Hum. Genet. 47, 279--290 (1979) © by Springer-Verlag 1979

Characterisation of the Isoenzymes of Phosphoglucomutase (PGM) Determined by the First (PGMI) and Second (PGM2) Locus Observed by Isoelectric Focusing J. G, Sutton Home Office Central Research Establishment, Aldermaston, Reading, Berkshire RG7 4PN, UK

Summary. The existence of four alleles of phosphoglucomutase ( P G M 0 in human red cell lysates has previously been demonstrated by isoelectric focusing (Bark et al., 1976; Kfihnl et al., 1977; Sutton and Burgess, 1978). Experiments are now described in which the position of each of the first-locus (PGM~) and second-locus (PGM2) isoenzymes is defined, thus extending and confirming the original proposal made by Bark et al.

Introduction It was proposed by Bark et al. (1976) that the a and b isoenzymes observed by starch gel electrophoresis (Spencer et al., 1964) could be resolved further by isoelectric focusing into the isoenzymes a+, a n d / o r a - or b+, a n d / o r b - , respectively, which gave the phenotypes 1+, 1-1+, 1- and 2+, 2 - 2 + , and 2-. The frequency obtained for these new alleles in a number of population studies (Bark et al., 1976; Ktihnl et al., 1977; Sutton and Burgess, 1978) supported this hypothesis, which was further substantiated by the inheritance studies of these alleles in a number of family studies (Sutton and Burgess, 1978). As final confirmation of this hypothesis it is proposed to show that the a and b isoenzymes initially separated by starch gel electrophoresis, when isolated and run on an isoelectric focusing gel (pH 5.7), do indeed occupy the positions predicted on the basis of the proposals put forward by Bark et al. (1976). In addition, the position of the remaining PGM1 and PGM2 (e, f) locus isoenzymes will be investigated.

Materials and Methods

Collection of the Red Cell Lysates Fresh human red cell lysates were obtained by freezing and thawing according to the procedure described by Sutton and Burgess (1978). 0340-6717/79/0047/0279/$ 02.40

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The following PGM1 phenotypes were used in the work; PGM11 (1-1+, 1+) and PGM~2 ( 2 - 2 + and 2+). These phenotypes provided a source for all the new alleles determined by the first locus ( P G M 0 , and have the added advantage of giving starch gel electrophoretic patterns in which the various isoenzymes i.e., a, c, b, and d, attain the greatest separation. An example of the PGM~ 2 - phenotype was not available at any time during the work, whilst the only PGM~ 1donor gave blood infrequently. In practice it was found that polyacrylamide electrophoresis proved to be a better medium than starch for the initial separation, because its conductivity was very similar to that of the isoelectric focusing gel. The separation of the PGM~ and PGM2 isoenzymes by polyacrylamide gel electrophoresis was identical with that observed when the starch gel technique was used.

Preparation of the Polyacrylamide Gel A sandwich of two glass plates, each with the dimensions 20 cm × 15 cm × 2 mm thick was held together with a number of bulldog clips, but separated by a 1-mm rubber gasket. The gasket was cut at one point to allow the introduction of the acrylamide mixture. We dissolved 2.5 g Cyanogum 41 (BDH) and 3.5g sucrose (BDH) in 50 ml gel buffer (Spencer et al., 1964). To this we added 0.4 ml riboflavin (4 mg% in distilled water) and 75 tll pH 5 - 7 Ampholine (LKB product No. 1809-121). The solution was degassed in a 500-ml Buchner flask by means of an Edwards high vacuum pump (Speedivac Ed 75). Once all the air bubbles had been removed the solution was carefully introduced into the glass sandwich with a 50-ml pipette. The rubber gasket was then closed and the gel allowed to photopolymerise under ultraviolet light for 2 h. Ampholines are polyamino-polycarboxylic acids, which are normally used in the preparation of isoelectric focusing gels. In this instance they are used not for the purpose of gradient formation but to ensure rapid polymerisation by virtue of the low concentrations of tertiary amines they contain (Karlsson et al., 1973). Once polymerisation was complete the clips were removed and the sandwich of glass plates placed in a refrigerator at 4 ° C for 30 rain. The rubber gasket was then removed and the glass sandwich carefully prised apart with a metal spatula. Cooling to 4°C for 30 min aids the subsequent separation of the glass plates.

Preparation of the Gel Inserts The introduction of a protein mixture (red cell lysate) into a polyacrylamide gel was found to be most readily achieved with a starch gel insert. Slots some 6 cm long and 1-2 mm wide were cut in the acrylamide gel some 5 cm from the proposed anodic end of the gel. Into each slot was poured a cooled starch gel (Spencer et al., 1964), which was allowed to harden for 1 h before the lysate was introduced into the starch on cotton threads.

Running Conditions Electrophoresis was conducted at 4°C for 18 h at 110 V (10-12 mA), with the same electrophoretic apparatus as is normally used for a starch gel separation (Culliford, 1967; Spencer et al., 1964).

Isoenzyme Isolation When electrophoresis was complete, glass strips 20 cm long and 0.5 cm wide were laid on either side of the gel in such a position that the starch gel inserts extended approximately 1 cm beyond the edges of these strips. The overlay mixture (Culliford, 1967) was then poured along the outside edge of each strip so that the extremities of the isoenzyme bands could be developed. The gel was then incubated at 37° C in the dark and removed as soon as the isoenzyme pattern was just visible. The unstained area of the gel (centre) corresponding to each developed iso-

Characterisation of Isoenzymes of PGM

281

enzyme was carefully cut into 1-cm strips and removed with a microspatula. Each strip was then separately transferred to 1 ml 0.015% w/v solution of Ampholine (pH 5.7) in distilled water at 4° C for 10 min before being transferred to the surface of the electrofocusing gel.

Isoelectric Focusing The apparatus, preparation of the isoelectric focusing gels, and subsequent enzyme visualisation were as described by Sutton and Burgess (1978), except that a power pack (LKB 2103) with constant voltage, power, and current facilities was used.

Running Conditions The running conditions used in this work were essentially the same as those previously described (Sutton and Burgess, 1978), except that the gels were prefocused for 30min to ensure the formation of the pH gradient. The isolated isoenzymes (polyacrylamide strips) were carefully removed from the ampholine solutions with a microspatula and laid on the surface of the prefocused gel some 2 cm from the anode. Initially, the current was restricted to 3-4 mA (300-400 V) for 20 min before the red cell lysate controls were applied on Whatman 1M strips (1 cm x 2 mm). A low initial current was used when the isolated isoenzymes were applied to the gel, to prevent any possible denaturation that might be caused by overheating with a higher current. Once the control lysates had been applied, the current was increased to 6 mA and the voltage and power outputs set at 1300 V and 8 W, respectively. After 11/2h electrofocusing, the Whatman strips and the polyacrylamide gel sections were removed, by which time the haemoglobin from the control lysates had reached the cathode. Electrofocusing was continued for a further 31/2h before focusing was complete.

Results

Isoenzymes Determined by the First Locus (PGM1) Characterisation of the a and b Isoenzymes. The a isoenzyme initially separated by polyacrylamide gel electrophoresis was f o u n d to exist as one of three types designated as either a - a + , a - , or a+, c o r r e s p o n d i n g to the new p h e n o t y p e s 1 - 1 + , 1 - , a n d 1+ (Fig. 1). Similarly, the b isoenzyme could be one of three possible phenotypes, 2 - 2 + , 2+, or 2 - (Fig. 2). However, the position of the h o m o z y g o u s b - isoenzyme could n o t be illustrated, because a P G M I 2 - d o n o r was n o t available. Characterisation of the c and d Isoenzymes. Figure 3 shows the position occupied by the c isoenzyme from two P G M I 1 d o n o r s whose p h e n o t y p e s b y isoelectric focusing were PGM~ 1+ a n d PGM~ 1 - 1 + . No PGM1 1 - h o m o z y g o t e was available. The position of the d isoenzyme from a P G M I 2 ( 2 - 2 + ) d o n o r is shown in Figure 4; this does n o t include the 2+ or 2 - homozygotes. Isoenzymes Determined by the Second Locus (PGM2) Figures 3 a n d 4 show the presence of two other isoenzymes, one of which appears between the c+ a n d c - a n d the other between the d+ a n d d - isoenzymes.

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Characterisation of Isoenzymes of PGM

283

g

f

m

m

a

~

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2

1

j

~ 1-1.

a1-

1+

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Fig. 1. Characterisation of the a isoenzyme by isoelectric focusing (pH 5-7) from three different PGM11 lysates after their initial separation by polyacrylamide electrophoresis in a Tris, maleic acid, Mg2+, EDTA buffer system (pH 7.4)

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Fig. 2. Characterisation of the b isoenzyme by isoelectric focusing (pH 5-7) from two different PGMI 2 lysates after their initial separation by polyacrylamide electrophoresis in a Tris, maleic acid, Mg2+, EDTA buffer system (pH 7.4). A PGM12-2+ lysate was used as the control

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Characterisation of Isoenzymes of PGM

285 pH5

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pH 7

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I-I+

Controls

Fig.3. Characterisation of the c isoenzyme by isoelectric focusing from two different PGMI 1 lysates after their initial separation by polyacrylamide electrophoresis in a Tris, maleic acid, Mg 2÷, EDTA buffer system (pH 7.4). PGM11+ and PGM~ 1-1+ lysates were used as controls

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pH 7

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Fig. 4. Characterisation of the d isoenzyme by isoelectric focusing (pH 5-7) of a PGM~ 2 lysate after its initial separation by polyacrylamide electrophoresis in a Tris, maleic acid, Mg 2+, EDTA buffer system (pH 7.4). A PGM12-2+ lysate was used as the control

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Fig. 5

Characterisation of the PGM1 isoenzymes revealed no isoenzymes that focused in either of these positions. When the e isoenzyme from either a PGM1 1 or a PGM12 phenotype (polyacrylamide) was characterised by isoelectric focusing, however, it was found to focus between the c - and c+ isoenzymes of a PGM1 l - l + , but on the anodic side of the b+ isoenzyme (PGMI 2+ or 2-2+). The f isoenzyme obtained from either a PGM11 or a PGM~ 2 phenotype (polyacrylamide) focused between the d+ and d - isoenzymes of a P G M 1 2 - 2 + , and on the anodic side of the c+ isoenzyme (PGM~ l+ or l - l + ) . Attempts at characterising the g isoenzyme have not been successful. The position of the e and f isoenzymes isolated from a PGM~ 2+ phenotype are shown in Figure 5. The activity of these isoenzymes was found to be variable and they were frequently absent, especially from stored red cell lysates.

Discussion

These characterisation experiments have shown that the a and b isoenzymes and their associated c and d isoenzymes (starch or polyacrylamide) can be resolved into one or two components by isoelectric focusing, so giving ten PGM1 phenotypes (Bark et al., 1976) instead of the three originally proposed (Spencer et al., 1964).

Characterisation of Isoenzymes of P G M

287

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~

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Fig. 5. Characterisation of the e and f isoenzymes (PGM2) by isoelectric focusing (pH 5-7) from a PGM12 phenotype after its initial separation by polyacrylamide electrophoresis in a Tris, maleic acid, Mg 2+ , EDTA buffer system (pH 7.4). A PGM12+ lysate was used as a control

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Fig. 6. Proposed relationship between the first- and second-locus isoenzymes of phosphoglucomutase from h u m a n red cell lysate observed by isoelectric focusing (pH 5.7)

288

Fig. 8

J.G. Sutton

Characterisation of Isoenzymes of P G M

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Fig. 7. Isoelectric focusing patterns obtained from two PGM~ 1+ phenotypes (different donors) and one PGM~ 1 - 1 + phenotype showing the proposed sequence of the a - , a+ and c - and c+ isoenzymes in relation to the e and f isoenzymes determined by the PGM2 locus

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pH 7

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Fig.8. Isoelectric focusing patterns of the PGM~2+, P G M 1 2 - 2 + and P G M t 2 - phenotypes showing the proposed sequence of the b - , b+ and d - , d+ isoenzymes in relation to the e, f isoenzymes determined by the PGM2 locus

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Correlation of the PGMI and PGM2 Isoenzymes Comparison between the position of the various a and c isoenzymes observed by isoelectric focusing will show that they appear to move as isoenzyme pairs. The a+ isoenzyme was always paired with the c+ and not the c - isoenzyme. Similarly, the a - isoenzyme was always paired with the c - and not the c+ isoenzyme. An identical relationship was also observed between the b+, d + and b - and d isoenzymes. The proposed relationship between the PGM1 and PGM2 isoenzymes is shown diagramatically in Figure 6, whilst Figures 7 and 8 show the isoelectric focusing patterns of some of the phenotypes illustrating this relationship. Theoretically it should be possible to identify the different PGM~ phenotypes from the position occupied by the various c and d isoenzymes. In practice, however, it was found that the c - or c+ isoenzymes were often difficult to observe, since they focused so close to the larger e isoenzyme (PGM2) that they invariably only appeared as very faint bands. The d - and d+ isoenzymes were frequently accompanied by isoenzymes (possibly 'secondary'), which appeared predominantly between the d - and the e positions. Typing by means of the c - , c+ a n d / o r the d - , d+ isoenzymes is therefore difficult and not to be recommended, for only with the freshest of lysates were patterns free of secondary isoenzymes observed.

References Bark, J. E., Harris, M. J., Firth, M.: Typing of the common phosphoglucomutase variants using isoelectric focusing. A new interpretation of the phosphoglucomutase system. J. Forensic Sci. Soc. 16, 115--120 (1976) Culliford, B. J.: The determination of phosphoglucomutase (PGM) types in blood stains. J. Forensic Sci. Soc. 7, 131--133 (1967) Karlsson, C., Davies, H., Ohman, J., Anderson, U.-B.: Analytical thin layer gel electrofocusing in polyacrylamide gel. LKB Application Note 75, p6 (1973) Ktihnl, P., Schmidtmann, V., Spielmann, W.: Evidence for two additional alleles at the PGM~ locus (phosphoglucomutase--EC 2.7.5.1.). Humangenetik 35, 219--223 (1977) Spencer, N., Hopkinson, D. A., Harris, H.: Phosphoglucomutase polymorphism in man. Nature 742--745 (1964) Sutton, J. G., Burgess, R.: Genetic evidence for four common alleles at the phosphoglucomutase-1 locus (PGM1) detectable by isoelectric focusing. Vox. Sang. 34, 97--103 (1978) Received October 27, 1978

Characterisation of the isoenzymes of phosphoglucomutase (PGM) determined by the first (PGM1) and second (PGM2) locus observed by isoelectric focusing.

Hum. Genet. 47, 279--290 (1979) © by Springer-Verlag 1979 Characterisation of the Isoenzymes of Phosphoglucomutase (PGM) Determined by the First (PGM...
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