Alkaline phosphomonoesterase activity in insects—I. Activity in various enzyme preparations from the blowflies, Phormia regina and Sarcophaga bullata

7. Ins. Physiol., 1963, Vol. 9, pp. 383 to 389. Pergamon Press Ltd. Printed in Great Britain

ALKALINE

PHOSPHQMONOESTERASE

INSECTS-I.

ACTIVITY

TIONS

FROM

THE

BLOWFLIES,

SARCOPHAGA ERNEST Department

of Entomology,

North

ACTIVITY

IN VARIOUS

PHORMliZ

IN

PREPARA-

REGINA

AND

BULLATA” HODGSON

Carolina

(Received

ENZYME

State College,

Raleigh,

North

Carolina,

U.S.A.

12 December 1962)

Abstract-A

relatively non-specific alkaline phosphomonoesterase activity with a pH optimum of approximately 9.3 has been demonstrated in enzyme preparations derived from larvae and adults of the blowflies, Phormia regim and Sarcophaga bullata. The nitrogen-containing phosphate esters, phosphorylcholine, phosphorylethanolamine, and phosphorylserine, as well as other phosphomonoesters, are hydrolysed. Enzyme assays show that the larvae of both species exhibit three to four times the activity of the adults. In both species a shift in the distribution of activity between mitochondria and the S,,,,,, fraction can be seen between the larval and adult stages. In the larvae the mitochondria are about twice as active as the S,,,,,, fraction while in the adults the S~O.OOOfraction is the more active (ca. four times in the case of P. regina and ca. six times in S. buZEata). Comparison of the relative rates of hydroIysis of different phosphate esters by different fractions from the same insect indicate that the enzyme or enzyme complex of the mitochondria is different from that of the S1O,Ooofraction.

INTRODUCTION

ALKALINE phosphomonoesterase activity has been demonstrated in insects both histochemically (BRADFIELD, 1946; DAY, 1949) and in enzyme preparations (NAKAMURA, 1940 ; DRILHON and BUSNEL, 1945 ; FITZGERALD,1949 ; DENUCB, 1952). Alkaline phosphomonoesterases have been widely studied in vertebrate tissue and have been purified from a variety of vertebrate sources (STADTMAN, 1960). Investigations of these enzymes in insects have been few, and little or nothing is known of their substrate specificity, distribution within different sub-cellular fractions, or their variation from one developmental stage to another. No purification of insect phosphomonoesterases has been attempted. BAMANNet al. (1960) have described the presence in rat liver cells of two alkaline phosphomonoesterases, one present in the cell nuclei and the other in both mitochondria and microsomes. * Contribution from the Entomology Department, North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval of the Director of Research, Paper No. 1515 of the Journal Series. 383

384

ERNESTHODGSON

The hydrolysis by insect enzymes of nitrogen-containing phosphate esters such as phosphorylcholine, phosphorylethanolamine, and phosphorylserine has not been investigated although presumptive evidence of their synthesis in insects is available from the work of WYATT and KALF (1956) and WYATT (1958). In mammalian tissues, STRICKLANDet al. (1956) demonstrated that although all of these compounds were hydrolysed by alkaline phosphomonoesterases, very little hydrolysis by acid phosphomonoesterases was observed. This is at variance with the findings of ROCHE and BOUCHILLOUX(1947) who demonstrated hydrolysis of phosphorylcholine and phosphorylethanolamine at pH 5.6 in a variety of mammalian tissues. SMITH et aE. (1955) demonstrated the hydrolysis of phosphorylethanolamine by the acid phosphatase of mammalian prostate while a specific phosphoserine phosphatase, with a pH optimum of 5.9, has been described in rat and chick liver (NEUHAUSand BYRNE, 1958). Classification of phosphatases into substrate specific categories can be misleading. SCHMIDT (1961) pointed out, that in the case of prostatic acid phosphatase, the Km values for certain substrates could be different without any significant difference in maximum velocity. The present communication arose from a consideration of the hydrolysis of phosphorylcholine and related compounds by enzyme preparations from the blowflies, Phormia regina (Meigen) and Sarcophaga bullata Parker. METHODS Larvae and adults of P. regina and S. bullata were obtained from cultures at North Carolina State College, maintained and reared by the method of MCGINNIS et al. (1956). These cultures were derived from those at the Science Research Institute, Oregon State College. The larvae were removed from the medium in the third instar before the prepupal period and used immediately. Adults were used within 24 hr of emergence from the puparium. The following enzyme preparations were used. Whole homogenates were prepared by homogenization in five volumes of 0.25 M sucrose using either a Waring blendor or a Servall homogenizer. The resultant brei was passed through several layers of cheese cloth to remove gross debris. Mitochondria were prepared by centrifugation at 10,000 g for 10 min after a preliminary centrifugation to remove debris, whole cells, etc. The pellet of mitochondria was washed three times by resuspending in 0.25 M sucrose and centrifuging at 10,OOOg. The fraction designated SiO,saO was the supernatant from the first centrifugation at 10,000 g. All operations were carried out at 0-5°C. The alkaline phosphomonoesterase assays were carried out in the following medium: 1.6 ml ethanolamine buffer, 0.2 M, pH 9.3 ; 1-O ml substrate, 20 plmole/ml; 0.2 ml MgCl,, 0.5 M; enzyme as indicated; water to final volume. Total volume was 4-O ml in most experiments although some preliminary experiments were carried out in a total volume of 2.7 ml. In all experiments a series of different enzyme concentrations were used and activities calculated from those samples showing a linear relationship between hydrolysis and enzyme concentration. Following incubation at 37°C for 30 min an equal volume of 10 per cent

ALKALINE

PHOSPHOMONOESTERASE ACTIVITY

IN INSECTS-I

385

Inorganic trichloracetic acid was added and the protein removed by centrifugation. phosphate determination by the method of FISKE and SUBBAROW(1925) was carried In certain experiments using p-nitrophenyl out on aliquots of the supernatant. phosphate the proteins were removed by acetone precipitation and the absorbance due to thep-nitrophenate ion read directly at 400 mp. Protein was determined by the modified Biuret method of WEICHSELBAUM(1946). Under these conditions the maximum hydrolysis observed was 21 per cent and no inhibition from phosphate released was apparent. Controls were run and all experiments corrected for endogenous phosphate and non-enzymatic hydrolysis of the substrate. RESULTS

AND DISCUSSION

Preliminary experiments indicated that enzymatic hydrolysis of phosphorylcholine and phosphorylethanolamine in whole homogenates of P. r-egina or S. These preliminary experiments bullata occurs only under alkaline conditions. showed also that the pH optimum is 9.3, that added magnesium ions are stimulatory and that glycine buffer is inhibitory. These results are essentially in agreement with the findings of STRICKLAND et al. (1956) in mammalian tissues. TABLE

~-ALKALINE

PHOSPHOMONOESTERASE

ACTIVITY

OF WHOLE

HOMOGENATES

TOWARD

SEVERAL SUBSTRATES Enzyme

source

Substrate instar larvae

2.9 x 1O-3

Phosphorylserine

2.4 x 1O-3

l-1 x 10-3

Phosphorylethanolamine

3.2 x 1O-3

1.3 x IO-3

13.4 x 10-s

10.1 x 10-s

8.7 x 10-Z

1.3 x 10-s

10.5 x 10-s

5.2 x 1O-3

Phenyl

phosphate phosphate

Phenolphthalein activity

S. bullata third

instar larvae Phosphorylcholine

p-Nitrophenyl

All

P. regina third

expressed

phosphate

in pmole/min

1.2 x 10-a

per mg protein.

The results in Table 1 show these preparations to have a relatively nonspecific hydrolytic ability toward monoesters of phosphoric acid. In addition to the substrates shown whole homogenates of P. regina will hydrolyse c+glycerophosphate, /3_glycerophosphate, glucose-l-phosphate, glucose-6-phosphate and fructose-1,6diphosphate. Ribose-l-phosphate is not hydrolysed. Two characteristics are noteworthy, that the aliphatic nitrogen-containing phosphate esters are hydrolysed at a significantly slower rate than the aromatic phosphate esters and that the addition of a p-nitro group to phenyl phosphate causes a significant drop in the rate of hydrolysis. It is apparent that glucose-l-phosphate could have been split by phosphorylase action although this possibility has not been investigated further. 26

ERNESTHODGSON

386

In both species the enzyme activity decreases markedly from larval to adult stage (Table 2). The gross metabolic differences between these two stages are obvious, the larva is a rapidly growing, relatively sluggish stage requiring a high protein diet whereas the adult is virtually non-growing, extremely active and requires a high carbohydrate diet. In comparisons of larval and adult P. regina MCGINNIS et al. (1956) have shown differences in carbohydrate metabolism whereas Bieber et al. (1961) d emonstrated differences in the phospholipid content. TABLE

Z-ALKALINE

PHOSPHOMONOESTERASE

regina AND Enzyme

P. P. S. S. Substrate protein.

ACTIVITY

Sarcophaga

source

OF WHOLE

Enzyme

reginu third instar larvae regina adults bdata third instar larvae bullata adults

used was phenyl

phosphate.

HOMOGENATES

OF Phormia

bullata

10.7 3.8 12.3 3.3

All activities

activity

x x x x

10-a 1O-3 lO-3 IO-3

expressed

in pmole/min

per mg

Since the adult fly thorax is filled with flight muscle, the possibility exists that the decrease in enzyme activity is due primarily to the increase in muscle mass. This was tested by assaying a homogenate of P. regina thoraces and comparing this to the activity shown by a homogenate of heads and abdomens from the same insect. The activity of the heads and abdomens toward phenyl phosphate is TABLE

%-ALKALINE

PHOSPHOMONOESTRRASE

Enzyme

Substrate protein.

source

ACTIVITY

Cell fraction

OF

CELL

Enzyme

FRACTIONS

activity

P. regina third instar larvae

Mitochondria S 10,000

40.0 x 1 o-3 24.2 x lO-3

P. regina adults

Mitochondria S 1cl,000

2.7 x lO-3 10.0 x IO-3

S. bullata third instar larvae

Mitochondria S 10,000

17.3 x IO-3 8.3 x lO-3

S. bullata adults

Mitochondria S 10,000

2-1 x IO-3 13.5 x IO-3

used

was phenyl

phosphate.

All activities

expressed

in pmole/min

per mg

19 x 10-3 pmole/min per mg protein whereas that of the thorax is about one-tenth of this amount. While this explanation is somewhat complicated by the fact that the activity of the thorax homogenate toward p-nitrophenyl phosphate is about

ALKALINE

PHOSPHOMONOESTEBASE

ACTIVITY IN INSECTS-I

387

pmole/min per mg protein, it is apparent that the increase in mass of a relatively inert tissue accounts in large part for the decrease in activity shown in the adult stage.

5

TABLE

~-RELATIVE

ALKALINE

Enzyme

PH~SPHOM~NOESTBRASE

ACTIVITY OF CELL FRACTIONS

Ratio of activities Mitochondria : S1,,ooo

source

P. yegina thirdinstarlarvae P. regina adults 5'.bullata third instar larvae S. bullata adults

1 :0.61 1 : 3.70 1 : 0.48 1 : 6.43

Table 3 shows the alkaline phosphomonoesterase activity of different cell fractions. The S,,,,,, activity is not separated into microsomal and soluble although studies in progress suggest that both of these fractions have alkaline phosphomonoesterase activity with somewhat different characteristics. The activity of these fractions is higher than that of whole homogenates indicating that the nuclei and TABLE

~--RELATIVBRATESOFHYDROLYSISOFDIFFERENTPHOSPHATEESTERSBYCELLFRACTIONS

Substrates Phenyl phosphate : phosphoryl choline

Phenyl phosphate : glucose-l-phosphate

Phenyl phosphate : p-nitrophenyl phosphate

Phenyl phosphate : p-nitrophenyl phosphate

Enzyme source P. regina larval mitochondria P. regina larval S 10,000 P. regina larval mitochondra P. regina larval s 10,000

S. ‘bullata larval mitochondria S. bullata larval S 10,000 S. bullata adult mitochondria S. bullata adult S 10,ooll

Relative rates of hydrolysis

1

: 0.35

1

: 0.26

1

: 0.23

1

: 0.14

1

: 0.12

1

: 0.25

1 : 0.10 1

: 0.45

cell debris represent relatively inactive protein. In the larvae of either species the activity of the mitochondria is higher than that of the SIO,,,, fraction while in the adults the reverse is clearly true. This is illustrated in Table 4 which shows the relative activity of cell fractions.

388

ERNESTHODGSON

In view of the differences in quantity and particularly of subcellular distribution the possibility exists that the enzyme or enzymes characteristic of mitochondria fraction are different. An attempt was made to test this hypothesis and the &ll,OclO by comparing the relative rates of hydrolysis by mitochondria, of different phosphate esters, with the same ratio obtained using the S,,,OOofrom the same preparation. The results of these experiments are shown in Table 5. With two different pairs of substrates the mitochondria and S,,,,, of P. w&a larvae show different characteristics. Similarly using phenyl phosphate andp-nitrophenyl phosphate the same fractions derived from either the larvae or adults of S. bullata appear to differ from one another. These data can also mean that different components of an enzyme complex are present in different amounts in the two fractions. This problem is particularly difficult to resolve as in this type of preparation phosphatases from all the organs are present. Clearly further characterization of these enzymes is necessary to resolve this and other problems. Such investigations are being initiated. The close similarity of all the findings in flies from two different families indicate that these phenomena may be common to a number of insects, at least in the Diptera. Acknowledgements-This research was supported in part by a grant from the National Institutes of Health, U.S. Public Health Service, No. E 4465. The author gratefully acknowledges the technical assistance of Miss SALLY F. LAWS and the assistance of Mr. EUGENETERRY in maintaining the cultures. REFERENCES BAMANNE., G&SWEIN C., and RIEHLJ. (1960) Auftrennung dei “alkalischen Phosphatase” aus Leber in zwei Enzyme mit der Lokalisation im Zellkern sowie in den Mikrosomen bzw. in den Mitochondrien. Charakterisierrung der beiden Enzyme in einigen ihrer Haupteigenschaften. Naturwiss. 8,42. BIEBERL. L., HODGSONE., CHELDELINV. H., BROOKSV. J., and NEWBURGHR. W. (1961) Phospholipid patterns in the blowfly, Phormia regina (Meigen). J. biol. Chem. 236, 2590-2595. BRADFIELD J. R. G. (1946) Alkaline phosphatase in invertebrate sites of protein secretion. Nature, Lond. 157, 876-877. DAY M. F. (1949) The distribution of alkaline phosphatase in insects. Aust.J. Sci. Res. (B) 2, 31-41. DENUCBJ. M. (1952) Recherches sur le systeme phosphatique des glandes serigenes chez. le ver a soie (Bombyx mori L.). Experientia 8, 64-65. DRILHONA. and BUSNELR. G. (1945) Recherches sur les phosphatases d’insectes. Bull. sot. chim. Biol. 27, 415--118. FISKEC. H. and SUBBAROW Y. (1925) Th e calorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. FITZGERALDL. R. (1949) The alkaline phosphatase of the developing grasshopper egg. J.exp.Zool. 110,461-487. MCGINNIS A. J., CHELDELINV. H., and NEWBURGHR. W. (1956) Enzyme studies of various stages of the blowfly, Phormia retina (Meig.). Arch. biochem. biophys. 63, 427-436. MCGINNIS A. J., NEW~URGHR. W., and CHELDELIN V. H. (1956) Nutritional studies on the blowfly, Phomzia regina (Meig.). J. Nutrition 58, 309-323.

ALKALINE PHOSPHOMONOESTERASEACTIVITY IN INSECTS-I

389

NAKAMURA T. (1940) The phosphorus metabolism during the growth of the animal. The behaviour of various phosphatases and phosphoric acid compounds of Bombyx mwi L. during growth. Mitt. med. Akad. Kioto 28, 387416; 590-592. NEUHAUS F. C. and BYRNE W. L. (1958) 0-Phosphoserine phosphatase. Biochim. biophys.

Acta 28, 223-224. ROCHE J. and BOUCHILLOUX S. (1947) Sur les phosphatases hydrolysant la phosphorylcholine et la phosphorylcolamine. C.R. Sot. biol., Paris 141, 1068-1070. SCHMIDT G. (1961) Nonspecific acid phosphomonoesterases. In The Enzymes, Vol. 5, pp. 37-47. Academic Press. SMITH L. C., ZELLER E. A., and ROSSI F. M. (1955) Occurrence and enzymatic degradation of ethanolamine phosphate. Fed. Proc. 14, 283. STADTMAN T. C. (1961) Alkaline phosphatases. In The Enzymes, Vol. 5, pp. 55-71. Academic Press. STRICKLAND K. P., THOMPSON R. H. S., and WEBSTER G. R. (1956) Hydrolysis of phosphorylcholine and related esters by the phosphomonoesterases of animal tissues. Arch.

biochem. biophys. 64, 498-505. WEICHSELBAUM T. E. (1946) An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Amer. J. C&z. Path., Tech. Suppl. 10,

16-20. WYATT G. R. (1958) Phosphorus

compounds

in insect development.

4th int. Congr. Biochem.

12, 161-184. WYATT

G. R. and KALF G. F. (1956)

10th int. Congr. Ent. 2, 333.

Organic

components

of insect haemolymph.

PYOC.