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An isoelectrofocusing study of rabbit pyruvate kinases
Inr. J Biochrm.,
1976. Vol. 1. pp.
103 to 106. Prrgamon
Press.
Prmtrd
in Great
Brrtain
AN ISOELECTROFOCUSING STUDY PYRUVATE KINASES*
OF RABBIT
KENNETH H. IBSEN,? STEVENW. MARLES, TONY P. LOPEZ STEVEN E. WILSON AND JOANN R. BASAB~ Department
of Biological Chemistry, California College of Medicine, University of California, Irvine, CA 92717. U.S.A. (Rrcriord
15 January
1976)
Extracts of rabbit thigh muscle, liver, kidney cortex and spleen were subjected to isoelectrofocusing. In some cases isolated activity peaks were refocused. 2. Evidence for six variant forms of the K-isozyme were obtained. They had pI values of -5.4, -5.9, 7.0. 7.4. 8.3 and 8.8. Evidence was also obtained for the existence of two L-isozyme variants with pI values of 4.7 and 5.0. The M-isozyme had a pI value of 8.9. 3. These data were compared to isoelectrofocusing data obtained for other vertebrate species. The pleomorphism of the K and L-isozymes appears to be the rule.
Abstract-l.
INTRODUCI’ION
RESULTS AND DISCUSSION
The rabbit, like other mammalian organisms has three basic isozymes of pyruvate kinase (Whittell et
The first line of Table 1 summarizes the p1 values obtained for the various forms of pyruvate kinase found in extracts of rabbit organs. The variant forms isolated are grouped according to the isozyme they are suspected of conforming to. There are: six variants, suspected to be forms of the K-isozyme, having p1 values of _ 5.4, - 5.9,7.0, 7.4,8.3 and 8.8; two variants suspected to be forms of the L-isozyme, having p1 values of 4.7 and 5.0; and one M-isozyme variant with a p1 value 8.9. The rational for these assignments and a comparison with other studies, and other species, are discussed below.
ul., 1973; Osterman
& Fritz,
1973).
Although
it has
been suggested that these may result from epigenetic modification (Ibsen & Trippet, 1972), the body of available evidence suggests each basic isozyme is a homotetramer coded by a separate gene (for review see Ibsen et al., 19766). In this study the basic isozymes are called K, L and M. The K-isozyme is found as the major fetal enzyme as well as an enzyme of many adult tissues such as spleen and kidney. The L-isozyme is the major liver enzyme and the M-isozyme is the major skeletal muscle enzyme (Imamura & Tanaka, 1972; Whittel rt al., 1973; Osterman & Fritz, 1973 ; Ibsen & Krueger, 1973). The isoelectrofocusing properties of the three isozymes procured from extracts of rabbit skeletal muscle, liver, spleen and kidney cortex are described. Evidence was obtained for: six derived forms of the K-isozyme, two of the L-isozyme and one M-isozyme variant. These data were compared to results obtained from other species. METHODS AND
MATERIALS
Dutch-Belguin rabbits (Oryctolagus cuniculus) kindly donated by Dr. Roland Giolli, Department of Anatomy. California College of Medicine, were killed by injection of sodium pentobarbitol. Tissue preparations and electrofocusing was performed as described (Ibsen & Trippet, 1972). * Supported by National Institutes of Health Grant CA-07883. i-To whom to address correspondence.
Research
103
M-isozyme
Electrophoretic studies of rabbit skeletal muscle yields only M-isozyme (Whittell et al., 1973; Osterman & Fritz, 1973). Therefore, it may be concluded that the one peak of activity obtained by electrofocusing rabbit skeletal muscle extracts and having a p1 value of 8.9, is the M-isozyme. In a previous electrofocusing study of highly purified rabbit muscle enzyme Susor et al., (1973) obtained four bands of activity with p1 values of 8.6, 8.2, 7.2 and 7.0. However, since only one activity peak is found in both electrophoretic and electrofocusing studies of fresh rabbit muscle extracts, it seems probable the purified enzyme studied by Susor et al. was deamidated or otherwise altered. The p1 value of 8.9 is similar to that obtained for bovine and chicken M-isozyme but l-2pH units higher than reported for the rat, mouse or African frog enzymes (Table 1).
K. H.
104 Table
1. A comparison
IBSEN,
MARLES, T. P. LOPI-%, S. E. WILSON
S. W.
of the pI values obtained for the three basic as derived from various vertebrate
J. R. BASABI:
AND
pyruvate species
kinase
Isozymes
and
their
variants
SpCClC$ . 54
Rahhtt
. 54
74
i0
x3
xx
47
?.O
BO”l”C
x Yh
Th,s
KY
C4KlN
study 4AS Vl ‘il.
lY75
Punfied 7 .I Rat
il
‘4
llnpuhhshed.
64
hH
6,
65
6 0”
6 7”
74
7:
5 5‘
7x
54
1.7’
??
IIIS~N
55
‘.X
I \
(KI5S.
5 4’
61’ 60
Spleen
& TKIWI
11161s Cf ul.
MOUW
56
7 3’
66
6.9
76
7x
52
67
6.‘)
75
7x
5h
6 h
7.2
x2
X6
?7
PG C‘htcken
45
50
NALAML
i (1
h 1 (, 2
H,s. : ,, is
IllSI
u~tract
1972
I’)7511
KA rr ‘I/.
,972
1%‘) &
K~T~~AcH’
N “l ui..
YA\AGl H,s
I.
,,‘),I,
lY7Su
<‘I ‘il.
1971
& Krrra~c,,’
(1971,
8X
[IIS, N i’,
X8
(‘AKIN 4AS i'ld.
1Y?5
Purdicd Xanopu
laews
-47
. 5.0
-58
6.O
. 6.6
_ h.9
1’
-73
IllSipi
cr ui
(1Y6701
Usually major form(s) obtained from fresh extracts. h A set with values of 8.6, 8.2, 7.2 and 7.0 was obtained from a purified preparation (Susor et nl.. 1973). c Purified enzyme has been reported to have higher and variable pH values (Susor et al., 1973; Ibsen rt al., 19756).
a
d Presumed frbm liver pattern: “ Called a new hepatoma isozyme. ’ Gel isoelectrofocusing. 4 Obtained at various stage of purification. ” No L-isozyme demonstrated.
Two bands of activity corresponding to L- and K-isozyme were observed when rabbit liver extracts were electrophoresed (Whittell et al., 1973; Osterman & Fritz, 1973). Isoelectrofocusing four different liver extracts yielded two preparations which had the majority of activity at pH 4.7 and two at pH 5.0. In all four cases minor peaks were obtained at pH 7.4, 8.3 and 8.6. By analogy with previous electrofocusing studies performed on the L-isozyme from other species, it was considered probable that the two lower p1 forms represent a R, T conformation set of the L-isozyme (Table l), while as discussed below the higher p1 forms are K-isozyme variants. It has been shown for the rat enzymes that the lower pI form represents the R-form which may have bound Fru-1.6-P, while the higher pl form is the T-form (Hess & Kutzback. 1971; Ibsen & Trippet, 1972; Ibsen et ul., 1975h). Although only two L-isozyme variants were found from fresh extracts of liver from all species studied (Table 1). Hess & Kutzback (1971) obtained a third form as they purified the enzyme from pig liver. Higher p1 variants were also obtained from rat liver during purification (Ibsen et al., 19756). Presumably some tightly bound moiety is lost during purification; perhaps the enzymes are deacylated. Assuming, as supported below, the pH 7.4, 8.3 and 8.6 forms are all K-isozyme variants, the pH 4.7.-5.0, L-isozyme set represents 76.3 to 79.3:/, of the total activity. This compares to a value of 85.5593.8”i;; L-isozyme found when the isozymes were separated
by DEAE-cellulose (Osterman & Fritz, 1973). This difference may be related to the different methodologies used or to strain differences, since New Zealand white rabbits were used in the latter studies. In any case. the L-isozyme clearly accounts for the majority of activity. As summarized in Table 1, the p1 values of the L-isozyme are about 0.5-1.0 pH units lower than that of the mouse, rat or pig. Thus, rabbit L-isozyme has the lowest p1 value of all the species reported while rabbit M-isozyme has the highest p1 value. This suggests that the L- and M-isozymes from different species differ in a nonparallel manner.
Electrophoretic studies have been conducted on spleen extracts from several mammalian sources but apparently not from rabbit. In all species studied. only one activity peak, corresponding to K-isozyme. was obtained (Imamura & Tanaka, 1972; Whittell ef ul., 1973; Ibsen & Krueger, 1973). Isoelectrofocusing rabbit spleen extracts yielded 85595pd of the total activity as a single sharp and symmetrical peak with a p1 value of 7.47.5. The remaining activity was obtained as an ill defined smear of activity between pH 4.5 to 5.8. An almost identical electrofocusing pattern was obtained from bovine spleen extracts (Table 1) and except for the lower p1 value (6.8) from rat or mouse spleen extracts. Thus it may be concluded that the pH 7.4 activity band is a K-isozyme variant (Table 1). This confirms the suspicion that the pH
An isoelectrofocusing
study of rabbit pyruvate kinases
7.4 enzyme obtained in liver extracts is K-isozyme. Electrophoretic patterns obtained from rabbit kidney yielded five bands of activity (Whittell rt al., 1973; Osterman & Fritz, 1973). The most cathodal band corresponded to K-isozyme and the most anodal band corresponded to L-isozyme. Three distinct bands of activity could be discerned between the other two. Because of the relative positions of these bands of activity. it was assumed they were K-L hybrids. When rabbit kidney cortex extracts were electrofocused. five bands of activity were obtained. About ZOO/;of the total recovered activity was found to have a pl value 4.7-4.8; IS”<, a pI value of 5.9-6.0; 35:~ a pI value of 7.4; l@)” a pI value of 8.3; and 202, a pl value of 8.6. Equally complex patterns have been obtained from extracts of rat kidney cortex (Ibsen & Trippet. 1972). In these studies it was possible to show that the multiplicity of activity bands resulted from the fact that the K-isozyme existed in six different but intcrconvertible p1 forms as well as the fact that rat kidney cortex extracts contained both K- and L-isozymes. In order to see if a similar situation existed in rabbit kidney cortex extracts, a pH 7.4 peak (pH range 7.c7.6) previously isolated by electrofocusing was incubated for 20min at 32’C and then reelectrofocused. About IO?, of the total recovered activity had a pl value of 5.4; 2OU:, a pI value of 5.8; 6”” a pI value of 7.0; 13”Z, a p1 value of 7.4; 5% (a shoulder) a p1 value of 8.3 and 45”/ of p1 value of 8.8. Thus it may be concluded that the pH 5.4, 5.8. 7.0, 8.3 and 8.8 forms of the enzyme are derived from the pH 7.4 enzyme. Analogous results were obtained in studies conducted on K-isozyme from rat (Ibsen & Trippet. 1972; Ibsen c’t al., 1975h). mouse (Ibsen rt ul., 1975~) and chicken (Ibsen rt al., 19766). Since pH 4.8 enzyme was not generated from pH 7.4 enzyme and since pH 4.8 enzyme is the major liver enzyme, it may also be concluded that about 207; of the total activity in kidney cortex extracts represents L-isozyme. A similar distribution of K- and L-isozymes was obtained from rat kidney cortex (Ibsen & Trippet, 1972). These observations also confirm the concept that the pH 7.4, 8.3 and 8.8 enzymes found in liver extracts represent K-isozyme. Since these data suggest that there are no hybrid forms in rabbit kidney cortex extracts, they raise the question whether the extra bands formed during electrophoresis are indeed hybrids. That at least some are not hybrids is suggested by the following observations: the relative migratory rates of the activity bands are not symmetrically distributed (Whittell rt (II.. 1973; Osterman & Fritz, 1973); all the various p1 forms of the rat K-isozyme isolated by electrofocusing tend to be converted to the most cathodal. high p1 form when electrophoresed, but still yield distinguishable bands of activity (Ibsen & Krueger. 1973); the relative proportions of the activity bands obtained in the two published studies of rabbit kidney cortex enzyme appear to be vastly different (i.e. Whit-
105
tell cut u/.. 1973 vs Osterman & Fritz, 1973); and hybrids do not appear to be formed or broken under conditions encountered during extraction or electrofocusing (Ibsen et al.. 19766). Because of the demonstrated polymorphism of the pyruvate kinase isozymes it seems prudent to interpret the meaning of “extra” activity bands found on zymograms with caution. Table 1 compares p1 values obtained for K-isozyme from various sources by various investigators. In each case the assignment as K-isozyme is suggested by at least the tissue distribution. In many cases these data are further supported by other lines of evidence. The consistently repeating polymorphic pattern can be seen for all species and families studied. It can also be seen that there is again no obvious evolutionary trend with respect to the pI values.
REFERENCES
CAKD~NAS J. M.. BLACHLY E. G.. GCCOTTI P. L. & DYSON R. D. (1975) Properties of chicken skeletal muscle pvruvate kinase and a proposal of its evolutionary relationship to the other avian and mammalian isozymes. Biocher,~istr~ 14. 2247-2252. CRISS W. E. (1969) A new pyruvate kinase isolyme in hepatomas. Biockm. hiopl~ys. Res. Corm~un. 35. 901-905. HESS B. & KUTZBACH C. (1971) Identification of two types of liver pyruvate kinases. Hopp~-Srylrr’s Z. physiol. Chem. 352. 453-458. Iesr~ K. H.. BASABFJ. R. & Lorr:z T. P. (19750) Extraction of a factor from ehrlich ascites tumor cells that increases the activity of the fetal isozyme of pyruvate kinase in mouse liver. CLUICU RL’s. 35. 18&l 88. IBSEN K. H. & KRUFC;FK E. (1973) Distribution of pyruvatc kinase isozymes among rat organs. Archs Biochr~n. Biop/1y.s. 157. 509-5 13. IBSEN K. H.. MAKLES S.. Lo~>tz T. P. & BASAI~~ J. R. (1976~) Pyruvate kinase isozymes of Xe,lop~.s /arcis. the african clawed frog. To be published. lesr-x K. H.. M~KKAY L. & MAKL~S S. W. (1976/~) Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinase. Biochemistira 15, 1064-1073. IBSIX K. H. & TKIPP~T P. (1972) Interconvertible and noninterconvertible forms of rat pyruvate kinase. Biockr,,nistry 11. 4442-4450. IBSEN K. H.. TRIPPFT P. & BASAB~:J. (1975h) Properties of rat pyruvate kinase isozymes in isorvmesI Molrculur Strucrurc (Edited by MARKCKT C.. L.). Vol. I. pp. 543-559. Academic Press, NY. IMAMLIRAK. & TANAKA T. (1972) Multimolecular forms of pyruvatc kinase from rat and other mammalian tissues-1. Electrophoretic Studies. J. Bioch@rn. (Tokyo) 71. 1043~1051. NAKAMLIKAT.. Hosol K.. NISHIKAWA K. & HOKIO T. (1972) Effect of injection of chromatins from rhodamine sarcoma into rats on pl-isorymes of liver pyruvate kinase. Gunn 63, 239-250. OSTF.RMAN J. & FRITZ P. J. (1973) Pyruvate kinase isozymes: a comparative study in tissues of various mammalian species. Camp. Biochrru. Physiol. 44B. 1077- 1085.
106
K. H. IBSEN, S. W. MARLES, T. P. LOPEZ, S. E. WILSON AND J. R. BASABE
SUSOK W. A., KOCHMAN M. & RUTTER W. J. (1973) Structure determinations of FDP aldolase and the fine resolution of some glycolytic enzymes by isoelectric focusing. Ann. N.Y. Acad. Sci. 209, 328-344. WHITTELL N. M.. NC D. O., PRABHAKARARAO K. & HOLMES R. S. (1973) A comparative electrophoretic
analysis of mammalian pyruvate kinase isozymes. Cotnp, Biochrm. Phvsiol. 46B. 71-80. YANAGI S., K~MIVA T., IKEHARA Y. & ENDO H. (1971) Isozymes in the liver from mice given hepatocarcinogen and from tumor-bearing mice. Gum 62, 28%291.
1976. Vol. 1. pp.
103 to 106. Prrgamon
Press.
Prmtrd
in Great
Brrtain
AN ISOELECTROFOCUSING STUDY PYRUVATE KINASES*
OF RABBIT
KENNETH H. IBSEN,? STEVENW. MARLES, TONY P. LOPEZ STEVEN E. WILSON AND JOANN R. BASAB~ Department
of Biological Chemistry, California College of Medicine, University of California, Irvine, CA 92717. U.S.A. (Rrcriord
15 January
1976)
Extracts of rabbit thigh muscle, liver, kidney cortex and spleen were subjected to isoelectrofocusing. In some cases isolated activity peaks were refocused. 2. Evidence for six variant forms of the K-isozyme were obtained. They had pI values of -5.4, -5.9, 7.0. 7.4. 8.3 and 8.8. Evidence was also obtained for the existence of two L-isozyme variants with pI values of 4.7 and 5.0. The M-isozyme had a pI value of 8.9. 3. These data were compared to isoelectrofocusing data obtained for other vertebrate species. The pleomorphism of the K and L-isozymes appears to be the rule.
Abstract-l.
INTRODUCI’ION
RESULTS AND DISCUSSION
The rabbit, like other mammalian organisms has three basic isozymes of pyruvate kinase (Whittell et
The first line of Table 1 summarizes the p1 values obtained for the various forms of pyruvate kinase found in extracts of rabbit organs. The variant forms isolated are grouped according to the isozyme they are suspected of conforming to. There are: six variants, suspected to be forms of the K-isozyme, having p1 values of _ 5.4, - 5.9,7.0, 7.4,8.3 and 8.8; two variants suspected to be forms of the L-isozyme, having p1 values of 4.7 and 5.0; and one M-isozyme variant with a p1 value 8.9. The rational for these assignments and a comparison with other studies, and other species, are discussed below.
ul., 1973; Osterman
& Fritz,
1973).
Although
it has
been suggested that these may result from epigenetic modification (Ibsen & Trippet, 1972), the body of available evidence suggests each basic isozyme is a homotetramer coded by a separate gene (for review see Ibsen et al., 19766). In this study the basic isozymes are called K, L and M. The K-isozyme is found as the major fetal enzyme as well as an enzyme of many adult tissues such as spleen and kidney. The L-isozyme is the major liver enzyme and the M-isozyme is the major skeletal muscle enzyme (Imamura & Tanaka, 1972; Whittel rt al., 1973; Osterman & Fritz, 1973 ; Ibsen & Krueger, 1973). The isoelectrofocusing properties of the three isozymes procured from extracts of rabbit skeletal muscle, liver, spleen and kidney cortex are described. Evidence was obtained for: six derived forms of the K-isozyme, two of the L-isozyme and one M-isozyme variant. These data were compared to results obtained from other species. METHODS AND
MATERIALS
Dutch-Belguin rabbits (Oryctolagus cuniculus) kindly donated by Dr. Roland Giolli, Department of Anatomy. California College of Medicine, were killed by injection of sodium pentobarbitol. Tissue preparations and electrofocusing was performed as described (Ibsen & Trippet, 1972). * Supported by National Institutes of Health Grant CA-07883. i-To whom to address correspondence.
Research
103
M-isozyme
Electrophoretic studies of rabbit skeletal muscle yields only M-isozyme (Whittell et al., 1973; Osterman & Fritz, 1973). Therefore, it may be concluded that the one peak of activity obtained by electrofocusing rabbit skeletal muscle extracts and having a p1 value of 8.9, is the M-isozyme. In a previous electrofocusing study of highly purified rabbit muscle enzyme Susor et al., (1973) obtained four bands of activity with p1 values of 8.6, 8.2, 7.2 and 7.0. However, since only one activity peak is found in both electrophoretic and electrofocusing studies of fresh rabbit muscle extracts, it seems probable the purified enzyme studied by Susor et al. was deamidated or otherwise altered. The p1 value of 8.9 is similar to that obtained for bovine and chicken M-isozyme but l-2pH units higher than reported for the rat, mouse or African frog enzymes (Table 1).
K. H.
104 Table
1. A comparison
IBSEN,
MARLES, T. P. LOPI-%, S. E. WILSON
S. W.
of the pI values obtained for the three basic as derived from various vertebrate
J. R. BASABI:
AND
pyruvate species
kinase
Isozymes
and
their
variants
SpCClC$ . 54
Rahhtt
. 54
74
i0
x3
xx
47
?.O
BO”l”C
x Yh
Th,s
KY
C4KlN
study 4AS Vl ‘il.
lY75
Punfied 7 .I Rat
il
‘4
llnpuhhshed.
64
hH
6,
65
6 0”
6 7”
74
7:
5 5‘
7x
54
1.7’
??
IIIS~N
55
‘.X
I \
(KI5S.
5 4’
61’ 60
Spleen
& TKIWI
11161s Cf ul.
MOUW
56
7 3’
66
6.9
76
7x
52
67
6.‘)
75
7x
5h
6 h
7.2
x2
X6
?7
PG C‘htcken
45
50
NALAML
i (1
h 1 (, 2
H,s. : ,, is
IllSI
u~tract
1972
I’)7511
KA rr ‘I/.
,972
1%‘) &
K~T~~AcH’
N “l ui..
YA\AGl H,s
I.
,,‘),I,
lY7Su
<‘I ‘il.
1971
& Krrra~c,,’
(1971,
8X
[IIS, N i’,
X8
(‘AKIN 4AS i'ld.
1Y?5
Purdicd Xanopu
laews
-47
. 5.0
-58
6.O
. 6.6
_ h.9
1’
-73
IllSipi
cr ui
(1Y6701
Usually major form(s) obtained from fresh extracts. h A set with values of 8.6, 8.2, 7.2 and 7.0 was obtained from a purified preparation (Susor et nl.. 1973). c Purified enzyme has been reported to have higher and variable pH values (Susor et al., 1973; Ibsen rt al., 19756).
a
d Presumed frbm liver pattern: “ Called a new hepatoma isozyme. ’ Gel isoelectrofocusing. 4 Obtained at various stage of purification. ” No L-isozyme demonstrated.
Two bands of activity corresponding to L- and K-isozyme were observed when rabbit liver extracts were electrophoresed (Whittell et al., 1973; Osterman & Fritz, 1973). Isoelectrofocusing four different liver extracts yielded two preparations which had the majority of activity at pH 4.7 and two at pH 5.0. In all four cases minor peaks were obtained at pH 7.4, 8.3 and 8.6. By analogy with previous electrofocusing studies performed on the L-isozyme from other species, it was considered probable that the two lower p1 forms represent a R, T conformation set of the L-isozyme (Table l), while as discussed below the higher p1 forms are K-isozyme variants. It has been shown for the rat enzymes that the lower pI form represents the R-form which may have bound Fru-1.6-P, while the higher pl form is the T-form (Hess & Kutzback. 1971; Ibsen & Trippet, 1972; Ibsen et ul., 1975h). Although only two L-isozyme variants were found from fresh extracts of liver from all species studied (Table 1). Hess & Kutzback (1971) obtained a third form as they purified the enzyme from pig liver. Higher p1 variants were also obtained from rat liver during purification (Ibsen et al., 19756). Presumably some tightly bound moiety is lost during purification; perhaps the enzymes are deacylated. Assuming, as supported below, the pH 7.4, 8.3 and 8.6 forms are all K-isozyme variants, the pH 4.7.-5.0, L-isozyme set represents 76.3 to 79.3:/, of the total activity. This compares to a value of 85.5593.8”i;; L-isozyme found when the isozymes were separated
by DEAE-cellulose (Osterman & Fritz, 1973). This difference may be related to the different methodologies used or to strain differences, since New Zealand white rabbits were used in the latter studies. In any case. the L-isozyme clearly accounts for the majority of activity. As summarized in Table 1, the p1 values of the L-isozyme are about 0.5-1.0 pH units lower than that of the mouse, rat or pig. Thus, rabbit L-isozyme has the lowest p1 value of all the species reported while rabbit M-isozyme has the highest p1 value. This suggests that the L- and M-isozymes from different species differ in a nonparallel manner.
Electrophoretic studies have been conducted on spleen extracts from several mammalian sources but apparently not from rabbit. In all species studied. only one activity peak, corresponding to K-isozyme. was obtained (Imamura & Tanaka, 1972; Whittell ef ul., 1973; Ibsen & Krueger, 1973). Isoelectrofocusing rabbit spleen extracts yielded 85595pd of the total activity as a single sharp and symmetrical peak with a p1 value of 7.47.5. The remaining activity was obtained as an ill defined smear of activity between pH 4.5 to 5.8. An almost identical electrofocusing pattern was obtained from bovine spleen extracts (Table 1) and except for the lower p1 value (6.8) from rat or mouse spleen extracts. Thus it may be concluded that the pH 7.4 activity band is a K-isozyme variant (Table 1). This confirms the suspicion that the pH
An isoelectrofocusing
study of rabbit pyruvate kinases
7.4 enzyme obtained in liver extracts is K-isozyme. Electrophoretic patterns obtained from rabbit kidney yielded five bands of activity (Whittell rt al., 1973; Osterman & Fritz, 1973). The most cathodal band corresponded to K-isozyme and the most anodal band corresponded to L-isozyme. Three distinct bands of activity could be discerned between the other two. Because of the relative positions of these bands of activity. it was assumed they were K-L hybrids. When rabbit kidney cortex extracts were electrofocused. five bands of activity were obtained. About ZOO/;of the total recovered activity was found to have a pl value 4.7-4.8; IS”<, a pI value of 5.9-6.0; 35:~ a pI value of 7.4; l@)” a pI value of 8.3; and 202, a pl value of 8.6. Equally complex patterns have been obtained from extracts of rat kidney cortex (Ibsen & Trippet. 1972). In these studies it was possible to show that the multiplicity of activity bands resulted from the fact that the K-isozyme existed in six different but intcrconvertible p1 forms as well as the fact that rat kidney cortex extracts contained both K- and L-isozymes. In order to see if a similar situation existed in rabbit kidney cortex extracts, a pH 7.4 peak (pH range 7.c7.6) previously isolated by electrofocusing was incubated for 20min at 32’C and then reelectrofocused. About IO?, of the total recovered activity had a pl value of 5.4; 2OU:, a pI value of 5.8; 6”” a pI value of 7.0; 13”Z, a p1 value of 7.4; 5% (a shoulder) a p1 value of 8.3 and 45”/ of p1 value of 8.8. Thus it may be concluded that the pH 5.4, 5.8. 7.0, 8.3 and 8.8 forms of the enzyme are derived from the pH 7.4 enzyme. Analogous results were obtained in studies conducted on K-isozyme from rat (Ibsen & Trippet. 1972; Ibsen c’t al., 1975h). mouse (Ibsen rt ul., 1975~) and chicken (Ibsen rt al., 19766). Since pH 4.8 enzyme was not generated from pH 7.4 enzyme and since pH 4.8 enzyme is the major liver enzyme, it may also be concluded that about 207; of the total activity in kidney cortex extracts represents L-isozyme. A similar distribution of K- and L-isozymes was obtained from rat kidney cortex (Ibsen & Trippet, 1972). These observations also confirm the concept that the pH 7.4, 8.3 and 8.8 enzymes found in liver extracts represent K-isozyme. Since these data suggest that there are no hybrid forms in rabbit kidney cortex extracts, they raise the question whether the extra bands formed during electrophoresis are indeed hybrids. That at least some are not hybrids is suggested by the following observations: the relative migratory rates of the activity bands are not symmetrically distributed (Whittell rt (II.. 1973; Osterman & Fritz, 1973); all the various p1 forms of the rat K-isozyme isolated by electrofocusing tend to be converted to the most cathodal. high p1 form when electrophoresed, but still yield distinguishable bands of activity (Ibsen & Krueger. 1973); the relative proportions of the activity bands obtained in the two published studies of rabbit kidney cortex enzyme appear to be vastly different (i.e. Whit-
105
tell cut u/.. 1973 vs Osterman & Fritz, 1973); and hybrids do not appear to be formed or broken under conditions encountered during extraction or electrofocusing (Ibsen et al.. 19766). Because of the demonstrated polymorphism of the pyruvate kinase isozymes it seems prudent to interpret the meaning of “extra” activity bands found on zymograms with caution. Table 1 compares p1 values obtained for K-isozyme from various sources by various investigators. In each case the assignment as K-isozyme is suggested by at least the tissue distribution. In many cases these data are further supported by other lines of evidence. The consistently repeating polymorphic pattern can be seen for all species and families studied. It can also be seen that there is again no obvious evolutionary trend with respect to the pI values.
REFERENCES
CAKD~NAS J. M.. BLACHLY E. G.. GCCOTTI P. L. & DYSON R. D. (1975) Properties of chicken skeletal muscle pvruvate kinase and a proposal of its evolutionary relationship to the other avian and mammalian isozymes. Biocher,~istr~ 14. 2247-2252. CRISS W. E. (1969) A new pyruvate kinase isolyme in hepatomas. Biockm. hiopl~ys. Res. Corm~un. 35. 901-905. HESS B. & KUTZBACH C. (1971) Identification of two types of liver pyruvate kinases. Hopp~-Srylrr’s Z. physiol. Chem. 352. 453-458. Iesr~ K. H.. BASABFJ. R. & Lorr:z T. P. (19750) Extraction of a factor from ehrlich ascites tumor cells that increases the activity of the fetal isozyme of pyruvate kinase in mouse liver. CLUICU RL’s. 35. 18&l 88. IBSEN K. H. & KRUFC;FK E. (1973) Distribution of pyruvatc kinase isozymes among rat organs. Archs Biochr~n. Biop/1y.s. 157. 509-5 13. IBSEN K. H.. MAKLES S.. Lo~>tz T. P. & BASAI~~ J. R. (1976~) Pyruvate kinase isozymes of Xe,lop~.s /arcis. the african clawed frog. To be published. lesr-x K. H.. M~KKAY L. & MAKL~S S. W. (1976/~) Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinase. Biochemistira 15, 1064-1073. IBSIX K. H. & TKIPP~T P. (1972) Interconvertible and noninterconvertible forms of rat pyruvate kinase. Biockr,,nistry 11. 4442-4450. IBSEN K. H.. TRIPPFT P. & BASAB~:J. (1975h) Properties of rat pyruvate kinase isozymes in isorvmesI Molrculur Strucrurc (Edited by MARKCKT C.. L.). Vol. I. pp. 543-559. Academic Press, NY. IMAMLIRAK. & TANAKA T. (1972) Multimolecular forms of pyruvatc kinase from rat and other mammalian tissues-1. Electrophoretic Studies. J. Bioch@rn. (Tokyo) 71. 1043~1051. NAKAMLIKAT.. Hosol K.. NISHIKAWA K. & HOKIO T. (1972) Effect of injection of chromatins from rhodamine sarcoma into rats on pl-isorymes of liver pyruvate kinase. Gunn 63, 239-250. OSTF.RMAN J. & FRITZ P. J. (1973) Pyruvate kinase isozymes: a comparative study in tissues of various mammalian species. Camp. Biochrru. Physiol. 44B. 1077- 1085.
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SUSOK W. A., KOCHMAN M. & RUTTER W. J. (1973) Structure determinations of FDP aldolase and the fine resolution of some glycolytic enzymes by isoelectric focusing. Ann. N.Y. Acad. Sci. 209, 328-344. WHITTELL N. M.. NC D. O., PRABHAKARARAO K. & HOLMES R. S. (1973) A comparative electrophoretic
analysis of mammalian pyruvate kinase isozymes. Cotnp, Biochrm. Phvsiol. 46B. 71-80. YANAGI S., K~MIVA T., IKEHARA Y. & ENDO H. (1971) Isozymes in the liver from mice given hepatocarcinogen and from tumor-bearing mice. Gum 62, 28%291.