On the phosphoglucomutase activity in pea cotyledons

Plant Science Letters, 7 (1976)69--76

69

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

ON THE PHOSPHOGLUCOMUTASE ACTIVITY IN PEA COTYLEDONS

L. RIGGIO BEVILACQUA, R. MARTINUCCI and G. SERRATO VALENTI

Institute of Botany, University of Genopa, Corso Dogali 1-C, Genopa (Italy) (Received November 27th 1975) (Revision received and accepted March 30th, 1976)

SUMMARY

Phosphoglucomutase (PGM) activity increases in extracts of cotyledons of ungerminated pea seeds during incubation. The process occurs both at 4°C and at 20°C, is pH-dependent and impaired in an atmosphere of nitrogen. If extracts are dialyzed or carefully warmed, a decrease of PGM activity is observed during the subsequent incubation. This phenomenon is also present in extracts of cotyledons of germinating seeds. In extracts of cotyledons of seeds that have germinated for 3 days the increase reaches a maximum in 4 h at 4°C and is followed by a rapid decrease. When extracts of cotyledons from 7-day-old peas are incubated, there is no increase, but only a decrease in their PGM activity. Mixing experiments show that these results are not caused by different concentration of inhibitors or activators of the enzymatic reaction and suggest that the PGM protein may be activated by dialyzable and thermolabile substances acting together. This hypothesis must be considered in the interpretation of the cycloheximideinhibited increase of PGM activity in cotyledons of germinating peas.

INTRODUCTION

P G M activity is greatly enhanced during the germination of castor bean seeds [1]. The castor bean contains considerable reserves of oil;during germination the lipidsare converted to carbohydrates [2] and P G M is required for this conversion. In seeds containing large reserves of starch, P G M m a y play a role in its glycolytic utilization,when glucose~l-phosphate is formed by starch phosphorolysis. This m a y be the case of the pea seed, in which the rate of glycolysis [3] and the phosphorylase activity [4] increase during germination. In fact, we found that in the embryo axis and in the cotyledons of the pea, P G M activity increasesduring the firstdays of germination [5]. The process Abbreviations: PGM, phosphoglucomutase; Tris, tris-(hydroxymethyl)aminomethane.

70 is inhibited by protein synthesis inhibitors [5] and by separation of the axis from the cotyledons [6], but it is enhanced by indole-3-acetic acid, gibbereUic acid, and kinetin [7,8]. In the course of this work on PGM it was noticed that the activity is not stable. Not only a loss of activity, but sometimes also values higher than the initial ones were observed after storage of the extracts at 4°C. The present research investigates this phenomenon and discusses its relation to our previous results. MATERIAL AND METHODS

Material. Cotyledons of ungerminated and germinating seeds of pea (Pisum sativum L., cv. Alaska) were used. Sterilized seeds (HgCI2 2°/00, for 2 rain) were rinsed several times and soaked in deionized water for 24 h (first day of germination), then placed on moist vermiculite for the desired time, always in the dark and at 25°C. Preparation of extracts. Cotyledons separated from the embryonic axes were ground with a cold mortar and pestle, extracted with deionized water or with 0.1 M Tris--HCl buffer at pH 7.5, and then centrifuged at 4°C at 34 000 g for 10 rain. Supernatants were used for the determination of enzyme activity. Dialysis. When required, the supernatants were dialyzed against cold deionized water using the hollow fiber technique (a Bio-Fiber 50 beaker, with a nominal weight cut off of 5000). Incubation. Extracts were incubated in the refrigerator at 4°C, or at room temperature (20°C). When extracts in buffer were incubated, the pH value was that of the buffer (pH 7.5); if extracts were in water, their pH was 6.2--6.3 and was varied with solid NaI-ICOs. Mixing experiments. Aliquots of different extracts (see Table I) were mixed in the ratio 1:1. PGM was assayed immediately after mixing. Separation and re-addition of low molecular weight substances. A BioFiber 50 beaker was used to separate part (40% of the volume of the extract) of the dialyzable phase (A). The remaining extract was then dialyzed against deionized water for 20 rain (B). A and B and the mixture of the two, in the same ratio as in the initial extract, were incubated at 4°C. Determination ofPGM. PGM activity was determined by the method of Turner and Turner [9] at pH 7.5 in 0.05 M Tris--HCl buffer. P was determined according to the method described by Fiske and Subbarow [10]. In one experiment a 10-min preincubation with imidezole and Mg2÷, according to Robinson and Najjar was carried out [11]. RESULTS The activity of PGM in extracts of pea cotyledons in 0.1 M Tris--HC1 at pH 7.5 varies during incubation at 4°C. Extracts from cotyledons of un-

71

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0 ungerminated seeds [] 3 days of germination 7 days of germination

I 4

t,

I 8

I 16

I 2/,

I

I

8

I

I

24

16

hours of incubation

Fig.1. Changes in PGM activity of extracts of pea cotyledons during incubation (medium: 0.1 M Tris--HCl at pH 7.5; temp.: 4°C). l-a: absolute values, l-b: values expressed as percentage of initial activity.Each point represents the mean of 4 replicates. Standard deviation did not exceed + 4.8%.

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E

O pH 7.5 /3 p N 8.0

o

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hours of incubation

2~

~

~

1~

2;

hours o1 incubation

Fig.2. Influence of pH on PGM activity during incubation. (Cotyledons from ungerminated pea seeds. Extracts in water. Incubation at 4°C; pH varied with solid NaHCO 3 . ) Each point represents the mean of 4 replicates. Standard deviation did not exceed ± 3.8%. Fig,3. Effect of temperature on PGM activity during incubation. (Extract in 0.1 M Tris-HCl at pH 7.5 from cotyledons of ungerminated pea seeds.) Each point represents the mean of 4 replicates. Standard deviation did not exceed ± 2.4%.

72

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0 control

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0

control

V dmaiyzed e x t r a c t

2 rnin at 50°C

4

5O

2~ hours of , n c u b a t , o n

Fig.4. Effect of heating the extract on PGM activity during incubation. (Extract of ungerminated pea cotyledons in 0.1 M Tris--HCl at pH 7.5, incubated at 4°C.) Each point represents the mean of 4 replicates. Standard deviation did not exceed ± 3.2%. Fig.5. Effect of dialysis on PGM activity during incubation. (Extract of ungerminated pea cotyledons in deionized water dialyzed for 1 h using a Bio-Fiber 50 beaker. Incubation in 0.1 M Tris--HC! at pH 7.5 at 4°C.) Each point represents the mean of 4 replicates. Standard deviation did not exceed +- 2.6%.

150o~ E

h 100.

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Fig.6. Influence of an atmosphere of nitrogen on PGM activity during incubation. (Extract and incubation as in Fig. 5.) Each point represents the mean of 4 replicates. Standard deviation did not exceed ± 2.4%.

~moles of G 6-P f or m ed /m l/m in

Extract 1.09

.

Found

Calculated

A

.

.

1.83

B

.

2.56

C 3.58

D

2.92

2.84

A+B

3.65

3.60

A+C

4.67

4.76

A+D

6.14

6.24

C+D

Extracts in 0.1 M Tris--HCl at pH 7.5. PGM assayed immediately after mixing. Ungerminated pea seed cotyledons: not incubated (A) and incubated for 24 h at 4°C (B); cotyledons of 3 days' germination: not incubated (C) and incubated for 4 h at 4°C (D). Each value represents the mean of 3 replicates. Standard deviation did not exceed ± 3.1%.

PGM ACTIVITY OF MIXED EXTRACTS

TABLEI

74

germinated seeds (Fig. 1, a and b) show a continuous increase of PGM activity during 16 h incubation; a much lower (on a per cent basis, Fig. lb) increase, lasting only 4 h, is observed if the cotyledons are taken from seeds that have germinated for 3 days; no increase at all is detected after 7 days of germination. These increases are in all instances followed by a decrease. The increase is pH-dependent: not present at pH 6.2, it occurs from pH 6.5 to pH 7.5 (Fig. 2). In extracts of cotyledons of ungerminated seeds the increase also occurs at 20°C (Fig. 3). When extracts are warmed for 2 min at 50°C, PGM is only partially inactivated, but the increase during incubation disappears completely (Fig. 4). A similar result is obtained in extracts dialyzed for 1 h in the Bio-Fiber 50 beaker (Fig. 5). In an atmosphere of nitrogen the increase of PGM activity in extracts of ungerminated seeds is limited to the first 4 h of incubation (Fig. 6). Activities are found to be strictly additive when incubated extracts are mixed, before PGM determination, with extracts that have not been incubated; mixture of extracts from ungerminated and germinating seeds are also additive (Table I). When the low molecular weight substances of the extract are added to the same extract dialyzed for 20 min a positive

TABLE II EFFECT ON PGM ACTIVITY OF THE LOW MOLECULAR WEIGHT SUBSTANCES PRESENT IN THE EXTRACT A, extract of ungerminated pea seed cotyledons partially deprived of its low molecular weight substances; B, A plus separated low molecular weight substances. Each value represents the mean of 3 replicates. Standard deviation did not exceed ± 2.8%. Hours of incubation

A - ~moles of G 6-P formed/g protein/rain B - pmoles of G 6-P formed/g protein/min

0

16

24

47 47

53 69

56 69

TABLE III EFFECT OF PREINCUBATION IN IMIDAZOLE 4-10 -2 M AND Mg s+ 10 -s M AT pH 7.5 ON PGM ACTIVITY Extracts of ungerminated pea seed cotyledonm. Each value represents the mean of 4 replicates. Standard deviation did not exceed ± 2.6%. Preincubation (min)

pmoles of G 6-P formed/g protein/min

0

10

20

80

80

78

75

effect on the increase of PGM activity during incubation is evident (Table II). Preincubation with imidazole and Mg2÷ is without effect (Table III). DISCUSSION

Disappearance of one or more inhibitors, appearance of one or more activators of the enzymatic reaction or activation of the enzyme protein might be the cause of the increase of PGM activity occurring during storage of pea cotyledons extracts. The additive results of the mixing experiments (Table I) are not in favour of an effect due to activators or inhibitors. As the increase during storage is abolished either by heat (Fig. 4) or by dialysis (Fig. 5), one may infer that it requires both thermolabile and dialyzable substances. Moreover, the pH dependence (Fig. 2), the impairment in an atmosphere of nitrogen (Fig. 6), the effect of the readdition of separated low molecular weight substances to dialyzed extracts (Table II) suggest a modification of the enzyme protein determined by metabolic reactions carried out by thermolabile and dialyzable substances acting together. PGMs from various sources [12--16] have been isolated and intensively investigated. For rabbit muscle PGM the existence of a phosphoform and of a dephosphoform [17,18] is well known. According to a proposed phosphoenzyme mechanism, only the phosphoform is catalytically active [19]. Nucleotides inhibit [20], some amino acids [21] and extreme pHs [22] activate rabbit muscle PGM. Several genetically regulated isozymes have also been demonstrated [23--25]. Our results might be compatible with the hypothesis of a phosphorylation of a dephosphoform, that is accomplished, in the case of rabbit muscle PGM, by glucose-l,6-diphosphate [17]. Attempts to clarify this point have so far been unsuccessful. The observation that the ability of the extract to increase PGM activity during incubation is lost as germination progresses (Fig. 1), poses the problem of the relation of this phenomenon to the enhancement of PGM activity found in pea cotyledons in the first days of germination. In the light of the present results, it seems probable that the higher values of PGM activity found in the cotyledons of germinating pea seeds may arise from the activation of a preexisting inactive form. In fact, the intervention of activators or inhibitors of the enzymatic reaction may be excluded (Table I) and the effect, previously demonstrated [5] of protein synthesis inhibitors, usually interpreted as a de novo synthesis, is possibly only an indirect one. REFERENCES 1 M.P. Cornaggia, Giorn. Bot. Ital., 71 (1964) 503. 2 J.R. Murlin, J. Gen.Physiol., 17 (1933) 283. 3 S.P. Spragg and E.W. Yemm, J. Exp. Bot., 10 (1959) 409.

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