Characteristics of luciferases from a variety of firefly species: Evidence for the presence of luciferase isozymes

Insect Biochem., Vol. 11, No. 4, pp. 417-422, 1981. Printed in Great Britain.

0020-1790/81/040417-06502.00/0 © 1981 Pergamon Press Ltd.

C H A R A C T E R I S T I C S OF L U C I F E R A S E S F R O M A V A R I E T Y OF F I R E F L Y SPECIES : E V I D E N C E F O R THE P R E S E N C E OF LUCIFERASE ISOZYMES L. G. STRAUSE* and M. DELUCAt Departments of *Biology and tChemistry, University of California, San Diego, La Jolla, CA 92093, U.S.A. (Received 26 August 1980) Abstract--The properties ofluciferases from five species of adult fireflies have been compared. The pI values as determined by isoelectric focusing are essentially the same for all five enzymes. All of the enzymes are inhibited to a comparable extent by antibody raised against the enzyme from Photinus pyralis. These experiments suggest that the enzyme found in various adult species is similar. An isozyme of luciferase, distinct from the adult form, exists in the larval stage of the firefly, Photuris pennsylvanica. Michaelis constants, pH profiles, and molecular weights are similar for larval and adult luciferases. However, the isoelectric points and the antigenicity of the larval and adult luciferases are significantly different. Key Word Index." Luciferase, isozyme, fireflies, bioluminescence, antigenicity, isoelectric points

INTRODUCTION

MATERIALS AND METHODS

MULTIPLE enzyme forms, isozymes, have been shown to exist in a variety of organisms and to exhibit a high degree of tissue and cellular specificity (for reviews, see MASTERS and HOLMES, 1972; WHITT, 1975). A m o n g insects, the existence of isozymes frequently reflects changes occurring during development (LAUrER, 1961; WHITMORE et al., 1972; WAGNER and SELANDER, 1974; KOROCHKIN, 1975; NARANG and NARANG, 1975; THOMPSON, 1975; VERDEBRAT and WITT, 1975; VOLKNANDT and HARDENLAND, 1978). Studies of isozyme systems during ontogeny may therefore provide an insight into the regulation of protein synthesis during development. The luminescent system of the firefly, Photuris pennsylvanica, undergoes major physiological changes during metamorphosis (CASE and STRAUSE, 1978; STRAUSE et al., 1979; STRAUSE and CASE, 1980; STRAUSE, LINBERG and C A S E , 1981). The biochemical reaction underlying light emission and the characteristics of the enzyme, luciferase, have been studied extensively in the adult of another species, Photinus pyralis (for review see DELUCA, 1976). To date, luciferases from other developmental stages have not been studied. Objectives of the present study are: (1) to determine if an isozyme of luciferase, distinct from the adult form, exists in the larval stage of the firefly, P. pennsylvanica and (2) to compare luciferase from a variety of adult firefly species using isoelectric focusing (I.E.F.), kinetic studies, molecular weights and immunological cross-reaction as criteria.

Source of animals and determination Of luci]brase activity The species of the adult fireflies used in these experiments are: Photinus pyralis, collected in Tennessee; Photuris pennsylvaniea, provided by J. BUCKand reared from larvae; Photuris versicolor, provided by A. CARLSON; Pteroptyx tener, provided by J. BUCK;and Luciola cruciata, provided by Y. HANEDA. P. pennsylvanica larvae were collected in Baltimore, Maryland in autumn 1979. Larvae were maintained at 10°C on a 12 hr : 12 hr light : dark cycle. Pupation was induced and individual pupa staged as described by STRAUSEet al. (1979). Live specimens were frozen in a dry ice-acetone bath and lyophilized. Lyophilized light organs, isolated from whole insects, were weighed and suspended in 4 C , distilled water. Luciferase activity was assayed by measuring the peak light intensity obtained when excess ATP (0.1 ml of 0.02 M) is injected into a mixture containing 0.025 M glycyl glycine buffer, pH 7.8, 5.3 mM magnesium sulphate, 0.08 mM purified luciferin and 0.01 ml of the light organ suspension, final volume 0.51 ml. Light intensity was measured with an Aminco Chem-glow photometer and Aminco recorder, o-luciferin was synthesized according to the method of SETOet al. (1963). Partially purified luciferase, isolated following isoelectric focusing (see below), was used for antigenic determination, kinetic analysis and pH profiles of the enzyme, crude enzyme extracts were used in molecular weight studies.

* To whom all correspondence should be addressed.

Isoelectric.[ocusing ( I.E.F. ) Sucrose density gradient columns were used for I.E.F. of luciferase. After focusing, the column was eluted and 1 ml fractions were collected from 110 ml columns or 0.5 ml fractions from 20 ml columns. Isoelectric points (pl values) were obtained by the measurement of pH of the fraction with the maximum level of luciferase activity, determined as described above, after focusing and fractionation. Proteins differing in pI by as little as 0.01 ofa pH unit can be separated

417

418

L . G . STRAUSEand M. DELUCA

(VESTEKGERG, 1971). Two procedures (described below) were used depending on the quantity of lyophilized material available. The technique described by LUNDIN (1978) was modified for luciferase purification from P. pyralis adults and P. pennsylvanica larvae, of which a large quantity of material was available. Crude lantern extracts, corresponding to 250 mg of dried adult lanterns or 450-750 m g of dried larva tails, were ground in a mortar and suspended in 22 ml, 4°C, distilled water, maintaining p H 7.0 by the addition of 0.1 M N a O H . Centrifugation, 5900 g for 10 min, at 4°C, removed particulate matter from the extract. A m m o n i u m sulphate fractionation, to 50~o saturation, was used to precipitate out non-specific proteins. Luciferase precipitated out of the supernatant at 60~, saturated a m m o n i u m sulphate. The final protein precipitate collected by centrifugation, 5900 g for 10 rain, at 4'C, was dissolved in 3 ml cold, distilled water and the enzymatic activity was determined. In a 110 ml electrofocusing column, the dense and light solutions for the density gradient were made according to LUNDIN (1978). For I.E.F. of larval luciferase, 0.1 ml ampholine (LKB) pH 3.5-10 was added to the dense solution. A voltage of 1000 V was applied for 20-24 hr. Fractions, 1 ml, were collected and kept on ice. The luciferase activity and pH were measured for each fraction. Fractions containing luciferase, as evidenced by peak light intensities, were pooled and immediately neutralized. For all other species studied, crude lantern extracts, corresponding to less than 100 mg of dried lanterns, were ground in a mortar and suspended in cold, 0.1 M phosphate buffer, pH 7.8. Extracts were centrifuged, 5900g for 10 min, at 4'C, to remove particulate matter. Light activity of the supernatant was determined. The supernatant was loaded in the middle of the density gradient of a 20 ml electrofocusing column. The dense solution contained 8.6 ml distilled water, 5.6 g sucrose, 0.88 ml ampholine (LKB) pH 5-7 and 0.1 ml ampholine (LKB) pH 3.5-10. Distilled water was used as the light solution. Current of 1000 V was applied for 20-24 hr. Fractions were collected from the column and assayed as previously described. Protein concentrations applied to the columns and recovered in the peaks of light activity are recorded in mg/ml, based on a specific activity of 250,000 light units (L.U.)/mg. The presence of ampholines with the eluted protein resulted in a substantially decreased specific activity when using optical density readings at 280 nm, and also made accurate protein determinations impossible. Adult, P. pyralis,

luciferase, following I.E.F., had a specific activity of only 7800 L. U./mg according to O. D.: s 0 readings, whereas, using 5,5'-dithiobis-2-nitrobenzoic Acid (DTNB), which binds specifically to sulphydral groups (ELLMAN, 19593, the specific activity was calculated to be 254,600 U U . / m g . Therefore, since all luciferases except P. pyralis were available in limited quantities, the specific activity is assumed throughout the text to be 250,000 L.U./mg. The authors believe that this is an accurate assumption.

Antibody cross-reactions Antigenicity was determined by the percent inhibition of luciferase by antibody raised against adult, P. pyralis, luciferase. Purification of the antibody was accomplished by a m m o n i u m sulphate precipitation according to WILL1AMS and CHASE (19673. Two or three-fold molar excess of specific antibody, assuming 1",i specific antibody was contained in IgG fractions of the antisera (20 mg/ml) (WItMAMS and CHASE, 1967), was added to partially purified luciferase from adult P. pyralis and larval P. pennsylvanica lanterns, to a total volume of 0.4 ml. The titration curve was determined by maintaining a constant concentration of luciferase and increasing the concentration of antibody. The volume was brought up to 0.4 ml with pre-immune rabbit serum. Light intensity was measured immediately following the addition of antibody and at varying times up to 48 hr of incubation. A protein concentration of pre-immune rabbit serum or bovine serum albumin (BSA), comparable to the concentration of antibody, was added to the luciferase as a control. Inhibition of purified adult P. pyralis luciferase by the antibody was used as a second control, for each set of experiments.

Kinetics. p H profih, s and moh, cular weights The apparent Michaelis constants for partially purified luciferase from larva P. pennsvlvanica and adult P. pyralis were obtained from Lineweaver-Burk plots (LINEWEAVER and BunK, 19343. Kmvalues of luciferase for A T P were measured in the presence of excess luciferin (10 3M). K m values of luciferase for luciferin were measured with saturating ATP concentration (4 x 10 3M). pH profiles for partially purified luciferase, from larva P. pennsylvanica and adult P. pyralis, were determined over a pH range of 5.5-8.0 in 0.025 M phosphate, pH 7.5-9.0 in 0.025 M Tris, and pH 8.0-9.0 in 0.025 M glycyl glycine buffers at 2 5 C . The molecular weight of luciferase from a crude extract of larval P. pennsyh'anica was compared to purified luciferase

Table 1. Characterization of luciferases

Species and stages (n)

Protein onto I.E.F. column (mg/ml)

P. pyralis--adult (8)*

0.09 - 1.05

P. pennsylvanica--adult (1)

1.3 x 10

P. pennsylvanica

3

larvae

(8)*

0.001-0.06

P. versicolor--adult (1)

P. tener

1.6 × 10 - s adult

(3)

L. cruciata (4)

0.88-1.6 × 10 -3 adult 2 . 4 - 4 . 8 × 10 3

Protein off I.E.F. column (mg/ml) Major peak Minor peak (pl) (pI) 0.1-0.18 (5.6 - 6.1 ) 1.6 × 10 4 (5.95) 0 . 1 6 - 6 . 0 x 10 -3 (4.5-4.8) 0.05 × 10 s (6.0) 1.5-3.3 (5.3-5.6) 2 . 3 - 3 . 3 x 10 4 (5.7-6.0)

.003 - 0 . 1 3 (5.4 - 5.6) 9.6 x 10 s (4.75)

Percent yield (by light activity) from I.E.F. column 2 0 - 50

10 10-15 5

0.3-1.0 (5.0-5.3)

13-15 20

n = N u m b e r of enzyme purifications by I.E.F. * = O f the total number of I.E.F. columns, three are combined P. pyralis adult and P. pennsylvanica larva.

Firefly luciferase isozymes 3600

90-

3200

80-

2800

N"

419

70-

2400

x

60

2000

~

so

.~

30

1600 I.-

1200 800

20

400

10

0

I

4.0

I

I

4.2

I

I

4.4

I

I

I

46

4.8

I

I

5.0

5.2

5.6

pH UNITS

6.0

64

68

72

pH UNITS

Fig. 1. Elution pattern from an isoelectric focusing column containing both adult P. pyralis (0.096 mg/ml), and larval, P. pennsylvanica (0.0048 mg/ml), extracts. (a) Fraction numbers 35-50 containing the larval enzyme. (b) Fraction numbers 48-71 containing the adult enzyme. After I. E. F., the protein yield was 0.061 mg/ml adult luicferase and 0.007 mg/ml larval luciferase. Note change in scale for relative light units. from adult P. pyralis. Purified P. pyralis luciferase, 0.2 ml of 0.01 rag, was loaded onto a I cm i.d. x 55 cm S-200 Sephacryl column. Fractions, 0.5 ml, were collected and light intensity was determined. Crude larval extract, corresponding to 67 mg dry weight, was dissolved in 1 ml, 0.1 M phosphate buffer, pH 7.8 and centrifuged, 5900g for 10 rain at 4~C, to remove particulate matter. The supernatant, 0.2 ml, was loaded onto the same column and fractions collected at the same rate. A mixture of larval extract and purified adult P. pyralis, 0.2 ml each, were loaded onto the same column and fractions again collected at the same rate. Fractions from the three columns were compared according to peak light intensity.

of this value (Table 1). W h e n extracts of adult, P. pyralis a n d larval, P. pennsulvanica, are developed together in one column, two light activity peaks are found (Figs. l a and lb). The pl value for P. pennsylvanica larval luciferase is between pH 4.5

500

450

400

RESULTS c.O

The level of luciferase activity is determined by the peak light intensity o b t a i n e d in the s t a n d a r d assay. All species of adult fireflies studied a p p e a r to have similar luciferases based on pI values a n d reaction with a n t i b o d y to adult, P. pyralis, luciferase. However, a second form ofluciferase appears to exist in the larval stage of P. pennsylvanica. Because of the limited supply of P. pennsylvanica adults, the Michaelis constants, p H profiles a n d molecular weights of the larval luciferase are c o m p a r e d to those of the luciferase o f P . pyralis adults. According to the present results, luciferase from P. pennsylvanica and P. pyralis adults a p p e a r to be identical.

~ ==,,

350

~ __.

300

_> '~ ua ""

250

...I

200

150 100

Isoelectric Jbcusing The pI values of luciferases from different species and stages of d e v e l o p m e n t are determined by correlating light intensity with p H following I.E.F. The p l value of luciferase f r o m the adult, P. pyralis is between p H 5.8 a n d 6.1 with a m i n o r peak between p H 5.5 to 5.6 (DENBURG and MCELROY, 1970). U s i n g this species enzyme as a s t a n d a r d , luciferases from all o t h e r adult species tested have pI values within 0.5 p H units

0 ~

4.0

4.4

4.8

5.2

5.6

6.0

6.4

pH UNITS Fig. 2. Elution pattern from an isoelectric focusing column containing adult, P. pennsylvanica (0.0013 mg/ml), extract. After I. E. F., the protein yield was 9.6 x 10 5 mg/ml larval luciferase and 1.6 x l0 -4 mg/ml adult luciferase. Pattern spans fraction numbers 7-30.

420

L.G. STRAUSEand M. DELUCA

1000 900 -

90 a. 80 g-.-

~

-

b.

!

800

70

~ 700 z

60

l-- 600

50

~

z

5oo

I---

..~ 40~ 30 -

~

~

4oo

I---

=z/~

300

20-

~

200

10

~

100

0 5,4

5.6

I I I 5,8 6.0

I

I I I I 6,2 6,4

I

]

0

I 4,0

I

1 I I 4,4 4,8

pHUNITS

I

I I I I I 5.2 5,6 60

pHUNITS

Fig. 3. (a) Elution pattern from an isoelectric focusing column containing adult, P. pyialis (0.45 mg/ml), extract. After I.E.F., the protein yield was 0.1 mg/ml luciferase. Pattern spans fraction numbers 35-55. (b) Elution pattern from an isoelectric focusing column containing larval, P. pennsylvanica (0.06 mg/ml), extract. After I.E.F., the protein yield was 6.0 x 10 3mg/ml luciferase. Pattern spans fraction numbers 35-60. Note change in scale for relative light units. and

4.8, which differs considerably

from

from adult P. pyralis, 12.5 and 35% respectively, when comparable dry-weight extracts are used. This is most likely due to the instability of the enzyme at low p H values.

adult

P. pennsylvanica luciferase, which has a pI value of pH 5.95 with a secondary peak at p H 4.75 (Fig. 2). The luciferases of adult and larval extracts, developed by electrofocusing columns individually (Figs. 3a and b) or in combination, (Figs. la and b), gave similar results. The recovery of larval luciferase from the electrofocusing columns is low in comparison with luciferase

100

g

Antibody cross-reactions Adult and larval luciferases from P. penn,s:vhanica appear to be immunologically distinct. Activity of

48

90 8O 7O 60-

~.~.e

L48 -L24

I

21) [

2

[

4

6

1

I

I

I

l

l

I

I

I

I I~1

I

I

l

I

I

8 10 12 14 16 18 20 22 24 26 28 30 32 34 40 45 50 55 60 65 MOLAR RATIO OF IgG/LUCIFERASE

Fig. 4. Percent inhibition of luciferase activity when incubated with increasing amounts of antibody against adult, P. pyralis, luciferase. Luciferase activity, as measured by light emission (see text), is determined for the adult, P. pyralis, and larval, P. pennsylvanica, luciferases after 24 hr (A24, L24) and 48 hr (A48, L48) incubation. Molar concentrations of IgG were determined assuming 1% of the total protein was specific antibody (WILLIAMSand CHASE, 1967).

Firefly luciferase isozymes purified and partially purified luciferase from adult P. pyralis, in two to three-fold molar excess antibody, is inhibited 70-75~o in 24 hr and 78-80~o in 48 hr. Incubation of this luciferase in greater than 50-fold molar excess antibody resulted in inhibition between 95 to 98~o in 24 hr. However, under similar conditions, larval P. pennsylvanica luciferase activity is inhibited between 8 to 10}o in 24 hr and 13}~,in 48 hr. Incubation of larval luciferase in greater than 50-fold excess antibody increased the effective inhibition to between 40 to 50~o in 24 hr. In increasing amounts of antibody, the activity of luciferase from adult P. pyralis is inhibited 95')/0in the presence of five-fold molar excess. In similar conditions, larval P. pennsylvanica luciferase activity is inhibited between 40 to 50~o in 50-fold molar excess and the percent of inhibition did not increase with increasing amounts of antibody (Fig. 4). Luciferase activities from the other adult species are inhibited by a comparable amount.

Characteristics oJ P. pennsylvanica larval luciJerase Michaelis constants, pH profiles and molecular weights were determined in order to further characterize the larval luciferase. The apparent Michaelis constants for larval luciferase are 3.2 × 10-4M and 1.2 × 10-SM for ATP and luciferin, respectively. K values for the adult luciferase are 3.4 × 10 _4 M an~ 1.3 × 10-SM for ATP and luciferin, respectively. The K m values for P. pyralis adult luciferase are consistent with the findings of DENBURG et al. (1969) and LEE et al. (1970). The pH optimum in glycine, Tris and phosphate buffers is 8.0 for both adult and larval enzymes. The molecular weights of the adult and larval luciferases appear to be identical, 50,000 (TRAVISand MCELROY, 1966), as measured by the molecular sieve method using S-200 Sephacryl columns. When adult and larval extracts are loaded onto Sephacryl columns, a single peak in light intensity is obtained whether loaded individually or in combination. Therefore, although the K v a l u e s , pH optima and molecular weights of the adul't and larval luciferase are similar, the isoelectric points and antigenicity appear to differ,

DISCUSSION A variety of enzymes have been reported to change qualitatively and quantitatively during postembryonic development. Certain insect enzymes, such as esterases (LAUFER, 1961; ELDEFRAWL et al., 1970; BOOTH et al., 1973; BOOTHet al., 1975; VEDBRATand WHITT, 1975) and dehydrogenases (LA~TFER, 1961; NARANG and NARANG, 1975; BARNES et al., 1977), have been shown to exist in various forms based on electrophoretic patterns and effects of specific inhibitors. Malate dehydrogenase, of the mosquito Culex p. quinqueJhscitatus, has different pI values in larval, pupal and adult stages (NARANGand NARANG, 1975). The role of the enzyme luciferase and the substrate luciferin in bioluminescent reactions has been extensively studied. It appears that the native luciferin, from twenty different species, are the same as that of P. pyralis and that the luciferase is the same for all individuals of a given species (McELROY et al., 1965;

421

MCELROY and SELIGER,1966; MCELROY et al., 1969). These results were determined by cross-reactivity of enzymes and substrates. However, if a given enzyme can be separated into distinguishable types, by physical, chemical, immunochemical, or kinetic properties, each separate type constitutes an isozymic form (MARKERT, 1968). The concentration and localization ofluciferase and luciferin has been shown to change during postembryonic development of the firefly, P. pennsylvanica (STRAUSEe t al., 1979). These results were based on the assumption that luciferase, able to catalyze the same chemical reaction, was identical throughout development. The present study has shown that the luciferases, partially purified from five firefly species, were inhibited by antibody to adult P. pyralis luciferase by greater than 75~, when incubated in two to three-fold molar excess for 24 hr at 4°C. The pI values for these five luciferases were all between pH 5.3 and 6.1. The luciferase from larval. P. pennsylvank'a, and adult, P. pyralis, appear to have similar K mvalues, pH profiles and molecular weights. However, the pl values and the percent inhibition by antibody were substantially different. Activity of partially purified larval luciferase was inhibited by 8-10°~, when incubated for 24 hr in two or three-fold molar excess antibody at 4°C. The pI value for larval luciferase was between pH 4.5 and 4.8. The secondary peak in light intensity at pH 4.75, observed when luciferase is purified from P. pennsylvanica adults, by I.E.F. of 1.3 × 10- 3 mg/ml protein, can be accounted for by the frequent retention of larval light organs by newly emerged adults (STRAL'SE et al., 1979). The majority of these adults used for the enzyme preparation had larval light organs still present. These results suggest that luciferase isozymes exist in P. pennsylvanica and that they may be linked to postembryonic development. Insect hormones have been shown to act in regulating the synthesis of specific enzymes (WHITMOREet al., 1972; NARANGand NANG, 1975; THOMPSON, 1975; WHITT, 1975). Although the time course of adult and larval bioluminescence is dramatically different (BI'cK and CASE, 1961; BUCKet al., 1963: Bt'CK et al., 1965; CASE and BUCK, 1963; OERTEL and CASE, 1976), this is more likely a consequence of neural control and changes in luciferin and luciferase concentrations (CASE and STRAUSE, 1978; SRAVSEet al., 1979) than to the presence of a luciferase isozyme. However, the existence ofisozymes suggest that some level of genetic regulation occurs during postembryonic development and characterization of the luciferase from different pupal stages, currently in progresss, may provide insight as to the level at which regulation is exerted.

Acknowledgement We are grateful to Dr. J. CASE, Dr. F. HANSONand CAROLYNCEARLEYfor assistance in collecting firefly larvae. We thank STEVEALTERfor preparation of the antibody and Dr. P. RUSSELLfor the use of the isoelectric focusing columns. We thank Drs. W. D. MCELROY and P. RUSSELLfor comments on the text. Supported by National Science Foundation Grant PCM 7826221.

REFERENCES

BARNESC. S., CUPP E. W. and TOOMP. M. (1977) Lactate

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dehydrogenase isoenzyme in Aedes aegypti. Insect Biochem. 7, 387-391. BOOTH G. M., STRATTON C. L. and LARSENJ. R. (1975) Localization and substrate-inhibitor specificity of insect esterase isozyme. In Isozymes: Developmental Biology Vol. III (Ed. by MARKERTC. L.), pp. 731-728. Academic Press, London. BOOTH G. M., CONNORJ., METCALER. A. and LARSENJ. R. (1973) A comparative study of the effects of selective inhibitors on esterase isozymes from the mosquito Anapheles punctipennis. Comp. Biochem. Physiol. 44B, 1185-1195.

BUCKJ., and CASEJ. F. (1961 ) Control of flashing in fireflies: I. The lantern as a neuroeffector organ. Biol. Bull. mar. biol. Lab., Woods Hole 121, 234-256. BUCK J., CASE J. F. and HANSON F. E. (1963) Control of flashing in fireflies. Ill. Peripheral excitation. Biol. bull. mar. biol. Lab., Woods' Hole 125, 251-269. BUCK J., CASE J. F. and HANSON F. E. (1965) Control of flashing in fireflies. Proc. XIlIth Int. Congr. Ent.. p. 226. CASEJ. F. and BUCKJ. (1963) Control of flashing in fireflies. II. Role of the central nervous system. Biol. Bull. mar. biol. Lab.. Woods Hole 125, 234-250. CASE J. F. and STRAUSEL. G. (1978) Neurally controlled luminescent systems. In Bioluminescence in Action (Ed. by HERRING P.). pp. 331-366. Academic Press, London. DENBURGJ. L. and MCELROYW. O. (1970) Catalytic subunit of firefly luciferase. Biochemistry 9, 4619--4624. DENBURG J. L., LEE R. T. and MCELROY W. D. (1969) Substrate-binding properties of firefly luciferase. I. Luciferin-binding site. Archs. Biochem. Biophys. 134, 381-394. DELUCA M. (1976) Firefly luciferase. Adv. Enzymol. 44, 37--68. ELDEFRAWlM. E., TRIPATm R. K. and O'BRIEN R. D. (1970) Acetylcholinesterase isozymes from the housefly brain. Biochim. biophys. Acta. 212, 308-314. ELLMAN G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. KOROCHKIN L. (1975) Genetic control and developmental expression of esterase isozymes in Drosophila of virilis group. In Isozymes: Developmental Biology Vol. III (Ed. by MARKERT C. L.), pp. 99-117. Academic Press, London. LAUFERH. ( 1961) Forms of enzymes in insect development. Ann. N. Y. Acad. Sci. 94, 825-835. LEE R. T., DENBURG J. L. and MCELROY W. D. (1970) Substrate-binding properties of firefly luciferase. II. ATPbinding site. Arch. Biochem. Biophys. 141, 38-52. LINEWEAVERH. and BURK O. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658-666, LUNDIN A. (1978) Determination of creatine kinase isoenzymes in human serum by an immunological method using purified firefly luciferase. In Methods in Enzymology. Biolumineseence and ChemilumineScence Vol. LVii (Ed. by DELUCA M.II), pp. 56--65. Academic Press, New York. MARKERTC. L. (1968) Molecular basis for isozymes. Ann. N. Y. Acad. Sci. 151, 14-40. MASTERS C. J. and HOLMES R. S. (1972) Isoenzymes and ontogeny. Biol. Rev. 47, 309-361. MCELROY W. D., SELIGER H. H. and WHITE E. H. (1969) Mechanisms of bioluminescence, chemiluminescence and

enzyme function in the oxidation of firefly luciferin. Photochem. Photobio. 10, 153-170. MCELROY W. D. and SELIGER U. U. (1966) The colors of bioluminescence: Role of enzyme and substrate structure. In Molecular Architecture in Cell Physiology (Ed. b)' HAYASm and SZENT-GYORGYI) pp. 63-79. Prentice Hall, New Jersey .McELROVW. D., SELIGERH. H. and DELUCA M. (1965) Enzyme catalysis and color of light in bioluminescent reactions. In Evolving Genes and Proteins (Ed. by BRYSONV. and VOGELH.), pp. 319-340. Academic Press, New York NARANGS. and NARANGN. (1975) Malate dehydrogenase of a mosquito, Culex p. quinquefasciatus: developmental changes, polymorphism, and physiochemical characterization. Biochem. Genet. 13, 73-84. OERTEL D. and CASE J. F. (1976) Neural excitation of the larval firefly photocyte: slow depolarization possibly mediated by a cyclic nucleotide. J. exp. Biol. 65, 213-227. SETO S., OGURAK. and NISmYAMAY. (1963) A convenient synthetic method of 2-carbamoyl-6-methoxybenthiazole, one of the intermediates for the synthesis of firefly luciferin. Bull. Chem. Soc. Jap. 36, 331-333. STRAUSEL. G. and CASEJ. F. (1981) Neuro-pharmacological studies on firefly light organs during metamorphosis. J. Insect Physiol. 27, 5-15. STRAUSE L. G., DELUCA M. and CASE J. F. (1979) Biochemical and morphological changes accompanying light organ development in the firefly, Photuris pennsylvanica. J. Insect Physiol. 25, 339-347. STRAUSE L. G., LINaERG K. A. and CASE J. F. (1981) Ultrastructural study of firefly light organ degeneration during metamorphosis. Cell Tissue Res. Submitted. THOMPSON P. E. (1975) Selective expression of multiple hemoglobins in the development of Chironomus larvae. In Isozymes: Developmental Biology Vol. III (Ed. by Markert C. L.), pp. 119-129. Academic Press, London. TRAV1SJ. and MCELROYW. D. (1966) Isolation and sequence of an essential sulfhydryl peptide at the active site of firefly luciferase. Biochemistry 5, 43-95. VEDBRATS. S. and WHITT G. S. (1975) Isozyme ontogeny of the mosquito, Anopheles abbimas. In Isozymes: Developmental Biology Vol. I l i (Ed. by Markert C. L.), pp. 131-143. Academic Press, London. VOLKNANDT W. and HARDELAND R. (1978) Circadian rhythmicity of tyrosin amino-transferase activity in larval and prepupal fat body of Drosophila melanogaster. J. InterdiscipL Cycle Res. 9, 283-291. WAGNERR. P. and SELANDERR. K. (1974) Isozymes in insects and their significance. Ann. Rev. Entomology 19, 117-138. WHITMORE D. Jr., WHIXMOREE. and GILBERT L. I. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titer. Proc. natn. Acad. Sci. U.S.A. 69, 1592-1595. WHITT G. S. (1975) Isozymes and developmental bilogy. In lsozymes: Developmental Biology Vol. III (Ed. by MARKERT C. L.), pp. 1-8. Academic Press, London. WILLIAMS C. A. and CHASE M. W. (1967) Purification of antibody. In Methods' in hnmunology and lmmunochemistry: Preparation of Antigens and Antibodies Vol. I (Ed. by WILLIAMSC. A. and CHASEM. W.). pp. 307-385. Academic Press, New York.