using these values.

Kinetic Studies

Enzyme effectors were pre-incubated with the enzyme at 25°C for 10 min prior to assay. PEP concen-

trations used were determined enzymatically with commercial pyruvate kinase. K, values were determined from Lineweaver-Burk plots as described by Wilkinson (30).

Electrophoresis

Electrophoresis was carried out in 10% (w/v) starch gel made in 10 mM Tris-HCl, pH 7.8, contain-

ing 10% (w/v) sucrose; 5 mM MgCh, 5 mM KCl, 1 mM EDTA, 0.2 mM dithiothreitol. Gels were allowed to

230 FAULKNER AND JONES

0.4 r

0.3

I l r\\ M/T l

/)I \ . 0.1 .

\\\\ -1

0 0.2 0.4 0.6

L/l

FIG. 1. PKl and PK4 activity recovered after DEAE-cellulose chromatography. Samples contain-

ing varying proportions of PKl and PK4 were chro- matographed on DEAE-cellulose. Recovered activ-

ity of each isoenzyme is plotted as a proportion of the total activity applied to the column. The intercept with the abscissa represents the situation in which

no PK4 activity is present in the extract and hence gives (a) the percentage recovery of PKl while the intercept with the ordinate gives (b) the percentage

recovery of PK4.

set for 12 h after which samples of supernatants

from various tissue homogenates were inserted on filter paper wicks into slots cut in the gel. Horizon- tal electrophoresis was carried out at 2°C for 10 h, at

lo-15 V/cm CO-15 mA/cm*, cross-sectional area) using a Mini 68 Pherograph (Hormuther-Vetter, Hei- delberg, Wieslock). Both electrode reservoirs con-

tained 250 mM Tris-HCl pH 8 with 5 mM MgC12, 100 mM KCl, 1 mM dithiothreitol, and 0.1 mM FDP.

After electrophoresis gels were horizontally

sliced in half and activity detected by a modification of the method of von Fellenberg et al. (1). One cut surface of the gel was flooded with a solution contain-

ing 100 mM Tris-HCl, pH 7.5, 0.15 mM NADH, 0.5 mM PEP, 0.1 mM FDP, 1 mM ADP, 1OmM MgC12, 50 mM KCl, 5 U/ml lactate dehydrogenase. The other half of the gel was treated as above but ADP was omitted. Both halves were incubated at 30°C after being covered with a sheet of transparent polythene.

Pyruvate kinase activity was seen as dark zones (ADP dependent) on a blue fluorescent background when viewed under uv light. Photographs were taken every 10 min by placing the stained surface of the gel on photographic paper (Ilfobrom 5, Ilford Ltd.) and exposing to uv light for l-2 a.

Expression of Results

Results are expressed as the mean values 2 1 SD. The number of determinations is given in parenthe-

ses. Enzyme activities refer to pmoles of pyruvate producedlmin at 25°C.

Materials

PEP (tricyclohexylamine salt), FDP (tetrasodium

salt), ADP (disodium salt), ATP (sodium salt), NADH (disodium salt), and Tris were obtained from Sigma (London) Ltd.; muscle lactate dehydrogenase

(EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40) were obtained from Boehringer Corp. (London) Ltd. All

other chemicals were obtained from British Drug Houses, Poole, Dorset and were of Analar grade, or

the highest purity available.

RESULTS

Electrophoresis of Pyruvate Kinase Isoenzyme from Adult Guinea Pig Tissue

Four pyruvate kinase isoenzymes were detected after electrophoresis for 10 h at 10 V/cm, which in order of mobility are de- fined as PKl > PK2 > PK3 > PK4. The isoenzyme pattern of the various adult tissues was: blood cells, PK2 and PK4 (Fig. 10); liver, PKl and PK4; lung, PK4; kidney, PK3 and PK4; brain, PK3 and PK4; skeletal and cardiac muscle, PK3 (Fig. 2,

I I PKl

5 X -

mm- I PK3

l-l PK4

0 B C K Li Lu 5’

FIG. 2. Starch gel electrophoresispattern ofpyru- vate kinase isoenzymes from adult guinea pig tissues. Electrophoresis was performed as described in the Methods for 10 h at 10 V/cm. The following tissues were studied: B, brain; C, cardiac muscle; K, kidney; Li, liver; Lu, lung; S, skeletal muscle. The origin is represented by 0. Band X was present in control gels stained without ADP.


Plate 10. Electrophoresis of adult rat tissues gave essentially similar isoenzyme patterns to those previously reported (31).

The total pyruvate kinase activity in the adult guinea pig tissues studied is given in Table I. PKl represented 65% and PK4 35% of total adult guinea pig liver pyruvate kinase activity, comparable values obtained from the adult rat liver (Wistar strain) were: PKl, 76%; PK4, 24%.

Properties of the Pyruvate Kinase Isoenzymes from Adult Tissues

PKl

PKl from liver supernatant was eluted from DEAE-cellulose by 0.15 M KCl. The kinetic properties appeared similar to those described for the rat liver isoenzyme (4, 5, 9, 32). A sigmoidal saturation curve for PEP was obtained in the absence of FDP, with an apparent K, of l-2.2 mM (Hill coefficient, n = 1.7-2.1). The presence of FDP restored typical Michaelis- Menten kinetics resulting in a K, value of 40-80 PM (n = 1.0) for PEP. Alanine produced only a small inhibition of PKl activity in the guinea pig (30% at 10 mM). This is unlike rat PKl activity which shows 80% inhibition at 2-5 mM alanine (13).

PK3

Isoenzyme, PK3, from skeletal and cardiac muscle was partially purified by pas- sage through DEAE-cellulose to which it did not bind. Recoveries of 90-100% were obtained and kinetic properties were similar irrespective of the source of the prepara- tion. A hyperbolic saturation curve for PEP was obtained in the presence or absence of FDP with a K, of 44.2 2 7.4 pM

(5). Neither L-alanine nor ATP had an effect on the isoenzyme; L-phenylalanine gave 50% and complete inhibition at about 2 and >5 mM, respectively, in the absence of FDP. This inhibition was partially reversed by L-alanine or FDP. These properties are comparable to those described for the rat muscle isoenzyme (12,16,17-33).

PK4

PK4 from liver was separated from the other isoenzyme present by column chromatography. In crude preparations PK4

activity remained unchanged over a period of weeks. The partially purified enzyme was extremely labile, about 50% of the activity being lost within 1 h. Although the lost activity could be partially recovered by incubation with FDP for up to 30 min the extent of reactiviation was variable. All kinetic studies were performed within an hour of elution of PK4 from the DEAE-cellulose.

In the absence of FDP, the kinetic behav- ior of PK4 with PEP was complex and nonlinear Hill plots (n = 0.4-2.0) were obtained; the apparent K, for PEP was 2.0 ? 0.7 mM (4). With FDP typical Michae- lis-Menten kinetics were obtained with K, values for PEP of 51 -+ 22.3 pM (4) (Fig. 3).

Amino acids, especially L-alanine and L-

phenylalanine, inhibit rat PK4 from various tissues (14, 16, 41, 46). Both these amino acids inhibited the guinea pig isoenzyme; apparent K/s for alanine and phenylalanine were 0.8 and 0.2 mM, respectively (Fig. 4). ATP had no effect on the activity in the presence of excess free Mg’+.

PK4 isolated from guinea pig lung was kinetically and electrophoretically identical to that obtained from liver.

Pyruvate Kinase in the Developing Guinea Pig

The total activity and isoenzyme distribution of pyruvate kinase in various tissues of fetal and neonatal animals have been measured.

Pyruvate Kinase in the Liver

Total liver enzyme activity increased 2- fold between day 30 and 55 of gestation (P < 0.001) then fell by almost half as term approached (P < 0.001). The neonatal activity increased by approximately 50% after birth (PC 0.001) and was similar to that in the adult (Fig. 5). Three isoenzymes were detected in the fetal liver after electrophoresis of crude supernatant (Fig. 7a, plate la). Both PKl and PK4 were present in the livers of all fetuses examined. In addition, a third isoenzyme was identified in the younger fetal livers which had the same mobility as PK2 from blood cells.

The relative proportions of PKl and PK4


(a) Liver

BI 3OF 42F 55F 62F 2N

(b) Kidney

30F 42F 51F 62F 2N M PLATE 1. Starch gel electrophoresis of pyruvate kinase isoenzymes from fetal, neonatal and

adult guinea pig tissues. (a-e) Details as for Fig. 7. (0 Details as for Fig. 2.


30F 35F 42F

(d) Cardiac Muscle

51F 62F 2N M

3OF 35F 42F 50F 62F 67F 2N M PLATE l-Continued


(e) Skeletal Muscle

50F 56F 62F 67F 2N M

(f) Adult Tissues

B C K Li Lu S PLATE l-Continued


TABLE I

ACTIVITY OF PYRUVATE KINASE IN THE ADULT

GUINEA PIG TISSUES”

Tissues Total pyruvate kinase activity (pmol/min/g

wet wt)

Brain 35.4 + 8.9 (12)

Cardiac muscle 32.2 + 7.8 (12) Kidney 24.6 k 8.5 (12) Liver 8.9 c 1.3 (12)

Lung 27.8 + 10.1 (12) Skeletal muscle 121.3 ? 30.9 (12)

Liver PK4 2.9 k 0.76 (12) Liver PKl 5.4 2 0.85 (13)

a Total enzyme activity was determined on tissue extracts in the presence of 0.1 mM FDP as described in Methods. PKl and PK4 activities were deter-

mined after DEAE-cellulose chromatography of liver extracts.

PK4

0.5 0.1 mM FDP

,/ --‘no FDP

A” 0 /”

LLL-L.11.U 0 2 4 6 8 10

PEP (mMI

FIG. 3. Effect of PEP concentration on PK4 activity. Reaction rates were determined as described in

Methods in the absence (01 and presence of 0.1 mM FDP (0) with PK4 isolated after DEAE-cellulose chromatography of liver extracts.

were followed throughout gestation by chromotographic separation of the two isoenzymes. The proportion of the total liver activity represented by PK4 and PKl is shown in Fig. 6. Throughout most of the period of gestation studied PKl-type activity represented about 60% of the total then showed a significant rise (P <0.002) to

about 80% just prior to term. However as PK2 from blood cells was eluted in the same fraction as PKl and PKl from fetal liver contained detectable PK2 (Fig. 10a)

100 _ - -~ 1 L-alanine + 0.1 mM FDP

I- ‘1~

+ 0.1 mM FDP

L-alanine

- c---

L-phenylalanine

4 5

Amino acid fmM)

FIG. 4. Effect of L-alanine and phenylalanine on PK4 activity. Reaction rates were determined as

described in Methods but without FDP in the presence of L-phenylalanine (0) or L-alanine (0) and with 0.1 mM FDP in the presence of L-phenylalanine (A) or L-alanine (A) with PK4 isolated after DEAE-

cellulose chromatography of liver extracts.

r Liver

14 b

12 I

10 c .

8-. *.

. .

6 -*.

. -. . .

. -9 4r I :.

-. * . -. * .

0 L^LLL--Il U~.-l+U 30 40 50 60 10 M

fetal neonatal Age ldaysi

FIG. 5. Total pyruvate kinase activity in the liver during development. The total enzyme activity

was determined on liver extracts in the presence of 0.1 mM FDP as described in Methods. M, maternal liver.


100

t

PK4

80

60 -:

t *

8, . :D:

! :g:.. * . . ~ ;.: * .‘.*

:: . . .:.i,..-. :- :..

* t . . 8, . . . ..,

9, .

id 1w P 5 PKl

“., , , ij , 30 40 50 60 0 10 M

fetal neonatal Age (days)

FIG. 6. Relative proportions of PKl and PK4 activity in the liver during development. The enzyme

activity was determined in the presence of 0.1 mM FDP after DEAE-celluldse chromatography of liver

extracts aa described in Methods. (a) PKl, (b) PK4. M, Maternal liver.

(Plate la) the 60% contribution made before 50 days may be elevated by the presence of PK2. The percentage of PKl and PK4 activity in the neonatal liver is the same as in the adult, this represents a significant fall in the proportion of PKl (P < 0.002) and rise in the proportion of PK4 (P < 0.01) immediately after birth.

Kinetic properties of the isoenzymes from fetal liver were studied as described for the adult above. No significant differ- ences were observed. (Table II).

Pyruvate Kinase in the Kidney

Total enzyme activity in the fetal kidney doubled ZP < 0.001) between 30 days to term although a large variation occurred. At term and during neonatal life the activity was comparable to that found in the adult kidney (Fig. 8). PK4 was the only isoenzyme detected in the fetal kidney before 60 days. Thereafter, and in neonatal and adult kidney, significant amounts of PK3 were detected (Fig. 7b, Plate lb).

Pyruvate Kinase in the Brain

Between 30 and 55 days the total activity in the fetal brain did not change significantly. It then rose to about twice that activity by term (P < 0.02). No significant change in activity was observed at birth (Fig. 9).

Electrophoresis of crude supernatant from the brain of fetal, neonatal and adult guinea pig gave up to five bands of pyruvate kinase activity with mobilities in the region of PK3 and PK4 (Fig. 7c, Plate lc). The PK3-like bands predominated in the adult brain while that of the fetus showed a change from predominantly PKClike bands as term approached.

Pyruvate Kinase in the Muscle

The hind leg skeletal muscle activity of the fetus remained constant between 30 and 55 days and then increased about 6- fold by term (P < 0.001). This activity remained unchanged for the first ten days of neonatal life, being approximately 40% of the adult muscle value (Fig. 10). Activ- ity comparable to that of the adult was present in 30-day-old neonates. Pyruvate kinase activity in fetal cardiac muscle did not change significantly between 30 days gestation to term, its mean activity was 32.3 r 14.9 (40) units/g. The activity in the neonatal heart was 29.1 + 11.1 (26) units/g.

Electrophoresis of crude supernatants from skeletal and cardiac muscle gave up to five bands of pyruvate kinase activity which, like those from brain, had mobilities in the regions of PK3 and PK4. During development there was a change from PKClike to predominantly PKS-like bands (Fig. 7d and e, Plate Id and e).

Electrophoresis of a combination of extracts obtained from adult skeletal muscle (PK3) and lung (PK4) or liver (PKl and PK4) gave no indication of more than two bands in the regions of PK3 and PK4 mobility.

Fetal (35 and 55 days) brain and muscle pyruvate. kinase from crude tissue extracts and after DEAE-cellulose chromatography was not significantly different in kinetic properties from that of adult PK3, despite the presence of bands of activity in the

PYRUVATE KINASE DURING DEVELOPMENT

TABLE II

SOME KINETIC PROPERTIES OF PKl AND PK4 ISOENZYMES FROM FETAL AND ADULT LIVER”

237

PKl PK4

Fetal Adult Fetal Adult

K,, PEP (mM) 0.1 mM FDP 0.04 (0.05) 0.04-0.08 0.066 (0.085) 0.064 No FDP 1.9 (2.2) 1.00-2.2 2.7 (2.9) 1.5-2.5

Apparent Ki ATP, (mM) 0.8 (0.4) 0.4-0.8 No inhibition

L-Alanine (mM) >lO 0.8 (0.9) 0.8 L-Phenylalanine (mM) >lO 0.14 (0.2) 0.2

a Enzyme activity was determined in absence of FDP (unless otherwise stated) on PKl and PK4 isolated by DEAE-cellulose chromatography of liver extracts as described in Methods. Isoenzymes were prepared from the livers of 50- and (IO-day-old fetuses).

region of PK4 after electrophoresis of the fetal supernatants. They were not significantly affected by 0.1 mM FDP or 5 mM L-

alanine but almost completely inhibited by 5 mM L-phenylalanine; the K, for PEP was approximately 50 PM.

DISCUSSION

The kinetic properties of the guinea pig pyruvate kinase isoenzymes with two ex- ceptions are comparable to those previously reported for the rat, rabbit, mouse, ox, and human (34-40). PKl has been reported to be strongly inhibited by alanine however a maximum of only 30% inhibition was observed with a guinea pig liver isoenzyme. ATP inhibition of PK4 has been observed in the rat (13, 14) although this can be partially reversed by Mg2+ (16, 18). PK4 from guinea pig liver showed no inhibition by ATP which compares with PK4 isolated from rat kidney cortex (41).

All fetal guinea pig tissues except cardiac muscle show a progressive rise in total activity during development. No changes were observed immediately before or after birth or during the first ten days of postnatal life. In rat tissues the developmental changes in total activity, with the exception of skeletal muscle, which also showed a progressive rise, were variable and large changes in activity were noted just before and after birth (21, 23, 42).

Developmental patterns for pyruvate kinase isoenzymes in rat liver show a clear increase in the activity of PKl and fall in

PK4 immediately after birth. Isoenzyme changes reported in the fetal rat liver have not been consistent. Substantial activities of PK4 have been repeatedly found; however PKl has either not been found (22) or has been observed in low (23) or high activity (21). Two studies quantitated the developmental changes in PKl and PK4 of the rat. Ostermann et al. (21) observed a fall in the activity of both as term approached with PKl always higher than PK4; these changes in PKl activity in the fetal liver were not confirmed by electrophoresis. Middleton and Walker (23) showed a pro- gessive fall in fetal rat liver PK4 activity from a value substantially higher than PKl to one below that of PKl during the five days before birth. They also showed that PKl activity rose during this period. However the kinetic method used in their analysis has limitations because of recent evidence on FDP activation of PK4. Elec- trophoreses of fetal rat liver extracts have also demonstrated the existence of bands between PKl and PK4 that have been called PK3 or hybrids of PKl and PK4 isoenzymes (19, 22). Such intermediate bands have also been identified as PKw (21, 31).

The reported presence of low PKl and high PK4 activity, as found in tumor cells (22, 43-45), has led to the suggestion that the fetal liver cells are in an undifferen- tiated state (22). However the fetal rat liver has a large population of haemopoietic cells (46) which makes comparison

-1-m-1 PKI

~ ---- PK2

I !

--I x

I -111-11 PK4

w m m m m m PK4

~ 0 8, 30F 42F 52 62F ZN M 1

30F 4% 51F 62F 2N M 0

Aqe ldaysi Age (days1

CC) Brain td) Cardiac Muscle

i 0

3OF 35F 48 51F 62F 2N M ’ 3OF 35F 48 50F 6ZF 67F 2N M

Age (days1

(el Skeletal Muscle

Age (days1

t

I PK4

I 5OF 56F 62F 67F ZN M

0

FIG. 7. Starch gel electrophoresis pattern of pyruvate kinase isoenzymes from guinea pig tissues during development. Electrophoresis was performed as described in the Methods for IO h at lo-15 V/cm. (a) liver (Bl-PK from blood cells), (b) kidney, Cc) brain, (d) cardiac muscle, (e) skeletal muscle. F, fetal; N, neonatal; M, maternal tissues. Band X was present in control gels stained without ADP.

238


5o [ Kidney

40

-8 10 I.;.’ y: *I~ :

. .

* 0 L-u AAL

30 40 50 60 0 10 M fetal neonatal

Age (daysl

FIG. 8. Total pyruvate kinase activity in the kid-

ney during development. The total enzyme activity was determined on kidney extracts in the presence

of 0.1 mM FDP as described in Methods. M, maternal kidney.

difficult. In contrast the fetal guinea pig liver has substantial PKl activity. Contri- butions to this activity from haemopoietic cells may occur early in gestation, but after about 50 days the presence of much smaller amounts of haemopoietic tissue (47) suggests that for the last 15 days of gestation the hepatic cells have a high PKl activity. Thus a low PKl activity is not necessarily characteristic of fetal hepatic tissue. We have also observed significant PKl activity (determined by DEAE- cellulose chromatography and electrophoresis) in the livers of 12- to l&week human fetuses (48).

The proportions of PKl and PK4 in the adult guinea pig livers were comparable to those we observed in the adult rat liver (Wistar) and those reported by Tanaka et al. (3,4) for the Sprague-Dawley and Van Berkel et al. (49) for the Wistar strain of rat. Recently much lower PK4 activity has been reported for the Sprague-Dawley (21, 22) and Wistar (23) strains of rat.

PKl isolated from pig and ox liver (39, 50) and PK3 isolated from skeletal muscle (50) each have a molecular weight of approximately 250,000 and have been re- solved into four subunits of molecular weight of approximately 60,000. Cardenas

and Dyson (39) have observed hybridisa- tion between PK3 and PKl subunits prepared from ox tissues and found the theo- retical maximum number of hybrids based on a tetrameric structure of identical subunits for pyruvate kinase. Electrophoresis of extracts from the developing brain and skeletal and cardiac muscle of the fetal

60 r

Brain ” .

.

50 L .

0 b--ad IA

40 50 60 0 10 M

fetal neonatal

Age (days1

FIG. 9. Total pyruvate kinase activity in the

brain during development. The total enzyme activity was determined on brain extracts in the presence

of 0.1 mM FDP as described in Methods. M, maternal brain.

160 Skeletal Muscle

I 18 *

,

n I *

. . . .

. *. ( *’ “U_U1-11lLL 1 1

30 40 te:aY

60 0 10 M neonatal

Age (days)

FIG. 10. Total pyruvate kinase activity in skele-

tal muscle during development. The total enzyme activity was determined on hind limb skeletal muscle in the presence of 0.1 mM FDP as described in Methods. M, maternal tissue.


guinea pig shows up to five bands of pyruvate kinase activity in the region of PK3 and PK4. It is possible that they represent the in uivo formation of PK3 and PK4 hybrids. No evidence was found for five forms in the adult tissues and this may be related to the low activity of PK4 in these compared with fetal tissues. If hybrids are formed in uiuo, then this suggests that the subunits of the two isoenzymes arc formed in the same cell and are allowed to freely interact. No hybrids of PKl with other isoenzymes were observed.

The biochemical significance of PKl and PK4 found in the liver has been discussed in relation to the allosteric properties of PKl allowing large changes in the rate of glycolysis, and the reported absence of such properties for PK4 leading to little control of glycolysis by this isoenzyme (51). Furthermore the occurrence of PKl together with glucokinase in parenchymal cells, where it is thought to control the net rate of gluconeogenesis and glycolysis, and of PK4 together with hexokinase in Kupf- fer cells, that exhibit high rate of glycolysis, has added weight to this view (34, 49, 52). However, the recent reports on the allosteric nature of PK4 and the virtual absence of glucokinase in any of the guinea pig tissues studied (unpublished observations) or in the fetal rat liver (22) make the interpretation of the precise reg- ulatory functions of PK4 difficult. The developmental pattern for these isoenzymes suggests that the control of glycolysis and gluconeogenesis by pyruvate kinase could occur to the same extent in developing as in adult liver. Any changes in the control are likely to occur at the substrate level. In brain and muscle tissue the predominance of PK3 supports the view that pyruvate kinase exerts little control over the high rates of glycolysis these tissues are capable of achieving. During fetal life the appear- ance of PK4 and PK3-PK4 hybrids suggests that in these developing tissues pyruvate kinase may exert a larger measure of control over glycolysis, although some of these changes may be related to changes in cell population, e.g., fall in gliabneuronal during brain development (53).

ACKNOWLEDGMENTS

We were grateful to Dr. G. S. Dawes for his

interest and encouragement and Mrs. Paula Webb

for expert technical assistance. The work was supported by a grant for the Medical Research Council.

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