Blomquist, Göran

Hartman, Annika

Johansson, Göte

Shanbhag, Vasant

Elsevier - Publisher Connector 

Three forms of enolase separated by counter-current distribution 

Volume 22, number 1 FEBS LETTERS April 1972 
THREE FORMS OF ENOLASE SEPARATED BY COUNTER-CURRENT DISTRIBUTION 
Vasant SHANBHAG, Goran BLOMQUIST, Gate JOHANSSON and Annika HARTMAN 
Department of Biochemistry, University of Umeii, 90187 Ume& Sweden 
Received 20 January 1972 
Revised version received 10 February 1972 
1. Introduction 
The existence of enzymes in multiple forms 
(isoenzymes) is well documented [ 1 ] . The enzyme 
enolase (EC 4.2.1 .ll) has been shown to be electro- 
phoretically heterogeneous [2]. At least 3 forms 
exist in yeast [3,4] and in muscle of several species 
of trout [5] . Some have proposed that this may be an 
artifact resulting from the preparation of the enzyme 
[2,61. 
The separation of the enzymes of the glycolytic 
system in bakers yeast has been studied by extraction 
as well as counter-current distribution using aqueous 
two-phase systems containing polyethylene glycol 
carrying ionisable groups and dextran [7] . In this 
communication we present results that demonstrate 
the presence of 3 forms of enolase in bakers yeast, 
2. Materials and methods 
Dextran used was “Dextran 500” (M.W. = 5 X IO’) 
obtained from Pharmacia, Uppsala, Sweden. Trimethyl- 
amino-polyethylene glycol, TMA-PEG, was prepared 
from polyethylene glycol “Carbowax 6000” 
(Mn = 6 X 1 03) obtained from Union Carbide, USA 
[7] This contains 0.10 meq TMA groups per g. 
Enolase, type III, from yeast, was obtained from 
Sigma Chemical Co., St. Louis, USA. The salts and 
other chemicals used were of analytical reagent grade. 
2.1. Assay for enolase 
The enolase activity was measured according to the 
method of Warburg and Christian [8] . 100 ~1 of the 
solution to be assay was mixed with 2 ml of the 
North-Holland Publishing Company - Amsterdam 
substrate solution having the following composition: 
2.5 X 10e3 M sodium DL-2-phosphoglycerate 
prepared according to Kiessling [9] ,5 X 1 O-’ M 
EDTA 4 X 10m3 M MgSO,, 0.02 M potassium phos- 
phate buffer, pH 6.8. The change in absorbance at 
240 nm and 25” was measured using 1 cm cells in a 
Unicam SP 800 spectrophotometer and recorded on a 
Servogor recorder. Activity is expressed as the initial 
change in Ai: per min. 
2.2, Preparation of yeast extract 
Fresh bakers yeast obtained from Jastbolaget 
(Sollentuna, Sweden) was mixed with crushed dry ice 
in 1: 1 ratio and the mixture was homogenized in a 
Turmix mixer. The homogenate was spread on a plastic 
plate to allow the evaporation of dry ice. The yeast 
homogenate was then centrifuged for 20 min at 
15,000 g. The turbid supernatant was collected and 
recentrifuged for 60 min at 160,000 g. The clear 
supernatant so obtained was dialysed for 5 hr at 10” 
against 14 or 20 mM K,HPO,-KH,PO, buffer 
solution of the required pH, which was 5.5 in the 
case of extraction experiments and 6.5 in the case of 
counter-current distribution. All the other steps were 
carried out at 3”. 
2.3. Preparation of enolase from bakers yeast 
The yeast extract obtained as under sect. 2.2 was 
subjected to fractionation steps according to Malm- 
Strom [2]. The precipitate obtained in the last 
ethanol precipitation step was dialysed against 14 
mM phosphate buffer at 3”. The dialysate after 
removal of precipitated material was used in counter- 
current distribution experiments. 
105 
Volume 22, number 1 FEBS LETTERS April 1972 
2.4. Preparation of two-phase system 3. Results and discussion 
The two-phase systems used in these experiments 
contained 6.6% (wt/wt) dextran 500,6.4% (wt/wt) 
TMA-PEG and 5 mM potassium phosphate buffer at 
the appropriate pH. Phase systems were prepared by 
mixing required quantities of stock solutions of the 
polymers and the dialysed yeast extract or enolase 
solution. Blank systems were prepared by adding an 
equivalent quantity of buffer solution in place of the 
yeast extract. 
2.5. Extraction with two-phase system 
The extraction of enolase as a function of pH was 
studied as follows: A phase system containing 25% 
(wt/wt) of dialysed yeast extract was prepared at 
pH = 5 ..5 and the pH of the system was raised in 
discrete steps by careful addition of 2 M NaOH under 
stirring. At each step the system was centrifuged to 
speed up the phase separation. Samples were with- 
drawn from the upper phase and the concentration of 
enolase measured in terms of its enzyme activity. The 
pH in each step was measured with aid of a glass 
electrode that was directly immersed in the upper 
phase. In calculating the per cent of enolase activity 
in the upper phase at each pH, corrections were made 
for the loss in enzyme activity due to withdrawal of 
samples from the upper phase for analysis. The total 
amount of enolase in the system was determined by 
taking an assay of the mixed system at the starting 
PH. The entire procedure required not more than 2 hr. 
The extraction profile for enolase as a function of 
pH is shown in fig. 1, along with results obtained with 
similar experiments on CO-hemoglobin. It is seen that 
the curve for enolase consists of 3 distinct steps, which 
are absent in the case of hemoglobin. In systems con- 
taining PEG carrying ionisable groups, the partition of 
a protein is essentially dependent on charge. There 
will be an extreme change in partition near the iso- 
electric point of the protein. Thus, the concentration 
of hemoglobin in the upper phase changes from 10% 
to 80% between pH 7-9. By analogy, for a protein 
which consists of multiple forms which differ in their 
isoelectric points, the extraction curve would consist 
of a number of steps each corresponding to one form. 
By this criteria one can suspect from the extraction 
profile for enolase that the enzyme consists of at least 
3 different forms. The amount of enzyme extracted 
in the 3 steps are in the proportion 3:3:4. 
% in upper phase 
2.6. Counter-current distribution 
The counter-current distribution was carried out at 
pH 6.5. The apparatus used was an automatic thin- 
layer counter-current distribution apparatus with 60 
cavities [lo] . The first cavity was filled with 1.4 ml 
of the phase system containing yeast extract or solu- 
tion of enolase and the rest of the cavities were filled 
with 0.7 ml of each of the 2 phases obtained from the 
blank system. The settling time was 5 min, and the 
shaking time was 30 sec. The temperature was 20”. 
After 60 transfers the content of each cavity was 
diluted with 1.4 ml water, collected and then assayed 
for enolase activity. The total time required for the 
counter-current distribution experiment and activity 
measurements was 10-I 1 hr. 
Fig. 1. Extraction profiles as a function of pH for enolase in 
yeast extract (w ) and CO-hemoglobin prepared from 
human blood ( -). The ordinate records the amount of 
protein in the upper phase as per cent of the total amount of 
that protein in the system. Enolase concentration is measured 
in terms of activity (see sect. 2.5), and the concentration of 
CO-hemoglobin in terms of absorbance at 500 nm. 
PH 
106 
Volume 22, number 1 FEBS LETTERS April 1972 
ENOLASE ACTIVITY in &?03hln 
10 
t 
Table 1 
Relative proportions of three forms of yeast enolase obtained 
in different preparations. 
-- 
10 - 
5- 
0 
I 
10 
t 
I n c 
‘t Ji 
0 _ L./’ I 1. _I I 
0 10 20 30 40 50 60 
TUBE NUMBER 
Fig. 2. Counter-current distribution of enolase in aqueous 
phase systems containing 6.6% (wt/wt) dextran “SOO”, 6.4% 
(wt/wt) TMA-PEG and 5 mM potassium phosphate buffer. 
a) Enolase in yeast extract; b) enolase prepared from yeast; 
c) enolase obtained from Sigma Chemicals Co., USA. See sect. 
2.6 for details. (---) Experimental curves; (- - - - -) theoretical 
curves corresponding to each peak. 
Method 
-- 
Per cent of component 
--__- -- 
1 II III 
Counter-current distribution 
of yeast extract. 
41 29 30 
Countercurrent distribution 
of enolase prepared by the 
method of Malmstriim [ 21. 
50 33 17 
Electrophoretic separation of 
yeast enolase. Data from [4]. 41 29 20 
Electrophoretic separation of 
yeast enolase. Data from [ 111. 56 20 24 
The results of the counter-current distribution of 
yeast extract at pH 6.5 are shown in fig. 2a. The 
enolase activity is distributed over 3 distinct peaks. 
This again indicates that enolase is not homogeneous, 
but consists of at least 3 forms which differ in their 
partition characteristics at the pH used in the experi- 
ment. In this case the lower the isoelectric point the 
higher will be the partition coefficient. Analysis of 
the 3 experimental peaks has been carried out and the 
best fitting theoretical curves corresponding to each 
of the peaks are also shown in fig. 2a. The counter- 
current distribution of enolase prepared in this 
laboratory from yeast, shown in fig. 2b, also yields 3 
peaks, while only a single peak is obtained with the 
commercial preparation, fig. 2c. This peak corresponds 
to peak I in fig. 2a and 2b. According to manufacturers 
this product is prepared essentially per Westhead and 
McLain [6] , whose method is designed to yield only 
a single fraction. 
The relative proportions of the 3 forms have been 
calculated from the area under the peaks. This is with 
the assumption that the concentration of enolase is 
proportional to its activity at each point of the counter- 
107 
Volume 22. number 1 FEBS LETTERS April 1972 
current distribution curve. The results are given in 
table 1. These agree with the results obtained in the 
extraction experiments, sect. 2.5. The comparatively 
longer time required for a counter-current distribution 
experiment does not therefore seem to influence the 
relative proportions of the 3 forms. The relative pro- 
portions of 3 forms of enolase obtained by gel electro- 
phoresis reported by Pfleiderer et al. [4] and Geidan 
and Zhagat [ 111 are included for comparison. Since 
the molecular weight of the 3 forms of enolase is the 
same [2,4], their separation by counter-current 
distribution or by gel electrophoresis will depend on 
the charge carried out by each form under the condi- 
tions of the experiment. Thus, the 3 forms correspond- 
ing to the peaks I, II and III in the counter-current 
distribution curves, fig. 2, will be in order of negative 
charge or decreasing isoelectric points. When separated 
by gel electrophoresis, the form with lowest isoelectric 
point is most likely to be the one nearest the anode. 
On this basis we assume that the 3 enolase forms 
referred to as I, II and III in this paper correspond, 
respectively, to the forms III, II and I of [4], and 3,2 
and 1 of [ 1 l] . The fractionation of biological sub- 
stances by multistage distribution between the phases 
of aqueous polymer systems is a very mild method 
[IO] . It involves neither precipitation nor adsorption 
at active surfaces, e.g. ion exchangers. It is most likely 
that the 3 forms detected with this method are 
orginally present in the cell. The 3 forms of enolase 
may be more or less sensitive towards treatment such 
as precipitating agents, pH or adsorbents. Further, 
their solubility under a given condition may not be 
the same, since the 3 forms differ in their isoelectric 
points [3]. These 2 factors may cause a change in the 
relative proportions of the forms in purified enolase as 
compared to the state in the cell. This may be avoided 
if the 3 forms of enolase are first isolated by counter- 
current distribution method used in this work. Each 
form can then be further purified by conventional 
methods and characterized. Such work is in progress. 
Acknowledgements 
We wish to thank Prof. Per-Ake Albertsson for 
encouragement. This work has been financed by a 
grant from the Swedish Board for Technical Develop- 
ment . 
References 
[l] A.L. Latner and A.W. Skillen, Isoenzymes in Biology and 
Medicine (Academic Press, London-New York, 1968). 
[2] B.G. Malmstriim, Arch. Biochem. Biophys. 70 (1957) 
58. 
[3] W.A. Susor, M. Kochman and W.J. Rutter, Science 165 
(1969) 1260. 
[4] G. Pfleiderer, A. Neufahrt-Kreiling, R.W. Kaplan and 
P. Fortnagel, Biochem. Z. 346 (1966) 269. 
[5] F. Wold and H. Tsuyuki, Science 146 (1964) 535. 
[6] E.W. Westhead and G. McLain, J. Biol. Chem. 239 (1964) 
2464. 
[7] G. Johansson, A. Hartman and PA. Albertsson, in 
preparation. 
[8] 0. Warburg and W. Christian, Biochem. Z. 210 (1942) 
384. 
(91 W. Kiessling, Ber. 68 (1935) 243. 
[lo] P.A. Albertson, Partition of Cell Particles and Macro- 
molecules, 2nd Ed. (Almqvist and Wiksell, Stockholm, 
Wiley, New York, 1971). 
[ll] M. Geidan and R. Zhagat, Biokhimiya 35 (1970) 1051. 
108 


file:///data/remote/core/dit/data/elsevier/pdf/2ef/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS8wMDE0NTc5MzcyODAyMzF4.pdf

Three forms of enolase separated by counter-current distribution

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector