ARCHIVES
OF
BIOCHEMISTRY
Pyruvate
AND
Kinase
ANNE Nufield
Institute
fbr Medical
BIOPHYSICS
170,
228-241
(1976)
lsoenzymes in Tissues Guinea Pig FAULKNER
Research,
University Received
AND
COLIN
of Oxford, England February
Headley
of the Developing
T. JONES Way,
Headington,
Oxford
OX3
9DS
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 (l7). 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 attributed to this isoenzyme. Recent work has shown that PK4 is also regulated by
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
1 Abbreviations: PKl, PK2, PK3, and PK4, pyruvate kinase isoenzymes; FDP, fructose 1,8diphosphate; PEP, phosphoenolpyruvate. 228 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
PYRUVATE
KINASE
DURING
PK4 is the major isoenzyme in developing livers (19, 21-23). However without information 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 investigation 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
lactate dehydrogenase and 1 mM ADP. was started by the addition of ADP components and followed continuously crease in E,,, on an S.P. 1800 recording tometer (Pye Unicam Ltd., Cambridge).
The reaction to the other as the despectropho-
DEAE-cellulose Chromatography vate Kinase Isoenzymes
of Pyru-
Pyruvate kinase isoenzymes were separated and partially purified by chromatography (5) on DEAEcellulose (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 equilibrating 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 and PK4
Animals
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 muscle; 10% w/v, skeletal muscle), using a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. Homogenates were centrifuged at 180,OOOg for 45 min at 2”C, and the supernatant used for all experiments. 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
of PKl
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
METHODS
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). Gestational age was determined with an estimated accuracy 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
229
DEVELOPMENT
(T) = L/u MIT
+ M/b
= 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 concentrations 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, containing 10% (w/v) sucrose; 5 mM MgCh, 5 mM KCl, 1 mM EDTA, 0.2 mM dithiothreitol. Gels were allowed to
230
FAULKNER 0.4
AND
JONES
Expression
r
Results are expressed as the mean values 2 1 SD. The number of determinations is given in parentheses. Enzyme activities refer to pmoles of pyruvate producedlmin at 25°C.
r\\.
0.3
I
M/T
l
Materials
l
/)I\ 0.1
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.
. \\\\
-1 0
0.2
of Results
0.4
RESULTS
0.6
L/l
FIG. 1. PKl and PK4 activity recovered after DEAE-cellulose chromatography. Samples containing varying proportions of PKl and PK4 were chromatographed on DEAE-cellulose. Recovered activity 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. Horizontal 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, Heidelberg, Wieslock). Both electrode reservoirs contained 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 containing 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.
Electrophoresis of Pyruvate Isoenzyme from Adult Guinea
Kinase Pig Tissue
Four pyruvate kinase isoenzymes were detected after electrophoresis for 10 h at 10 V/cm, which in order of mobility are defined 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 ofpyruvate 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.
PYRUVATE
KINASE
DURING
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 Isoenzymes from Adult PKl
Kinase Tissues
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 MichaelisMenten 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 passage 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 preparation. 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
231
DEVELOPMENT
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 behavior 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 Michaelis-Menten kinetics were obtained with K, values for PEP of 51 -+ 22.3 pM (4) (Fig. 3). Amino acids, especially L-alanine and Lphenylalanine, 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 Pig
Guinea
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 2fold 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
232
FAULKNER
AND
JONES
(a) Liver
BI
3OF
42F 55F
62F
2N
(b) Kidney
30F 42F 51F PLATE 1. Starch gel electrophoresis adult guinea pig tissues. (a-e) Details
of pyruvate as for Fig.
62F
2N
M
kinase isoenzymes from fetal, 7. (0 Details as for Fig. 2.
neonatal
and
PYRUVATE
30F
35F
KINASE
42F
DURING
233
DEVELOPMENT
51F 62F
2N
M
(d) Cardiac Muscle
3OF 35F 42F 50F PLATE
l-Continued
62F 67F 2N
M
234
FAULKNER
AND
JONES
(e) Skeletal Muscle
50F 56F
62F
67F 2N
M
(f) Adult Tissues
B
K
C PLATE
Li
l-Continued
Lu
S
PYRUVATE TABLE ACTIVITY
KINASE
I
OF PYRUVATE KINASE IN THE ADULT GUINEA PIG TISSUES” Tissues
Total pyruvate kinase activity (pmol/min/g wet wt)
Brain Cardiac muscle Kidney Liver Lung Skeletal muscle Liver PK4 Liver PKl
35.4 32.2 24.6 8.9 27.8 121.3 2.9 5.4
+ + k c + ? k 2
8.9 (12) 7.8 (12) 8.5 (12) 1.3 (12) 10.1 (12) 30.9 (12) 0.76 (12) 0.85 (13)
DURING
about PK2 same liver
235
DEVELOPMENT
80% just prior to term. However as from blood cells was eluted in the fraction as PKl and PKl from fetal contained detectable PK2 (Fig. 10a) 100
_ I-
-
L-alanine
-~ 1
+ 0.1 mM FDP
‘1~
+ 0.1 mM FDP
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 determined after DEAE-cellulose chromatography of liver extracts.
L-alanine - c---
PK4 L-phenylalanine
0.5
0.1 mM FDP
4 Amino
A”
--‘no
,/ 0
FDP
/”
acid
5
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 DEAEcellulose chromatography of liver extracts. Liver
r 14 b
12 I 10c 0
LLL-L.11.U 2
. 4
6 PEP
8
10
8-.
*. .
(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 lO >lO
0.064 1.5-2.5
No inhibition
0.8 (0.9) 0.14 (0.2)
0.8 0.2
in absence of FDP (unless otherwise stated) on PKl and PK4 isolated of liver extracts as described in Methods. Isoenzymes were prepared fetuses).
region of PK4 after electrophoresis of the fetal supernatants. They were not significantly affected by 0.1 mM FDP or 5 mM Lalanine 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 exceptions 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 progessive 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. Electrophoreses 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 undifferentiated state (22). However the fetal rat liver has a large population of haemopoietic cells (46) which makes comparison
PKI
-1-m-1
~
----
PK2
I
--I
x
!
-111-11 I
8,
PK4
~ 62F ZN
30F 42F 52
0
M
wmmmmm
PK4
1 30F 4%
51F
62F
2N
0
M
Age (days1
Aqe ldaysi td) Cardiac
CC) Brain
Muscle
i 3OF 35F 3OF
35F
48
51F
62F
2N
M
48
50F
6ZF
67F
2N
M
0
’ Age (days1
Age (days1 (el Skeletal
Muscle
t
PK4
I
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
PYRUVATE 5o [
KINASE
DURING
239
DEVELOPMENT
and Dyson (39) have observed hybridisation between PK3 and PKl subunits prepared from ox tissues and found the theoretical 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
Kidney
40
60
Brain
”
r
10 I.;.’
-8 *I~
y: .
.
50
. . .
L
:
* 0 L-u
AAL 30
40
50 fetal
60
0 10 neonatal
M
Age (daysl
8. Total pyruvate kinase activity in the kidney 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. FIG.
difficult. In contrast the fetal guinea pig liver has substantial PKl activity. Contributions 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 DEAEcellulose 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 resolved into four subunits of molecular weight of approximately 60,000. Cardenas
0
IA
b--ad
40
50
0
60
Age
FIG. 9. Total pyruvate brain during development. ity was determined on brain of 0.1 mM FDP as described nal brain. 160
Skeletal
. . I
“U_U1-11lLL
kinase activity in the The total enzyme activextracts in the presence in Methods. M, mater-
Muscle
. *.
(
*’
*
,
. .
*
30
M
(days1
I 18
n
10
neonatal
fetal
40
60 te:aY
0 neonatal
1
10
1
M
Age (days)
FIG. 10. Total pyruvate kinase activity in skeletal 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.
240
FAULKNER
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 Kupffer 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 regulatory 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 appearance 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 fall in in cell population, e.g., gliabneuronal during brain development (53).
AND
JONES 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. REFERENCES 1. VON FELLENBERG, R., RICHTERICK, R., AND AEBI, H. (1963) Enzym. Biol. Clin. 3,240-250. 2. KOLER, R. D., BIGLEY, R. H., JONES, R. T., RIGAS, D. A., VANBELLINGHEN, P., AND THOM~ SON, P. (1964) Cold Spring Harb. Symp. Quad. Biol. 29, 213-220. 3. TANAKA, T., HARANO, Y., MORIMURA, H., AND MORI, R. (1965) Biochem. Biophys. Res. Commun. 21, 55-60. 4. TANAKA, T., HARANO, Y., SUE, F., AND MORIMURA, H. (1967) J. Biochem. (Tokyo) 62, 7191. 5. SUSOR, W. A., AND RUTTER, W. J. (1968) Biochem. Biophys. Res. Commun. 30, 14-20. 6. CRISS, W. E. (1969)Biochem. Biophys. Res. Commun. 35,901-905. 7. WHITTEL, N. M., NG, D. 0. K., PRABHAKARARAO, K., AND HOLMES, R. S. (1973) Comp. Biochem. Physiol. 46B, 71-80. 8. TANAKA, T., SUE, F., AND MORIMURA, H. (1967) Biochem. Biophys. Res. Commun. 29, 444449. 9. TAYLOR, C. B., AND BAILEY, E. (1967) Biochem. J. 102, 32C-33C. 10. WEBER, G., LEA, M. A., AND STAMM, N. B. (1968) Aduan. Enz. Reg. 6, 101-127. 11. SEUBERT, W., AND SCHONER, W. (1971) Current Topics Cell Reg. 3, 237-267. 12. IBSEN, K. H., AND TRIPPET, P. (1972) Biochemistry 11, 4420-4450. 13. IMAMURA, K., TANIUCHI, K., AND TANAKA, T. (1972) J. Biochem. (Tokyo) 72, 1001-1015. 14. WALKER, P. R., AND POTTER, V. R. (1973) J. Biol. Chem. 248, 4610-4616. 15. BOYER, P. D. (1962) in The Enzymes (Boyer, P. D, Lardy, H., and Myrback, K., eds.), Vol. VI, pp. 95-113, Academic Press, New York. 16. JIM~~NEZ DE A&A, L., ROZENGURT, E., DEVALLE, J. J., AND CARMINATTI, H. (1971) Biochem. Biophys. Acta 238, 234-326. 17. KAYNE, F. J., ANDPRICE, N. C. (1972)Biochemistry 11, 4415-4420. 18. IBSEN, K. H., AND TRIPPET, P. (1973) Arch. Biochem. Biophys. 150, 730-744. 19. RUTTER, W. J. (1969) in Foetal Autonomy (Wolstenholme, G. E. W., and O’Connor, M.), pp. 59-76, J. & A. Churchill, London. 20. SUSOR, W. A. (1970) Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 729. 21. OSTERMANN, J., FRITZ, P. J., AND WUNTCH, T. (1973) J. Biol. Chem. 248, 1011-1018.
PYRUVATE
KINASE
DURING
22. WALKER, P. R., AND POTTER, V. R. (1972) Advan. Enz. Reg. 11, 339-364. 23. MIDDLETON, M. C., AND WALKER, D. G. (1972) Biochem. J. 127, 721-731. 24. BALLARD, F. J. (1970) in Physiology of the Perinatal Period, Vol. I (U. Stave) pp. 417-440, Appleton Century Crofts: Meredith Corp. N. Y. 25. JONES, C. T. (1974) Biochem. J. Submitted for publication. 26. JONES, C. T., AND ASHTON, I. K. (1974) Biochem. J. Submitted for publication. 27. JONES, C. T. (1973) in Foetal and Neonatal Physiology. Proceedings of the Barcroft Centenary Symposium. pp. 403-409, Cambridge University Press, Cambridge. 28. ELVIDGE, H. (1972) J. Inst. Animal Technicians 23,111-117. 29. BUTCHER, T., AND PFLEIDERER, G. (1955) Methods Enzymol. 1,435-440. 30. WILKINSON, G. N. (1961) Biochem. J. 80, 324332. 31. IMAMURA, K., AND TANAKA, T. (1972) J. Biochem. (Tokyo) 71, 1043-1051. 32. CARMINATTI, H., JIM~NEZ DE A&A, L., RECONDO, E., PASSERON, S., AND ROZENGURT, E. (1968) J. Biol. Chem. 243, 3051-3056. 33. CARMINATTI, H., JIM~~NEZ DE A&A, L., LEIDERMAN, B., AND ROZENGURT, E. (1971) J. Biol. Chem. 246, 7284-7288. 34. CRISP, D. M., AND POGSON, C. I. (1972)Biochem. J. 126, 1009-1023. 35. IRVING, M. G., AND WILLIAMS, J. F. (1973) Bioehem. J. 131, 287-301. 36. IRVING, M. G., AND WILLIAMS, J. F. (19731 Biochem. J. 131, 303-313. 37. IMAMURA, K., TANAKA, T., NISHINA, T., NAKASHIMA, K., AND MIWA, S. (1973) J. Biochem. (Tokyo) 74, 1165-1175.
DEVELOPMENT
241
38. KAYNE, F. J., AND PRICE, N. C. (1973) Arch. Biochem. Biophys. 159, 292-296. 39. CARDINAS, J. M., AND DYSON, R. D. (1973) J. Biol. Chem. 248,6938-6944. 40. CARDINAS, J. M., DYSON, R. D., AND STRAND HOLM, J. J. (1973) J. Biol. Chem. 248, 69316937. 41. COSTA, L. JIM~~NEZ DE A&A, L., ROZENGURT, E., BADE, E. G., AND CARMINATTI, H. (1972) Biochem. Biophys. Acta 239, 128-136. 42. HOMMES, F. A., AND WILMINK, C. W. (1968) Biol. Neonate 13, 181-193. 43. TAYLOR, C. B., MORRIS, H. P., AND WEBER, G. (1969) Life Sci. (oxfird) 8, 635-644. 44. BALINSKY, D., CAYANIS, E., AND BERSOHN, I. (1973) Biochemistry 12, 863-870. 45. WEINHOUSE, S. (1973) Fed. PFOC. Amer. Sot. Exp. Biol. 32, 2162-2167. 46. OLIVER, I. T., BLUMER, W. F. C., AND WITHAM, I. J. (1963) Comp. Biochem. Physiol. 10, 3338. 47. PARAT, M. (1923) Bull. Biol. Fr. Belg. 57, 364399. 48. FAULKNER, A., AND JONES, C. T. (1975) Fed. EUF. Biochem. Sot. Lett., 53, 167-169. 49. VAN BERKEL, J. C., KOSTER, J. F., AND HULSMANN, W. C. (1972) Biochem. Biophys. A& 276, 425-429. 50. KAYNE, F. J. (1973) in The Enzymes, 3rd ed., (Bayer, P. D.), Vol. VIII, p. 353-382, Academic Press, London. 51. NEWSHOLME, E. A., AND GEVERS, W. (1967) Vit. Horn. 25, l-87. 52. SAPAG-HAGAR, M., MARCO, R., AND SOLS, A. (1969) Fed. EUF. Biochem. Sot. Lett. 3, 68-71. 53. MCILWAIN, H., AND BACHELARD, H. S. (1971) in Biochemistry and the Control Nervous System, 4th ed., pp. 406-441. Churchill Livingstone, Edinburgh.
OF
BIOCHEMISTRY
Pyruvate
AND
Kinase
ANNE Nufield
Institute
fbr Medical
BIOPHYSICS
170,
228-241
(1976)
lsoenzymes in Tissues Guinea Pig FAULKNER
Research,
University Received
AND
COLIN
of Oxford, England February
Headley
of the Developing
T. JONES Way,
Headington,
Oxford
OX3
9DS
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 (l7). 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 attributed to this isoenzyme. Recent work has shown that PK4 is also regulated by
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
1 Abbreviations: PKl, PK2, PK3, and PK4, pyruvate kinase isoenzymes; FDP, fructose 1,8diphosphate; PEP, phosphoenolpyruvate. 228 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
PYRUVATE
KINASE
DURING
PK4 is the major isoenzyme in developing livers (19, 21-23). However without information 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 investigation 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
lactate dehydrogenase and 1 mM ADP. was started by the addition of ADP components and followed continuously crease in E,,, on an S.P. 1800 recording tometer (Pye Unicam Ltd., Cambridge).
The reaction to the other as the despectropho-
DEAE-cellulose Chromatography vate Kinase Isoenzymes
of Pyru-
Pyruvate kinase isoenzymes were separated and partially purified by chromatography (5) on DEAEcellulose (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 equilibrating 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 and PK4
Animals
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 muscle; 10% w/v, skeletal muscle), using a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. Homogenates were centrifuged at 180,OOOg for 45 min at 2”C, and the supernatant used for all experiments. 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
of PKl
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
METHODS
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). Gestational age was determined with an estimated accuracy 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
229
DEVELOPMENT
(T) = L/u MIT
+ M/b
= 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 concentrations 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, containing 10% (w/v) sucrose; 5 mM MgCh, 5 mM KCl, 1 mM EDTA, 0.2 mM dithiothreitol. Gels were allowed to
230
FAULKNER 0.4
AND
JONES
Expression
r
Results are expressed as the mean values 2 1 SD. The number of determinations is given in parentheses. Enzyme activities refer to pmoles of pyruvate producedlmin at 25°C.
r\\.
0.3
I
M/T
l
Materials
l
/)I\ 0.1
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.
. \\\\
-1 0
0.2
of Results
0.4
RESULTS
0.6
L/l
FIG. 1. PKl and PK4 activity recovered after DEAE-cellulose chromatography. Samples containing varying proportions of PKl and PK4 were chromatographed on DEAE-cellulose. Recovered activity 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. Horizontal 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, Heidelberg, Wieslock). Both electrode reservoirs contained 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 containing 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.
Electrophoresis of Pyruvate Isoenzyme from Adult Guinea
Kinase Pig Tissue
Four pyruvate kinase isoenzymes were detected after electrophoresis for 10 h at 10 V/cm, which in order of mobility are defined 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 ofpyruvate 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.
PYRUVATE
KINASE
DURING
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 Isoenzymes from Adult PKl
Kinase Tissues
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 MichaelisMenten 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 passage 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 preparation. 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
231
DEVELOPMENT
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 behavior 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 Michaelis-Menten kinetics were obtained with K, values for PEP of 51 -+ 22.3 pM (4) (Fig. 3). Amino acids, especially L-alanine and Lphenylalanine, 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 Pig
Guinea
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 2fold 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
232
FAULKNER
AND
JONES
(a) Liver
BI
3OF
42F 55F
62F
2N
(b) Kidney
30F 42F 51F PLATE 1. Starch gel electrophoresis adult guinea pig tissues. (a-e) Details
of pyruvate as for Fig.
62F
2N
M
kinase isoenzymes from fetal, 7. (0 Details as for Fig. 2.
neonatal
and
PYRUVATE
30F
35F
KINASE
42F
DURING
233
DEVELOPMENT
51F 62F
2N
M
(d) Cardiac Muscle
3OF 35F 42F 50F PLATE
l-Continued
62F 67F 2N
M
234
FAULKNER
AND
JONES
(e) Skeletal Muscle
50F 56F
62F
67F 2N
M
(f) Adult Tissues
B
K
C PLATE
Li
l-Continued
Lu
S
PYRUVATE TABLE ACTIVITY
KINASE
I
OF PYRUVATE KINASE IN THE ADULT GUINEA PIG TISSUES” Tissues
Total pyruvate kinase activity (pmol/min/g wet wt)
Brain Cardiac muscle Kidney Liver Lung Skeletal muscle Liver PK4 Liver PKl
35.4 32.2 24.6 8.9 27.8 121.3 2.9 5.4
+ + k c + ? k 2
8.9 (12) 7.8 (12) 8.5 (12) 1.3 (12) 10.1 (12) 30.9 (12) 0.76 (12) 0.85 (13)
DURING
about PK2 same liver
235
DEVELOPMENT
80% just prior to term. However as from blood cells was eluted in the fraction as PKl and PKl from fetal contained detectable PK2 (Fig. 10a) 100
_ I-
-
L-alanine
-~ 1
+ 0.1 mM FDP
‘1~
+ 0.1 mM FDP
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 determined after DEAE-cellulose chromatography of liver extracts.
L-alanine - c---
PK4 L-phenylalanine
0.5
0.1 mM FDP
4 Amino
A”
--‘no
,/ 0
FDP
/”
acid
5
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 DEAEcellulose chromatography of liver extracts. Liver
r 14 b
12 I 10c 0
LLL-L.11.U 2
. 4
6 PEP
8
10
8-.
*. .
(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 lO >lO
0.064 1.5-2.5
No inhibition
0.8 (0.9) 0.14 (0.2)
0.8 0.2
in absence of FDP (unless otherwise stated) on PKl and PK4 isolated of liver extracts as described in Methods. Isoenzymes were prepared fetuses).
region of PK4 after electrophoresis of the fetal supernatants. They were not significantly affected by 0.1 mM FDP or 5 mM Lalanine 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 exceptions 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 progessive 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. Electrophoreses 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 undifferentiated state (22). However the fetal rat liver has a large population of haemopoietic cells (46) which makes comparison
PKI
-1-m-1
~
----
PK2
I
--I
x
!
-111-11 I
8,
PK4
~ 62F ZN
30F 42F 52
0
M
wmmmmm
PK4
1 30F 4%
51F
62F
2N
0
M
Age (days1
Aqe ldaysi td) Cardiac
CC) Brain
Muscle
i 3OF 35F 3OF
35F
48
51F
62F
2N
M
48
50F
6ZF
67F
2N
M
0
’ Age (days1
Age (days1 (el Skeletal
Muscle
t
PK4
I
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
PYRUVATE 5o [
KINASE
DURING
239
DEVELOPMENT
and Dyson (39) have observed hybridisation between PK3 and PKl subunits prepared from ox tissues and found the theoretical 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
Kidney
40
60
Brain
”
r
10 I.;.’
-8 *I~
y: .
.
50
. . .
L
:
* 0 L-u
AAL 30
40
50 fetal
60
0 10 neonatal
M
Age (daysl
8. Total pyruvate kinase activity in the kidney 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. FIG.
difficult. In contrast the fetal guinea pig liver has substantial PKl activity. Contributions 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 DEAEcellulose 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 resolved into four subunits of molecular weight of approximately 60,000. Cardenas
0
IA
b--ad
40
50
0
60
Age
FIG. 9. Total pyruvate brain during development. ity was determined on brain of 0.1 mM FDP as described nal brain. 160
Skeletal
. . I
“U_U1-11lLL
kinase activity in the The total enzyme activextracts in the presence in Methods. M, mater-
Muscle
. *.
(
*’
*
,
. .
*
30
M
(days1
I 18
n
10
neonatal
fetal
40
60 te:aY
0 neonatal
1
10
1
M
Age (days)
FIG. 10. Total pyruvate kinase activity in skeletal 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.
240
FAULKNER
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 Kupffer 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 regulatory 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 appearance 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 fall in in cell population, e.g., gliabneuronal during brain development (53).
AND
JONES 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. REFERENCES 1. VON FELLENBERG, R., RICHTERICK, R., AND AEBI, H. (1963) Enzym. Biol. Clin. 3,240-250. 2. KOLER, R. D., BIGLEY, R. H., JONES, R. T., RIGAS, D. A., VANBELLINGHEN, P., AND THOM~ SON, P. (1964) Cold Spring Harb. Symp. Quad. Biol. 29, 213-220. 3. TANAKA, T., HARANO, Y., MORIMURA, H., AND MORI, R. (1965) Biochem. Biophys. Res. Commun. 21, 55-60. 4. TANAKA, T., HARANO, Y., SUE, F., AND MORIMURA, H. (1967) J. Biochem. (Tokyo) 62, 7191. 5. SUSOR, W. A., AND RUTTER, W. J. (1968) Biochem. Biophys. Res. Commun. 30, 14-20. 6. CRISS, W. E. (1969)Biochem. Biophys. Res. Commun. 35,901-905. 7. WHITTEL, N. M., NG, D. 0. K., PRABHAKARARAO, K., AND HOLMES, R. S. (1973) Comp. Biochem. Physiol. 46B, 71-80. 8. TANAKA, T., SUE, F., AND MORIMURA, H. (1967) Biochem. Biophys. Res. Commun. 29, 444449. 9. TAYLOR, C. B., AND BAILEY, E. (1967) Biochem. J. 102, 32C-33C. 10. WEBER, G., LEA, M. A., AND STAMM, N. B. (1968) Aduan. Enz. Reg. 6, 101-127. 11. SEUBERT, W., AND SCHONER, W. (1971) Current Topics Cell Reg. 3, 237-267. 12. IBSEN, K. H., AND TRIPPET, P. (1972) Biochemistry 11, 4420-4450. 13. IMAMURA, K., TANIUCHI, K., AND TANAKA, T. (1972) J. Biochem. (Tokyo) 72, 1001-1015. 14. WALKER, P. R., AND POTTER, V. R. (1973) J. Biol. Chem. 248, 4610-4616. 15. BOYER, P. D. (1962) in The Enzymes (Boyer, P. D, Lardy, H., and Myrback, K., eds.), Vol. VI, pp. 95-113, Academic Press, New York. 16. JIM~~NEZ DE A&A, L., ROZENGURT, E., DEVALLE, J. J., AND CARMINATTI, H. (1971) Biochem. Biophys. Acta 238, 234-326. 17. KAYNE, F. J., ANDPRICE, N. C. (1972)Biochemistry 11, 4415-4420. 18. IBSEN, K. H., AND TRIPPET, P. (1973) Arch. Biochem. Biophys. 150, 730-744. 19. RUTTER, W. J. (1969) in Foetal Autonomy (Wolstenholme, G. E. W., and O’Connor, M.), pp. 59-76, J. & A. Churchill, London. 20. SUSOR, W. A. (1970) Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 729. 21. OSTERMANN, J., FRITZ, P. J., AND WUNTCH, T. (1973) J. Biol. Chem. 248, 1011-1018.
PYRUVATE
KINASE
DURING
22. WALKER, P. R., AND POTTER, V. R. (1972) Advan. Enz. Reg. 11, 339-364. 23. MIDDLETON, M. C., AND WALKER, D. G. (1972) Biochem. J. 127, 721-731. 24. BALLARD, F. J. (1970) in Physiology of the Perinatal Period, Vol. I (U. Stave) pp. 417-440, Appleton Century Crofts: Meredith Corp. N. Y. 25. JONES, C. T. (1974) Biochem. J. Submitted for publication. 26. JONES, C. T., AND ASHTON, I. K. (1974) Biochem. J. Submitted for publication. 27. JONES, C. T. (1973) in Foetal and Neonatal Physiology. Proceedings of the Barcroft Centenary Symposium. pp. 403-409, Cambridge University Press, Cambridge. 28. ELVIDGE, H. (1972) J. Inst. Animal Technicians 23,111-117. 29. BUTCHER, T., AND PFLEIDERER, G. (1955) Methods Enzymol. 1,435-440. 30. WILKINSON, G. N. (1961) Biochem. J. 80, 324332. 31. IMAMURA, K., AND TANAKA, T. (1972) J. Biochem. (Tokyo) 71, 1043-1051. 32. CARMINATTI, H., JIM~NEZ DE A&A, L., RECONDO, E., PASSERON, S., AND ROZENGURT, E. (1968) J. Biol. Chem. 243, 3051-3056. 33. CARMINATTI, H., JIM~~NEZ DE A&A, L., LEIDERMAN, B., AND ROZENGURT, E. (1971) J. Biol. Chem. 246, 7284-7288. 34. CRISP, D. M., AND POGSON, C. I. (1972)Biochem. J. 126, 1009-1023. 35. IRVING, M. G., AND WILLIAMS, J. F. (1973) Bioehem. J. 131, 287-301. 36. IRVING, M. G., AND WILLIAMS, J. F. (19731 Biochem. J. 131, 303-313. 37. IMAMURA, K., TANAKA, T., NISHINA, T., NAKASHIMA, K., AND MIWA, S. (1973) J. Biochem. (Tokyo) 74, 1165-1175.
DEVELOPMENT
241
38. KAYNE, F. J., AND PRICE, N. C. (1973) Arch. Biochem. Biophys. 159, 292-296. 39. CARDINAS, J. M., AND DYSON, R. D. (1973) J. Biol. Chem. 248,6938-6944. 40. CARDINAS, J. M., DYSON, R. D., AND STRAND HOLM, J. J. (1973) J. Biol. Chem. 248, 69316937. 41. COSTA, L. JIM~~NEZ DE A&A, L., ROZENGURT, E., BADE, E. G., AND CARMINATTI, H. (1972) Biochem. Biophys. Acta 239, 128-136. 42. HOMMES, F. A., AND WILMINK, C. W. (1968) Biol. Neonate 13, 181-193. 43. TAYLOR, C. B., MORRIS, H. P., AND WEBER, G. (1969) Life Sci. (oxfird) 8, 635-644. 44. BALINSKY, D., CAYANIS, E., AND BERSOHN, I. (1973) Biochemistry 12, 863-870. 45. WEINHOUSE, S. (1973) Fed. PFOC. Amer. Sot. Exp. Biol. 32, 2162-2167. 46. OLIVER, I. T., BLUMER, W. F. C., AND WITHAM, I. J. (1963) Comp. Biochem. Physiol. 10, 3338. 47. PARAT, M. (1923) Bull. Biol. Fr. Belg. 57, 364399. 48. FAULKNER, A., AND JONES, C. T. (1975) Fed. EUF. Biochem. Sot. Lett., 53, 167-169. 49. VAN BERKEL, J. C., KOSTER, J. F., AND HULSMANN, W. C. (1972) Biochem. Biophys. A& 276, 425-429. 50. KAYNE, F. J. (1973) in The Enzymes, 3rd ed., (Bayer, P. D.), Vol. VIII, p. 353-382, Academic Press, London. 51. NEWSHOLME, E. A., AND GEVERS, W. (1967) Vit. Horn. 25, l-87. 52. SAPAG-HAGAR, M., MARCO, R., AND SOLS, A. (1969) Fed. EUF. Biochem. Sot. Lett. 3, 68-71. 53. MCILWAIN, H., AND BACHELARD, H. S. (1971) in Biochemistry and the Control Nervous System, 4th ed., pp. 406-441. Churchill Livingstone, Edinburgh.
