Biochem. J. (1987) 242, 667-671 (Printed in Great Britain)

Nature of the subunits of the 6-phosphofructo-1-kinaseisoenzymes from rat tissues

George A. DUNAWAY* and Thomas P. KASTENDepartment of Pharmacology, Southern Illinois University, School of Medicine, Springfield, IL 62708, U.S.A.

The nature of the PFK (6-phosphofructo- 1 -kinase) isoenzymes in many rat tissues was examined byimmunological and chromatographic techniques and by measurement of their subunit compositions. It wasrevealed that, except for diaphragm and skeletal muscle, these complex isoenzymic populations containeddifferent amounts of the three subunit types and were nearly tissue-specific. Apparently this tissue specificityis due to different concentrations of the tetramers, which in turn are controlled by the types and amounts

of each subunit that are available to associate randomly.

INTRODUCTION

It is well known that PFK (EC 2.7.1.11) is a keyregulatory enzyme which is exquisitely sensitive to thecellular microenvironment through complex responses toa variety of reversibly bound modulators. Further, PFKactivity as well as isoenzyme types are affected byalterations in nutritional, hormonal, developmental andpathological states. Tissue-specific changes in PFKactivity and the nature of the PFK isoenzyme poolcontribute significantly to the diversities of glycolytic andgluconeogenic rates which have been observed indifferenttissues (for review see Dunaway, 1983).

Three distinct subunit types have been indicated inmammalian tissues (Tsai & Kemp, 1973; Gonzailez et al.,1975; Kahn et al., 1979; Meienhofer et al., 1979; Voraet al., 1980; Foe & Kemp, 1984; Dunaway & Kasten,1985a,b, 1986). The nature of the PFK isoenzymes inmammalian tissues has been classically examined bykinetic, immunological, chromatographic and/or electro-phoretic techniques (for review see Dunaway, 1983).These types of studies clearly indicated the complexity ofthe PFK isoenzyme pools, but the subunit compositionof the native isoenzyme pools is potentially subject tomisinterpretation unless directly measured. More recentstudies have remedied this problem by employing theSDS/polyacrylamide-gel electrophoresis method ofLaemmli (1970) to evaluate subunit compositionsdirectly and consequently to appreciate better the natureof the native PFK isoenzyme pools (Foe & Kemp, 1984;Dunaway & Kasten, 1985a,b). We have employed thistechnique to study the PFK isoenzymes in liver, muscle,heart and brain from different rat strains; and, althoughstrain differences were not noted, it was found that ineach tissue the pool of PFK isoenzymes was composedof differing amounts of the three types of subunits(Dunaway & Kasten, 1986). In the studies reportedherein, we extend these studies to other rat tissues andsuggest a uniform explanation for the different propertiesand/or types of PFK isoenzymes which have beenreported in various rat tissues.

EXPERIMENTAL

Wistar rats were maintained as previously described(Dunaway & Kasten, 1986). After decapitation andexsanguination, each of the tissues or organs was rapidlyremoved and homogenized in 50 mM-Tris/phosphate,pH 8.0, containing 25 mM-NaF, 1 mM-ATP,10 mM-dithiothreitol, 0.1 mM-EDTA, 0.5 mM-phenyl-methanesulphonyl fluoride, 1 mM-p-aminobenzamide,leupeptin (250 ,g/1), 0.1 mM-tosyl-lysylchloromethanehydrochloride, bestatin (500,tg/l) phosphoramidon(1 mg/l), pepstatin (1 mg/l) and 0.1 mM-tosylphenyl-alanylchloromethane hydrochloride. PFK activity was

assayed as previously described (Kasten et al., 1983).The immunological techniques, immunoblotting pro-

cedures and SDS/polyacrylamide-gel electrophoresismethodologies were conducted as previously described(Dunaway & Weber, 1974; Thrasher et al., 1981;Dunaway & Kasten, 1986).

Determination of subunit contentsPFK-containing supernatant fluids which were pre-

pared from each tissue were directly chromatographedon Cibacron Blue F3GA gel as described by Kasten et al.(1983). Nearly complete recovery of activity wasobtained, and the partially purified isoenzymes hadspecific activities of approx. 50 units/mg of protein. Thepartially purified isoenzymes (0.2 unit/gel track) weresubjected to electrophoresis on 60%-acrylamide slab gelsand silver-stained as described by Dunaway & Kasten(1985a,b). The PFK subunits for each tissue weretentatively identified by their electrophoretic mobilities,with brain PFK subunits as standards, and each subunitwas quantified by assuming equal intensity of absorption.Also, subunit identity was confirmed by immunoblottingwith subunit-specific antibodies. The amount of subunitprotein in each band was determined with a Zeinehsoft-laser scanning densitometer (SL-504-XL; BiomedInstruments, Fullerton, CA, U.S.A.), which was inter-faced with an Apple Ile computer. Data were analysedwith Zeineh electrophoresis software (Videophoresis I).

Abbreviations used: PFK, 6-phosphofructo-1-kinase; QAE, quaternary aminoethyl.* To whom reprint requests should be addressed.

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Table 1. Characterizatdon of PFK isoenzymes in different tssue by immunotitration with subunit-pecific IgG and by stepwise elutionfrom QAE-Sephadex

Total PFK activity in supernatant fluid was assayed as described by Dunaway & Kasten (1986). The supernatant fluids for eachtissue were incubated with graded amounts of subunit-specific IgG for 2 h at 37°C and then centrifuged. The amount of specificIgG and the maximum percentage titration of total activity were determined and recorded below. Also, each supernatant fluidwas loaded on a QAE-Sephadex column, which was then washed sequentially with 100 ml of buffer containing 150 mM-NaCland then 100 ml of buffer containing 300 mM-NaCl. This method completely resolves mixtures of the homotetramer of the M(150 mM-NaCI) and L subunits (Dunaway & Kasten, 1985a,b).

Percentage of PFKLoss of PFK activity activity eluted from

(%O) with: QAE-Sephadex by:Total PFK

activity Anti-L Anti-M Anti-C 150 mM- 300 mm-Tissue (units/g) IgG IgG IgG NaCl NaCI

Skeletal muscleDiaphragmBrainHeartThyroidIntestinal muscleTestisIntestinal mucosaSpleenKidneyLungLiverAdipocyte

80.280.725.623.59.79.210.86.36.35.55.24.00.5

00

2010625075806752688850

999976953840241650572070

00

9018779192948374776

28

10010060952819459

21231050

00

405

728196959179779050

Standard curves for the L and M subunits weredeveloped by plotting the densitometric areas versus theamounts of L4 and M4. The linear range was found to be50-600 ng. The subunit amounts were converted intopmol/g by assuming M, values of the L, M and Csubunits to be 80000, 85000 and 87500.

RESULTS AND DISCUSSIONThe PFK isoenzymes in many different types of organs

and tissues were examined by immunotitration withsubunit-specific antibodies and by column chromato-graphy on QAE-Sephadex with step-gradient elution. Inthe 150 mM-NaCl wash, M4 and M-rich hybrids wereeluted, whereas the more anionic hybrids were eluted by300 mM-NaCl (Dunaway & Kasten, 1985a,b). Therationale for using these methods has been previouslyjustified (Dunaway & Kasten, 1986). The resultsreported for muscle, liver, heart and brain essentiallyconfirm our earlier work (Dunaway & Kasten, 1986), butare included so that all tissues examined can be directlycompared.As shown in Table 1, PFK activities in skeletal muscle

and diaphragm were only affected by anti-M-subunitIgG and were exclusively eluted from QAE-Sephadex inthe 150 mM-NaCl wash. Collectively, these data implythat only M4 is present in these tissues. In contrast, thePFK activity in the other tissues or organs was titratedto various degrees by each type of subunit-specific IgGand exhibited a variety of elution patterns fromQAE-Sephadex. These data provide insight into thenature of the native isoenzymes in each tissues andsuggest that the brain isoenzymes were primarily M-Chybrids, that the heart isoenzymes were largely the M-richforms, that the isoenzymes in testis, intestinal mucosa

and spleen were primarily L-C hybrids, that thetetramers containing the L-type subunit were the majorforms in liver, and that in thyroid, intestinal muscle,kidney, lung and adipocytes extensive and complexhybridization of the three types of subunits hadoccurred. Clearly, the isoenzyme pools in most tissues,except for skeletal muscle and diaphragm, exhibitedcomplex, diverse and nearly characteristic patterns.With partially purified isoenzymes, the subunits in each

tissue were resolved by SDS/polyacrylamide-gel electro-phoresis (Fig. 1) and identified both by comparison withthe brain standard and from immunoblots of gels whichwere identical with those shown in Fig. 1. These dataconfirmed that only the M-type subunit and, conse-quently, M4 was present in skeletal muscle anddiaphragm. However, the PFK isoenzymes in othertissues and organs exhibited various contents of two orthree subunits. If proteolytic antagonists were used, theapparent Mr values ofeach type of subunit in each tissue,except intestinal mucosa, were very similar, 80000+ 500,85000+500 and 87500+600 respectively for the L, Mand C subunits. With respect to the standard curve, thelinear regression coefficients for each subunit Mr were noless than 0.995. Further, for all tissues, except intestinalmucosa, no proteins other than those which migratedidentically with the standard subunits reacted detectably,as measured by immunoblotting with correspondingsubunit-specific antibodies. Characterization of thesubunits from intestinal mucosa by immunoblotting (seeFig. 2) revealed that the two subunits, of Mr 82000 and86000, reacted exclusively with the anti-(C-type subunit)antibodies. As shown by scanning of the silver-stainedgel, the two C-type subunits produced 53% of the totalabsorption. Individually, the higher- and lower-Mr formswere 22% and 31 % of the total absorption respectively.

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Rat 6-phosphofructo-1-kinase isoenzymes

SkeletalBrain muscle Diaphragm Heart Thyroid

IntestinalBrain mucosa

C

M UIIONL

__~0am*

Spleen Kidney Lung

-.... wus

Liver Adipocyte

mt.:am*:-sam

Fig. 1. Resolution of subunits in each tissue

The 6% -acrylamide gels were developed by silver staining. For more details see the Experimental section.

Silver stain Anti-L Anti-M Anti-CMUC MUS BR MUC MUS BR MUC MUS BR MUC MUS BR

C

M

L;...|.;..11..

Fig. 2. Characterization of the subunits by immunoblotting

The silver-stained gels are shown on the left, and Anti-L, Anti-M and Anti-C indicate the type of antibody used to detect eachimmunoblotted subunit. MUC, MUS and BR indicate isoenzymes isolated from intestinal mucosa, intestinal muscle and brainrespectively.

Interestingly, nearly equal proportions were obtained byimmunoblotting. Assuming that maximum silver stainingwas obtained, this suggests that the higher-Mr formreacts better with the anti-(C subunit) antibodies. Also,another subunit, of Mr 76500, reacted specifically withthe L-type-subunit antibodies; and no protein from theintestinal-mucosa extract could be detected with anti-(M-type subunit) antibodies, although traces of M-typesubunit were suggested by immunotitration with anti-(M-type subunit) antibodies. As revealed by immuno-blotting, omission of proteolytic-enzyme inhibitorsduring purification of PFK isoenzymes can lead to theformation of a spectrum of immunoreactive proteinsexhibiting decreased Mr values (G. A. Dunaway &T. P. Kasten, unpublished work). However, the lowerMr values of the subunits found in the isoenzymes frommucosal cells of the small intestine cannot be so readilyexplained. That is, the proteolytic inhibitors that wereused blocked decrease in the Mr of PFK subunits fromall other tissues studied, including the intestinal musclefrom which the mucosal cells were isolated; and, in theintestinal-mucosal cells, only the lower-Mr forms of eachsubunit and none of the native subunits were detected. Itis possible that in this tissue the subunits with the lowerMr value could exist in vivo. However, it cannot beeliminated that a proteolytic enzyme in extracts ofintestinal-mucosal cells was not completely antagonizedby the inhibitors that were employed, and that thealtered subunits were exclusively formed. Although the

lower-Mr forms of the C-type subunit were previouslyunknown, this is not the first report of the detection ofaltered L-type subunits. It has been reported that the Mrof the L-type subunit from human leucocytes wasdecreased by 2000 compared with the same subunit fromhuman liver or red blood cells, which had an apparentMr of 74000-75000 (LaGrange et al., 1981). Also,comparable Mr losses have been obtained after limitedproteolysis in vitro ofPFK subunits from rabbit skeletalmuscle and yeast (Taucher et al., 1975; Kemp & Foe,1983).Each subunit was quantified from silver-stained gels,

and the results are shown in Table 2. Also, subunitpercentages were confirmed from immunoblots whichhad been developed with subunit-specific antibodies(Table 2). Since these percentages were not different fromthose from the silver-stained gels, the partially purifiedPFK isoenzyme preparations did not contain significantamounts of proteins which co-migrated with any of thePFK subunits. Consequently, the subunit contentsobtained from the silver-stained gels were validmeasurements.The subunits in each tissue vary with respect to the

types expressed and amount(s) present in the PFKisoenzyme pools. For example, the skeletal muscle anddiaphragm exhibited only the M-type subunit and thehighest concentrations of this subunit, or for that matterany type of subunit, in any of the other isoenzyme pools.Interestingly, the amount of the M-type subunit in

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Intestinalmuscle Testis

MO:''

669

:.... .::

G. A. Dunaway and T. P. Kasten

Table 2. Quantification of the PFK subunits in each tissue

The percentages of each subunit were determined from silver-stained polyacrylamide gels and from transblots which weredeveloped with subunit-specific antibodies. The silver-stained gels shown in Fig. 1 were scanned, and the percentages of eachsubunit in each tissue determined as described in the Experimental section. Polyacrylamide gels identical with those shown inFig. 1 were transblotted, and subunits were detected as described in the Experimental section. By scanning of the developedtransblots, the absorbance of each subunit, which was detected by the appropriate subunit-specific antibodies, was measured.The percentages of each subunit were calculated by dividing the absorbance for each subunit by the total absorbance andmultiplying by 100. ND indicates not detected. At least five separate preparations for each tissue were processed, and the resultsare expressed as means+ S.D.

L-type subunit M-type subunit C-type subunit

Silver-stained Silver-stained Silver-stainedgel Immuno- gel Immuno- gel Immuno-

blot blot blotTissue (pmol/g) (% ) (%) (pmol/g) (%) (%) (pmol/g) (%o) (%O)

Skeletal muscleDiaphragmBrainHeartThyroidIntestinal muscleTestisIntestinal mucosaSpleenKidneyLungLiverAdipocyte

NDND

158+16123+13185 + 20140+14231 +24141+14114+1297+8106+10143+149+2

NDND1311403247473837437538

NDND1610373350504040427837

3819+ 2003843 +200597 +60850+ 80134+12105+106+1ND45+479+ 867+ 740+412+2

10010049762924

lND1530272151

10010046793022NDND1027262253

NDND

463 +47145+15143+14193+18257 +26159+17141+1386+ 874+ 78+1.23 +0.2

NDND3813314452534733304

11

NDND381133455050503332010

isoenzymes from intestinal mucosa was very low toundetectable, which suggests that the M-type-subunitgene may not be expressed in these tissues. Of theisoenzyme populations in the various tissues where theM-type subunit was detected, the M-type subunitcontents varied over a very wide range, 12-3843 pmol/g.The isoenzvmes in liver contained the highest percentageof L-type subunit, but the actual amount of this subunitin the liver PFK isoenzymes was not remarkablydifferent from that in isoenzymes from other tissues,except for muscle and diaphragm, which had none. Whenpresent, the L-type subunit in the isoenzyme poolsexhibited a range of 9-231 pmol/g. Except the muscleand diaphragm, which expressed no C-type subunit, aconcentration range of 3-463 pmol/g for the C-typesubunit in the various isoenzyme pools was noted.Clearly, tissues and organs are capable ofproducing eachtype of subunit over a wide range, which conveysconsiderable variability to the types of isoenzyme poolsthat can exist. In order to understand better how thenative isoenzyme pools in each tissue are established, therole of the regulatory processes that control geneexpression, mRNA splicing and subunit concentrationmust be examined.On the basis of the data presented in Table 2, it is

possible to suggest a uniform explanation of earlier workwith PFK isoenzymes. For example, the minor hepaticPFK isoenzyme (PFK-L1) was only partially titratedwith the anti-(L-type subunit) antibody and had kineticproperties which were different from L4 and M4 enzymes(Dunaway & Weber, 1974). Since the work presentedherein indicates the presence of all three subunits inhepatic PFK isoenzymes, this 'isoenzyme' is probablyunresolved isoenzymes which are composed of L, M and

C subunits. Other investigators have studied PFKisoenzymes in a variety of rat tissues and have reporteddifferent physical, kinetic and regulatory properties. Forexample, Tejwani & Mousa (1981) have reported that ratlung PFK had different kinetic and regulatory propertiescompared with L4 and M4 enzymes. Rat thyroid PFKactivity has been reported to exhibit different kinetic andregulatory properties compared with M4 enzyme (Meldo-lesi & Laccetti, 1979). These observations are notsurprising, considering the existence of nearly equalamounts of all three subunits in the isoenzymes fromlung and thyroid. If the native PFK isoenzymes wereformed by random subunit interactions, then a highproportion of the complex hybrids containing all threesubunits would be expected to exist in these PFKisoenzyme pools. From testes of mature rats, two PFKisoenzymes have been reported which are distinct fromL4 and M4 (Lynn & Gomes, 1979). Since very lowamounts of M-type subunits are found in testes, theseisoenzymes were probably a mixture of the fivehomotetramers and hybrids containing the L and Csubunits. Also, it has been suggested that a unique PFKisoenzyme (S-type PFK) exists in rat spleen, which canbe kinetically, immunologically and electrophoreticallydiscriminated from L4 and M4 (Bittner et al., 1978; Levinet al., 1982). Since all three subunits are found in PFKisoenzymes from spleen, it is very likely that the S-typePFK is composed of homotetrameric and hybrid PFKisoenzymes. Some very interesting studies by Kellett andassociates suggested the presence of an unusual PFKisoenzyme in the mucosal cells of the rat small intestine(Khoja et al., 1983; Khoja & Kellett, 1983). This'isoenzyme', PFK-D in their terminology, was kinetic-ally, electrophoretically and chromatographically dis-

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Rat 6-phosphofructo-1 -kinase isoenzymes 671

tinct from the L4 and M4 isoenzymes. On the basis of ourdata, this is not surprising, since PFK-D is very likelyPFK isoenzymes which are composed of varyingamounts of the lower-Mr forms of the L- and C-typesubunits.

In rat tissues, we have demonstrated that total PFKactivity, immunological and chromatographic propertiesof the native isoenzymes, and the subunit compositionsof the PFK isoenzyme pools, are considerably varied.Except for skeletal muscle and diaphragm, the PFKisoenzyme pools in rat tissues are clearly not one or moreof the homotetramers of the three subunit types. On thecontrary, they are postulated to be complex mixtures ofdifferent amounts of homotetrameric and heterotetra-meric isoenzymes, which are formed randomly dependingon the types and amounts of each subunit that areexpressed.

We thank Tammy Kissel for typing this manuscript. Thiswork was supported by N.I.H. (grant HD 16666) and by theCentral Research Committee of Southern Illinois UniversitySchool of Medicine.

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Received 18 November 1986/2 January 1987; accepted 7 January 1987

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