Participation of hemocyte proteinase in dissociation of the fat body on pupation of Sarcophaga peregrina (flesh fly)

Insect Biochem. Vol. 20, No. 5, pp. 443-449, 1990 Printed in Great Britain.All rights reserved

0020-1790/90$3.00+ 0.00 Copyright © 1990PergamonPress plc

REGULATORY PROPERTIES OF 6-PHOSPHOFRUCTO-1-KINASE OF THE MID-GUT OF THE GRASSHOPPER, POEKILOCERUS BUFONIUS SAMIRM. KHOJAl, ALl A. AL-ROBAI2 and ZARRAGI. AL-FIFI2 *Department of Biochemistryand 2Departmentof Biology, Faculty of Science, King Abdulaziz University, P.O. Box 9028, Jeddah-21413, Saudi Arabia (Received 26 June 1989; revised and accepted 10 February 1990) Abstract--The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, gublet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofrueto-l-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-I displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P2 whereas the inhibition of the enzyme by high concentrations of ATP was relievedby fru-2,6-P2. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P2 and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effeetor. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK- 1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P2, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate. Key Word Index: PFK, regulatory properties, mid-gut, Poekilocerus bufonius

ATP, citrate or phosphocreatine; and activation by ADP, AMP, Pi, glucose-l,6-bisphosphate or fructose-2,6-bisphosphate (Uyeda, 1979; Hers and van Schaftingen, 1982; Khoja, 1986). Recently, McCune et al. (1989) reported that aurintricarboxylic acid was shown to be a very potent inhibitor of rabbit liver PFK-I, giving 50% inhibition at 0.2/~M. This inhibition by aurintricarboxylic acid was shown to be reversed by the addition of allosteric activators of PFK- 1. The present investigation examines the regulatory properties of PFK-1 obtained from the mid-gut of the grasshopper, Poekilocerus bufonius, to provide some information on the carbohydrate metabolism in this insect.

INTRODUCTION

The grasshopper, Poekilocerus bufonius, is found in many areas of Saudi Arabia. It constitutes one of the most distinctive species of insects that feeds on milkweeds, Calotropis procera, which contain cardiac glycosides (Euw et al., 1967; Duffy, 1980). A viscous secretion from a bilobed gland is discharged by disturbed adults which completely protects the insect from a wide variety of both invertebrate and vertebrate predators (Euw et al., 1967). In insects, it is widely accepted that the mid-gut is the principal site of digestion and absorption (Wigglesworth, 1972; Romoser, 1981). The ultrastructure of the mid-gut of P. bufonius has been investigated by A1-Robai et al. (1987) who identified three types of epithelial cells in the mid-gut, namely, normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myeline-like figures and the small basal cells with small and round nuclei, nidi. It is currently considered that 6-phosphofructo-1kinase (PFK-1: EC 2.7.1.11) plays an important role in the regulation of carbohydrate metabolism in mammalian, bacterial, yeast and plant systems. The salient features of the regulation of PFK-1 include a sigmoidal substrate saturation curve for fructose6-phosphate; inhibition by high concentrations of Address all correspondence and reprint requests to: Dr S. M. Khoja, P.O. Box 6781, Jeddah-21452, Saudi Arabia. Abbreviations used: PFK- I, 6-phosphofructo- 1-kinase (EC 2.7.1.11); fru-2,6-P2, fructose-2,6-bisphosphate. ,s 20/s--A

MATERIALS AND METHODS

Animals and chemicals Adult female grasshoppers, Poekilocerus bufonius, were collected from the Gizan area (southern region of Saudi Arabia) and stored at the Department of Biology, King Abdulaziz University, Jeddah, Saudi Arabia with free access to food (milkweeds). Biochemicals were purchased from Sigma Chemical Co. and were used without further purification. All chemicals were AnalR grade obtained from BDH. Electron microscopy The grasshoppers P. bufonius were dissected and the mid-gut was removed and fixed in 4*/o glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for 3 h, washed in the same buffer and then transferred to 1% OsO4. After 2 h of post-fixation at 4°C, the blocks were rinsed in 0.1 M cacodylate buffer, dehydrated in a graded series of acetone and embedded in epoxy resin. Thick sections (I #) were stained

443

444

SAMIR M. KHOJA et al. Table h Body weight, tissue weight and total activities of PFK-I of the grasshopper Poekilocerus bufonius. Values are given as mean _+SD with number of insects given in parentheses. For experimental details see Materials and Methods section Total activities of PFK-1 Tissue

Insect weight (g)

Tissue weight (mg)

Mid-gut Hind-gut Leg m u s c l e

5.54+0.50(10) 5.85_+0.52(8) 5.25+0.50(8)

59_+11(10) 2.80_+0.21(6) 0.14_+0.01(6) 65_+ 8(8) 2.40_+0.41(5) 0.11+0.01(5) 112_+12(8) 20.30+1.50(5) 0.71+0.05(5)

with toluidine blue, while thin sections were stained by lead citrate and uranyl acetate for 30 min each and viewed with a Joel 100 × TEM. Cellulose acetate electrophoresis This was carried out at 4°C on strips of Whatman Cellogel (14.0 x 5.7cm) in electrophoresis buffer (5raM Tris-phosphate, pH 8.0, 5 mM ammonium sulphate, 5 mM 2-mercaptoethanol, I mM EDTA, 0.1 mM ATP, 1 mM phenylmethanesulphonyl fluoride and 1 mM 6-amino-nhexanoic acid). Electrophoresis of PFK- 1 prepared from the mid-gut, hind-gut and leg muscle of the grasshopper (5-10/~1, 1-2 units/ml) was performed at 300 V (2 mA/strip) for 2 h, after which enzyme activity was detected by use of the agar-gel staining technique described by Kemp (1971). Preparation of extracts The mid-guts of 4-5 grasshoppers were quickly removed and immediately frozen in liquid N2. The frozen tissues were homogenized directly in a Potter-Elvehjem homogenizer with 3 vol (v/w) of extraction buffer consisting of 50 mM Tris-chloride, pH 8.0, 100 mM ammonium sulphate, 30 mM potassium fluoride, 1 mM 2-mercaptoethanol, 1 mM EDTA and as proteinase inhibitors, 1 mM phenylmethanesulphonyl fluoride and l mM 6-amino-n-hexanoic acid. The homogenate was then centrifuged at 75,000g for 30 min in a Sorvall RC5C HS20 centrifuge at 4°C. The pellets were discarded and the particle-free supernatant was stored in ice until use. Expression of results 6-Phosphofructo-l-kinase activity is expressed as the formation of 1/~mol of fructose-l,6-bisphosphate per min at 37°C. Data on the regulatory properties of mid-gut PFK- 1 are presented as the activity ratio (v/V) where v is the suboptimal activity at pH 7.0 in the presence of I mM ATP and a given concentration of fructose-6-phosphate under the conditions defined by Jamal and Kellett (1983) and originally by Hussey et al. (1977) and V is the maximal activity at pH 8.0, where the enzyme is not subject to allosteric regulation, determined according to Ling et al. (1965). Protein concentrations were measured by using the method of Lowry et al. (1951) with bovine serum albumin as a standard. RESULTS

Ultrastructure o f the mid-gut epithelial cells The mid-gut epithelial cells were of the columnar type. At the luminal portion of these, a striated border composed of numerous parallel cylindrical microvilli having uniform diameters and spacing was frequently shown (Fig. 1). The cytoplasm of the mid-gut epithelial cells showed two distinct regions (Figs 1 and 2). The apical portion of the cytoplasm, which is adjacent to the microvilli base, is composed of moderate electron amorphous materials with very few mitochondria. In contrast, the intermediate and basal regions of the epithelial cell cytoplasm had

Units/g wet weight

Units/mg protein

numerous mitochondria which appeared in different profiles: tubular, spherical, cylindrical and oval (Fig. 1). The cytoplasm contains numerous vacuoles of varying size and devoid of any content (Fig. 1). In addition to the above mentioned cells, there are a group of small basal cells, nidi, with round nuclei located at the haemocoel side of the mid-gut. The distribution o f PFK-1 Table 1 shows the insect weight, tissue weight and the total activities of PFK-1 prepared from the mid-gut, hind-gut and leg muscle of the grasshopper. The highest concentration of enzyme is obtained in the leg muscle, whereas the enzyme concentrations of the mid-gut and hind-gut are slightly similar to each other being lowest in the hind-gut. Under identical conditions electrophoresis on cellulose acetate of the mid-gut, hind-gut and leg muscle PFK-1 of the grasshopper revealed single bands. The enzyme of the mid-gut and hind-gut showed identical mobilities and were slightly more anodic than the enzyme of the leg muscle. PFK-1 regulatory properties The regulatory properties of PFK-1 were determined at pH 7.0 because assays of this enzyme at pH 7.8 or higher showed a hyperbolic curve with respect to fructose-6-phosphate (Srivastava and Hubscher, 1966), and hence the regulatory properties cannot be determined. In order to prevent proteolysis during the assays of PFK-1, since the gut of insects contains high levels of endogenous proteinases, all studies were performed in the presence of proteinase inhibitors as previously described. Figure 3 shows the fructose-6-phosphate saturation curve of the mid-gut PFK-1 obtained from the grasshopper in the presence of 1 mM-ATP. The enzyme displayed a sigmoidal velocity curve with a Vmax of 90% of the activity at pH 8.0. The effect of fructose-2,6-bisphosphate (fru-2,6-Pj on the saturation curve of mid-gut PFK-1 in the presence of 1 m M ATP is also shown in Fig. 3. The affinity for fructose-6-phosphate was increased by fru-2,6-P2 only at # M levels, and as a result the binding was less cooperative and the apparent Km value was shifted from 0.7 m M in the absence of fru-2,6-P2 to 0.2 m M at 1/~M fru-2,6-P2. The ATP inhibition curve at 0.7 m M fructose-6phosphate of the mid-gut PFK-1 is presented in Fig. 4. It was observed that the enzyme activity was markedly inhibited by high concentrations of ATP. This inhibition of PFK-1 was released in the presence of 1 p M fru-2,6-P2 (Fig. 4). This release of A T P inhibition is reflected in a decrease in the Km values for fructose-6-phosphate.

Fig. 1. A low magnification electron micrograph of a section through two adjacent epithelial cells of the mid-gut showing the regions of the cell. The apical region possesses microvilli (Mv), which project into the lumen. The intermediate and basal regions contain a numerous number of mitochondria (m) and vacuoles (v). Scale: 2/~m

445

Fig. 2. Electron micrograph through the apical region of two adjacent cells showing the uniform diameter and spacing of microvilli. The apical cytoplasm composed of moderate electron amorphous material with very few mitochondria (m). Scale: 1.25 #M

446

PFK-1 of

Poekilocerus bufonius

447

160

1.O

, 8o

.~.V O. 5 V

v

o

J.

Ii]l 2 3

I

0

1 [Fructose 6-phosphate

I [ Fru-2,6-Pz]

] (rnM)

2 (pM)

Fig. 3. Effect of fru-2,6-P2 on the affinity of mid-gut PFK-1 for fructose-6-phosphate. Assays were performed in the presence of I mM ATP at pH 7.0 without fru-2,6-P2 (Q) and with 1 #M fru-2,6-P2 (©).

Fig. 5. Variation of mid-gut PFK-1 activity with [fru-2,6P2]. Assays were performed at pH 7.0 in the presence of 0.7 mM fructose-6-phosphate and I mM ATP either without AMP (O) or with 10/~M AMP (O).

Effect of activators

at 1 mM ATP and 0.7 mM fructose-6-phosphate at pH 7.0.

The effect of activators on the grasshopper mid-gut PFK-1 were studied in the presence of 0.7 mM fructose-6-phosphate and 1 mM ATP at pH 7.0 because the apparent Km value was obtained at these concentrations and the regulatory properties could be followed. Figure 5 shows the dose-response curve to fru-2,6-P2. Under the conditions chosen for the present experiment, the K0.5 was obtained at 0.1 p M fru-2,6-P2 which clearly indicates that mid-gut PFK-1 is highly stimulated by fru-2,6-P2. The synergism between fru-2,6-P2 and A M P is also shown in Fig. 5 in the presence of 0.7mM fructose-6-phosphate, 1 mM ATP and 1 0 p M AMP. The effect of adenine nucleotides activators is illustrated in Fig. 6. PFK-1 was most activated with AMP, whereas c-AMP was observed to be the least effective activator of PFK-1. The extents of activation at 0.2 mM nucleotides were 72% for AMP, 60% for A D P and 49% for c-AMP. Therefore, the order of activation of mid-gut PFK-1 by these adenine nucleotides is A M P > A D P > c-AMP. It was observed that ammonium ion has no effect on mid-gut PFK-1

Effect of inhibitors It has been reported that PFK-1 purified from honey-bee flight muscle is inhibited by glucose1,6-bisphosphate and fructose- 1,6-bisphosphate (Wegener et al., 1986). In our investigation we studied the effect of glucose-l,6-bisphosphate on the activity of mid-gut PFK-1 of the grasshopper at 0.7 mM fructose-6-phosphate and 1 mM ATP, but the enzyme activity was not affected by this metabolite. The effect of citrate on PFK-1 was also examined in this study in the presence of 0.7 mM fructose-6-phosphate and 1 mM ATP but there was no effect of citrate (up to 3 mM) on enzyme activity even when the concentration of ATP was increased to 2 mM. DISCUSSION

The regulation of PFK-1 has been studied in different tissues of various species by several workers (Ramaiah, 1974; Newsholme et al., 1977; Uyeda,

"~

100

1.0

_o°

-•0.5

I

I

0

1 [ATP]

2 (raM}

I

I

3

4

Fig. 4. Effect o f fru-2,6-P 2 on the i n h i b i t i o n of m i d - g u t

PFK-1 by ATP. Assays were performed in the presence of 0.7 mM fructose-6-phosphate at pH 7.0 without fru-2,6-P2 (O) and with 1/~M fru-2,6-P2 (C)).

0

0

I 0.1 [ Nu¢l.eotldes'l (mM)

I 0.2

Fig. 6. Effect o f adenine nucleotides on the activity o f m i d - g u t P F K - 1 . A s s a y s were p e r f o r m e d in the presence o f

0.7 mM fructose-6-phosphate and 1 mM ATP at pH 7.0 (O) ADP, (A) AMP, (lq) c-AMP.