14 Biochimica et Biophysica Acta, 746 (1983) 14-17 Elsevier

BBA 31643

INSECT FAT BODY PHOSPHORYLASE KINASE IS Ca2+-INDEPENDENT AND ACTS EVEN AT 0°C

YOICHI HAYAKAWA and HARUO CHINO *

Biochemical Laboratory, Institute of Low Temperature Science, Hokkaido University, Sapporo 060 (Japan)

(Received February 4th, 1983)

Key words: Phosphorylase kinase; Ca 2 +; Cold activation," (Insect fat body)

Fat body glycogen phosphorylase in some overwintering insects is known to be activated by cold and, therefore, this enzyme acts as a key enzyme that regulates the production of glycerol or trehalose from glycogen during winter. In this paper we report the mechanism of phosphorylase activation by cold: the major phosphorylase kinase (EC 2.7.1.38) of fat body is bound to glycogen and functions at 0°C, whereas phosphorylase phosphatase does not; thus this may cause a slow but continuous accumulation of the active form of phosphorylase in the cold.

Introduction Materials and Methods

The conversion of glycogen into sugar alcohols such as sorbitol and/or glycerol [1,2] or trehalose [3,4] in overwintering insects is known in many species and, indeed, diapausing (dormant) insects can be categorized on the basis of their accumula- tion of sugar alcohols or trehalose during diapause [4]. It is accepted that such naturally produced compounds contribute to protection against cold injury. In certain insects (e.g., diapausing pupae of the cecropia [5-7] or cynthia [4] silkmoth) the production of glycerol or trehalose from glycogen depends on exposure to cold. Fat body glycogen phosphorylase in these insects is activated by cold and, therefore, this enzyme acts as a key enzyme [6-8]; however, the mechanism of phosphorylase activation by cold remains unresolved [9]. This study has been designed to resolve this problem, and we have reached a conclusion that interprets the above mechanism.

* To whom correspondence should be addressed. Abbreviation: EGTA, ethylene glycol bis(fl-aminoethyl ether)- N, N'-tetraacetic acid.

The partial purification of phosphorylase kinase from fat body of diapausing pupae of the silk- moth, Philosamia cynthia, that had been kept at 18-20°C was achieved by the following procedure. Dissected fat body was homogenized in buffer (50 mM Tris-HCl, 50 mM glycerol 3-phosphate, 5 mM dithiothreitol, pH 7.5, 5 ml/g tissue) and the homogenate was centrifuged at 10000 x g for 10 min at 4°C. The infranatant between the top fat layer and pellet was then centrifuged at 100 000 x g for 90 min at 4°C. The resulting glycogen-rich pellet contained approximately 60% of the total phosphorylase kinase activity. This contrasts with reports on mammalian liver kinase in which only 4% activity is recovered in the pellet obtained after 140 000 x g centrifugation [ 10]. The glycogen-rich pellet was used as the major enzyme source for further characterization, although the supernatant enzyme was used in some experiments. The pellet was resuspended in homogenization buffer and subjected to precipitation by ammonium sulphate (40% saturation). The precipitate, collected follow- ing centrifugation at 12000 x g for 20 min, was dissolved in buffer and dialysed against 1 mM

0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Tris-HC1 buffer, p H 7.5, for several hours. The dialysate was applied to a Sephadex G-50 column (1.2 × 11 cm), equilibrated with homogenizat ion buffer, and eluted with the same buffer. The active fraction was collected, and an aliquot was saved for obtaining an Arrhenius plot. The remaining fraction was subjected to further gel filtration on a Sepharose CL-4B column ( 1 . 5 x 14 cm) equi- librated with homogenizat ion buffer. The column was eluted with the same buffer and 1-ml fractions were collected in each tube. The enzyme eluted in a single peak with most of the activity collected in fractions 12 to 16; these were used for further characterization of enzyme.

R e s u l t s a n d D i s c u s s i o n

An Arrhenius plot for phosphorylase kinase bound to the glycogen-rich pellet is illustrated in Fig. l, together with that for the supernatant phos- phorylase kinase. The former preparat ion yields two straight lines ( E a = 76 and 32 k J / m o l ) with an inflection point at about 5°C, and a similar Arrhenius plot was also observed for the enzyme obtained f rom the Sepharose CL-4B column (data not shown). By contrast, the supernatant enzyme displays a single straight line ( E a = 77 k J / m o l ) similar to that reported for supernatant phos- phorylase kinase obtained after 120000 x g centrifugation of fat body homogenate f rom cecropia diapausing pupae [9]. The lower activa- tion energy observed for the glycogen-bound en- zyme at 0 - 5 ° C implies that-this enzyme functions at such low temperatures. The Arrhenius plot determined for glycogen-bound phosphorylase kinase at the physiological concentrat ion (13 / tM) of ATP reported for the fat body [11] demon- strates a single straight line without inflection point, but, instead, with lower activation energy (37 k J / m o l ) at 0 - 2 5 ° C (Fig. lc); suggesting little difference in its activity between the lower and higher temperatures, and thus this supports the supposi t ion that the enzyme retains its function at low temperature in natural conditions.

The effect of divalent cations on glycogen- b o u n d phosphorylase kinase was tested. Table I indicates that the enzyme requires Mg 2÷, as re- por ted for mammal ian phosphorylase kinase [12,13], but is virtually independent of Ca 2÷ and

c

m

15

-4.0

-5.0

-6.0

-7.0

( B )

Ea -- 76 kJ/mol

Ea:77 k J/too t ! ~ c ~ Ea :

(C) ~ ~ . 32 kJ/mol ~

Eo -- 37 kJ/mol ~\

- 5.0

- 6.0

- 7.0

- 8.0

i i i i i

3.3 3.4 3.5 3.6 3.7 1/T x 10 ~ (K -1)

Fig. 1. Arrhenius plots for fat body phosphorylase kinase prepared through a Sephadex G-50 column. Open circles and left ordinate, enzyme bound with glycogen-rich pellet; closed circles and right ordinate, supernatant enzyme after 100000 x g centrifugation. Phosphorylase kinase was measured by the con- version of phosphorylase b to a with a slight modification of the method of Ashida and Wyatt [9]. The standard reaction mixtures for (A) and (B) contained in 0.16 ml the following: 15.6 mM Tris-HC1 buffer (pH 7.5), 15.6 mM glycerol 3-phos- phate, 1.56 mM dithiothreitol, 0.5 mM ATP, 1.56 mM mag- nesium acetate, 40 #g rabbit muscle phosphorylase b (Boeh- ringer) and 50 #l phosphorylase kinase preparation. The reac- tion mixture for (C) contained 13 #M ATP instead of 0.5 mM ATP. The mixture, without enzyme, was previously equilibrated to the required temperature (0-25°C) for 5 min and then the reaction was started by adding the enzyme preparation. The reaction was terminated after 15 rain by addition of 2 vol. of a chilled solution containing 100 mM NaF, 10 mM EDTA, 5 mM dithiothreitol, 0.1% bovine serum albumin in 20 mM triethanolamine-HCl/NaOH buffer, pH 7.0. The mixture was assayed for phosphorylase a, and the kinase activity was calcu- lated from the phosphorylase a formed. Phosphorylase was assayed in the direction of glycogen breakdown by the method described previously [8]. Each point represents the mean of four determinations with + S.D. shown as the vertical bar.

Mn2+; the addit ion of E G T A or Ca 2÷ has almost no effect when Mg 2÷ is present. The enzyme pre- parat ion previously dialysed against 5 m M E G T A for 8 h demonstra ted no essential difference in its

16

TABLE I

EFFECT OF DIVALENT CATIONS ON G L Y C O G E N - B O U N D PHOSPHORYLASE KINASE A N D THE RATIO OF KINASE ACTIVITIES AT 0°C A N D 25°C

The reaction mixture was essentially the same as that described in Fig. 1 except for the metal ions. The experiment was done twice; both values are given. One unit of phosphorylase kinase is defined as that which catalysed the conversion of one unit of phosphorylase b to a to form l /~mol of glucose l-phosphate per min. The linearity of reaction persisted for 60 min at 0°C and 15 min at 25°C. Activities were determined for the control experiment and for 60-min incubation before calculating the activity-ratio.

Reaction Additions Activity Activity at 25°C ** ( U / m g protein)

Temperature Time Activity at 0°C (°C) (rain) Ratio of (a) to (b)

Insect fat body phosphorylase kinase

0 0 1.56 m M Mg(C2H302) 2 3.1, 5.0 (control) 0 60 12.5 # M CaCl 2 3.2, 4.9 0 60 1.56 m M Mg(C2H302) 2 3.2, 5.4 0 60 1.56 m M MnC12 + 15.6 m M EGTA 3.1, 5.4 0 60 1.56 mM'Mg(C2H302) 2 19, 20 (a) 0 60 1.56 mM Mg(C2H302) 2 -4- 15.6 mM EGTA 16, 19 0 60 1.56 mM Mg(C2H302) 2 + 12.5 ~M CaC12 14, 18 0 60 1.56 m M Mg(C2H302) 2 + 1.56 m M CaCl 2 15, 15

25 15 1.56 m M Mg(CEH302) 2 23, 27 (b) 25 15 1.56 m M Mg(C2H302) 2 + 15.6 m M EGTA 24,26

Rabbit muscle phosphorylase kinase 0 0 1.56 m M Mg(C2H302) 2 + 11.1 /~M CaCl 2 5.5, 5.9 (control) 0 60 1.56 m M Mg(C2H302) 2 + l l . l /~M CaCI 2 9.3, l0 (a)

25 15 1.56 m M Mg(C2H302) 2 + 11.1 /~M CaCl 2 40, 43 (b)

5.25

36.3

8O =

~' 6o

g

.~ 4O

i 2o f.

0

/ II Mg2+ /

l / ' Q Q •

I I I I

0 60 120 180 Reaction time (min)

Fig. 2. Effect of Mg 2 + on glycogen-bound phosphorylase kinase activity with or without EGTA. The reaction mixture (0.7 ml) was essentially similar to that given in Fig. 1, and the reactions were carried out at 0°C. At appropriate intervals, 0.1 ml reaction mixture was pipetted and the reaction was terminated by adding 0.2 ml chilled solution as described for Fig. 1. The final concentration of magnesium acetate added was 2 m M . Open circles, no EGTA; closed circles, 15.6 m M EGTA. Each point represents the average of two determinations.

Ca 2+ sensitivity from the non-dialysed enzyme. The essential requirement for Mg 2+ and the lack of dependence on Ca 2÷ are further demonstrated in Fig. 2, which shows that phosphorylase kinase activity is expressed in the presence or absence of EGTA only when Mg 2÷ is added to the incubation mixture. Table I also reveals that the activity ratio (5.25) of the enzyme at 0°C and 25°C is much lower than that (36.3) of mammalian muscle phos- phorylase kinase, thus supporting the suggestion derived from the Arrhenius plot that glycogen- bound phosphorylase kinase can function at 0°C. This proposal is also vindicated by the results of Fig. 2, which indicate that the reaction proceeds steadily at 0°C in the presence of Mg 2+.

Fig. 3 provides evidence that phosphorylase phosphatase is involved in the mechanism of phos- phorylase activation by cold. When dissected fat body is incubated at 0°C, the level of phosphory- lase a increases to about 70% within 90 min. If incubated at 25°C, a prompt activation to about 30% (shock activation) is followed by a gradual

100

v t

/ o i I I

0 50 100

Incubation time (min)

Fig. 3. Activation of phosphorylase of fat body after incubation of the dissected tissue at 0°C and 25°C with or without NaF. Fat body was dissected quickly from diapausing pupae, rinsed gently with Ringer solution (0.15 M NaC1, 0.02 M KC1, 20 mM Tris-HCl buffer, pH 7.0), and incubated in a small beaker with the same Ringer solution at 0°C or 25°C. At the time in- dicated, the tissue was homogenized with a buffer (50 mM Tris-HCl, pH 7.8) containing 5 mM dithiothreitol, 50 mM NaF and 10 mM EDTA, and centrifuged at 10000× g for 10 rain at 4°C. The infranatant between the top fat layer and pellet was collected and subjected to the phosphorylase assay. Open trian- gles, 0°C ( - NaF); closed triangles, 0°C (+ NaF); open circles, 25°C ( - NaF); dosed circles, 25°C ( + NaF).

decrease. These data conf i rm previous reports [6-8]; however, if N a F is added to the incuba t ion mixture at 25°C, a cont inuous increase of phos- phorylase a, similar to that observed at 0°C, is obtained. These observations suggest that phos-

phorylase phosphatase as well as phosphorylase kinase funct ions at higher temperature but, unl ike the kinase, the phosphatase displays virtually no activity at low temperature. Indeed, practically no

17

activity was detected at 0°C for the phosphatase

par t ia l ly purif ied on a DEAE-cellulose co lumn (data not shown). Thus, we conclude that the

part icular na ture of g lycogen-bound phosphory- lase kinase pr imari ly causes the activation of phos-

phorylase by cold in diapausing insects.

Acknowledgement

The authors wish to thank Dr. R.G.H. Downer, Univers i ty of Waterloo, Canada, for reading the manuscript .

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