THE Jonmn~ OB BIOLOGKAL CHEMISTRY Vol. 245, No. 11, Issue of June 10, pp. 2927-2936, 1970

PrGded in U.S.A.

Regulation of Glycogen Metabolism in Insect Flight Muscle

PURIFICATION AND PROPERTIES OF PHOSPHORYLASES IN VITRO AND IN VIVO*

(Received for publication, November 21, 1969)

CHARLES C. CHILDRESS AND BERTRAM SACKTOR

From the Gerontology Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland 212.5’4

SUMMARY

The mechanism for controlling the rapid rate of glycogen- olysis in flight muscle of the blowfly, Phormia regina, was studied by isolating phosphorylase a and b from the muscle and determining the physical and kinetic properties of the puriiied enzymes and the relative amounts of the two forms of the enzyme in the muscle of the insect at rest and during flight.

A method is described for the purification of phosphorylase a and b from flight muscle. Sedimentation velocity and disc gel electrophoresis studies indicate homogeneous prepara- tions. The molecular weights of both phosphorylase b and a are about 100,000. p-Chloromercuribenzoate has no effect on the sedimentation coefficient. Ammo acid analyses of the insect muscle enzyme show high contents of half- cystine and lysine and a low content of arginine, relative to the amounts of these residues found previously in mam- malian muscle preparations. Other properties of the flight muscle enzyme, including pH optimum, stability, and pyridoxal phosphate content are described.

The apparent K, of phosphorylase a and b for glycogen, Pi, or AMP has been determined at various concentrations of cosubstrate and activator. From these kinetic data, plus previously determined concentrations of metabolites in flight muscle, conditions in uiuo were simulated. The level of glycogen in the muscle is sufficient to saturate the enzymes. The apparent K, of phosphorylase b for Pi, at 0.1 mu AMP, is about 100 m&r, well above the concentration found in the muscle. The apparent K, for AMP, at 8 mu Pi, is about 1.0 XIlM, approximately lo-fold that in the muscle, at rest. ATP strongly inhibits phosphorylase b at low concentrations of AMP.

The concentration of AMP in the muscle is lOO-fold its apparent K,,, for phosphorylase a. The apparent K, for Pi at simulated conditions in uivo is 8 mrvr, approximately the concentration found in the muscle. ATP does not inhibit the a form of the enzyme. Thus, at the levels in viva of substrates, activators, and inhibitors, phosphorylase a retains about 50% of its potential activity.

The activity of phosphorylase b is too low to account for the rate of glycogenolysis during flight. The activity of phos-

* A preliminary report of a portion of this work was presented at the Annual Meeting of the Federation of Societies for Experi- mental Biology and Medicine, Atlantic City, 1969.

phorylase a is adequate, if at least 50% of the total enzyme is in the a form. Measurements of the relative amounts of phosphorylase a and b at rest and during flight show that the conversion of the b to the a form is of this order of magnitude. It is concluded that in blowfly flight muscle the mechanism for controlling the intense rate of glycogenolysis concomitant with the initiation of flight is the conversion of phosphorylase b to phosphorylase a.

On initiation of flight, the metabolic rate of insects may in- crease loo-fold or more and exceed that of any other known biological system (1). During this rest to flight transition, an intense rate of glycogenolysis in the flight muscle of the blowfly is triggered (2). The mechanisms of the control in the muscle are being investigated. As part of this study, native glycogen from blowfly flight muscle was isolated and characterized, both struc- turally and biochemically (3). The present paper deals with the purification and properties of the phosphorylases from flight muscle of the blowfly, Phormiu regina. Heretofore, the only evidence for phosphorylase in insect muscle was inferred, as gly- cogen was shown to be oxidized by muscle homogenates (4) and depleted during flight (2, 5), or was shown histochemically by an increased deposition of glycogen in sections of locust muscle incu- bated with glucose-l-P and AMP (6). This paper presents a detailed kinetic characterization of the purified blowfly phos- phorylases. From these findings, the mechanism for regulating the intense rate of glycogenolysis that is coincident with the initiation of flight is suggested.

EXPERIMENTAL PROCEDURE

Blowflies, Phormiu regina, were maintained in laboratory cul- ture as described previously (3). Seven days following eclosion, flies, without regard to sex, were frozen in liquid nitrogen. The thoraces were isolated and maintained in the frozen state (-80”) until used for the purification of phosphorylase.

Measurements of phosphorylase activity were carried out in the direction of glycogen breakdown with a coupled assay system that was optimal for the insect enzyme. The reaction mixture contained, in the volume of 0.62 ml, the following final concen- trations: 40 InM Tris-acetate, 5 mM imidazole (pH 7.0), 5 mM magnesium acetate, 2 mu EDTA, 1.4 mM mercaptoethanol,

2927

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Insect Flight Muscle Phosphorylase Vol. 245, No. 11

5.35 X 1OF M glucose-l,B-di-P, 0.6 mM NADP, 80 mM Pi, 1.6 mM AMP, 2 mg of glycogen, 0.065 unit of phosphoglucomutase, and 0.28 unit of glucose-6-P dehydrogenase. Reduction of NADP was measured spectrophotometrically. The temperature was maintained at 30”. After temperature equilibration, reac- tions were started by the addition of enough phosphorylase to produce a rate not greater than 0.01 pmole of glucose-l-P formed per min. The reaction rates were linear and proportional to the phosphorylase concentration. When necessary, turbidity con- trols, without added enzymes, were used as blanks. One unit of activity represents the formation of 1 pmole of glucose-l-P per min at 30”.

Dilutions of crystalline glucose-6-P dehydrogenase and phos- phoglucomutase were prepared with a diluting fluid containing 20 mM Tris-acetate (pH 7.0), 5 mM imidazole, 1 InM magnesium acetate, and 0.002% bovine serum albumin. Phosphorylase preparations were diluted with a solution containing 0.1 InM

EDTA, 1.0 mM mercaptoethanol, 0.1% bovine serum albumin, and 10 mM Tris-acetate (pH 7.0), just before assay.

The procedures for initiating and terminating flights were the same as detailed earlier (2), except that, in determining the relative activities of phosphorylase a and b in the u&own fly, the insect was allowed to rest for 2 hours in the dark after it had been prepared as if for flight. Extracts for estimating phosphorylase a and 6 activities in the flight muscle in vivo were made by a modification of the method of Danforth, Helmreich, and Cori (7). FolIowing the termination of flight by plunging the fly into liquid Nz, the thoraces were isolated and powdered in a stainless steel homogenizer that had been previously cooled in liquid NY. Five thoraces, about 100 mg, wet weight, were used per extract. The temperature was then raised to -35”, in a Dry Ice-acetone bath, and 1 ml of a solution containing 60% glycerol, 5 mM EDTA, 20 mM NaF, adjusted to pH 7.0, was added, followed by homogenization. The temperature was increased to 0” and 4 ml of a mixture containing 5 mM EDTA, 20 mM NaF, 20 mM Tris, adjusted to pH 7.0, were added, followed by another homogenization. The extract was centrifuged at 15,000 x g for 10 min and the supernatant was used for the assay of phosphorylases. In assaying for phos- phorylase a, the concentration of AMP was 0.01 InM, ensuring activation of the a form but not of the b form of the enzyme. The total phosphorylase activity, a + b, was measured in the presence of 3 InM AMP.

Precautions were taken to remove contaminating AMP from the assay system when examining the kinetics of phosphorylase a. Glycogen solutions were found to contain as much as 2 mpmoles of AMP per mg, which were removed by batchwise treatment with Dowex I-Cl. Phosphorylase preparations used for kinetic studies were routinely passed through a column, 1 x 1 cm, of Dowex l-Cl to remove any bound AMP. Most commercial preparations of NADP contained significant amounts of AMP, ranging from 0.5 to 14.0 mlmoles per pmole of NADP. In the present experiments, the lowest concentration of AMP attained in the assay system was 0.3 PM. AMP was determined spectrophotometrically in the coupled assay system (8). The reaction mixture, in a volume of 0.62 ml, contained the following final concentrations: 40 mM Tris-HCl (pH 7.6), 10 mM ATP, 16 mM phosphoenolpyruvate, 5 mM MgC12, 100 mM KCl, 0.14 mM NADH, 1.25 units of pyruvate kinase, 3.6 units of lactic dehydrogenase, and 18 units of adenylate kinase.

Protein was assayed either by the method of Lowry et al. (9),

following dialysis of the samples against distilled water to remove interfering substances, or, in t,he ca,se of very pure samples, by the spectrophotometric method of Warburg and Christian (lo), as modified by Layne (11).

In preparation for amino acid analysis, flight muscle phos- phorylase was exhaustively dialyzed against water and hy- drolyzed in 6 N HCl at 103” for 24 hours. After removal of the HCl, aliquots were analyzed with a Phoenix automatic amino acid analyzer. Tryptophan was determined spectrophoto- metrically (12). Total amino nitrogen of the hydrolysates was measured with ninhydrin (13).

Sedimentation velocity measurements were carried out at 20” in a Beckman-Spinco model E anaIytica1 ultracentrifuge, according to Schachman and Edelstein (13) and Schumaker and Schachman (14). When the protein concentration of the phos- phorylase sample was above 1.5 mg per ml, the position of the boundary was measured from photographic plates of the schlieren patterns. At protein concentrations below 1.5 mg per ml, the absorption optical system at 280 rnp was used. Estimations of molecular weight of purified flight muscle phosphorylase were made from sedimentation equilibrium measurements as well as from sedimentation velocity and diffusion coefficient data, the latter obtained from a calibrated gel filtration column, according to the method of Ackers (15). The partial specific volume was determined by simultaneous sedimentation equilibrium runs in Hz0 and DzO, as described by Edelstein and Schachman (16). Actual soIvent densities were determined pycnometrically. The partial specific volume was also calculated from amino acid analysis by the method of McMeekin, Groves, and Hipp (17). Sepharose 6B was used for gel filtration chromatography. The gel was equilibrated with buffer containing 50 mM Tris-NC1 (pH 7.0) and 0.1 M KC1 and poured into a column, 1.5 X 100 cm. Buffer was allowed to flow by gravity at a rate of 3 ml per hour and l-ml fractions were collected. Elution volumes were measured to the peak of either enzyme activity or absorp- tion at 280 rnp with collection beginning as the sample entered the gel bed. Sedimentations in linear sucrose gradients, from 5 to 20% (w/v), were carried out in a SW-39 rotor in a Spinco model L centrifuge, as described by Martin and Ames (18j.

Electrophoresis was carried out in 7.5% acrylamide gel with a Shandon disc electrophoresis apparatus. The buffer system was 10 mM Tris-HCI (pH 8.0). Bromothymol blue was used as a “tracker dye.” Electrophoresis was continued until the dye had moved through the gel rod. Following electrophoresis, the gel rods were removed from the tubes and were stained for protein by immersing for 2 to 3 hours in a solution containing 0.4 mg per ml of Amido black 10D dissolved in methanol, water, acetic acid, 5: 5: 1. The gels were then rinsed and allowed to stand for 24 hours in several changes of 7% acetic acid or, alter- natively, they were destained electrolytically. For the localiza- tion of phosphorylase activity, the gel rods were immersed in a solution containing 0.04 mg per ml of phenazine methosulfate and 0.4 mg per ml of nitroblue tetrazolium in addition to the other reactants which were used routinely for the measurement of phosphorylase activity by the coupled assay system. The gels were allowed to stand in the dark for several hours until the bands became clear and were then washed by immersion in 7% acetic acid for several hours.

Crystalline hexokinase, glucose-6-P dehydrogenase, adenylate kinase, pyruvate kinase, lactic dehydrogenase, and phospho- glucomutase, and glucose-l, 6-di-P and NADP, containing low

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Issue of June 10, 1970 6. C. Childress and B. Xacktor 2929

amounts of contaminat,ing AMP, were obtained from Boehringer and Soehne. NADH, AMP, ATP, GTP, UTP, and CTP were purchased from P-L Biochemicals. Adenylic acid deaminase, EDTA, and imidazole, Grade III, were obtained from Sigma. Mercaptoethanol was a product of Eastman Organic Chemicals. Enzyme grade ammonium sulfate and “ultra pure” Tris were obtained from Mann. Whatman microgranular DEAE- cellulose, type DE-32, was obtained through Reeve Angel. Acrylamide gel, Cyanogum 41, was obtained from Fisher. Sepharose 6B was a gift from Pharmacia.

Rabbit muscle phosphorylase b was purified from frozen rabbit muscle, obtained from Pel-Freeze Biologicals, by the procedure of Fischer and Krebs (19). Phosphorylase b kinase was prepared from blowfly flight muscle. The acid precipitate fraction, obtained during the purification of phosphorylase from flight muscle, was free of phosphorylase activity and contained all of the kinase activity. The precipitate was extracted with several small volumes of 0.1 M NaHC03 followed by centrifuga- tion at 15,000 x g for 10 min. The clear supernatant was stored frozen and used as a partially purified preparation of phosphorylase b kinase.

Native flight muscle glycogen was isolated and assayed as described earlier (3).

The enzymic rate data were processed with a computer pro- gram for least square fit to a hyperbola as described by Cleland (20). In the cases in which the relationships between the recip- rocals of initial velocity and substrate concentration were non- linear, the data were processed directly from plots of substrate against rate without use of the computer program.

RESULTS

Purijication of Phosphorylases a and &Frozen thoraces, usually 300 g, were homogenized in a Waring Blendor with 5 volumes (w/v) of 5 mM mercaptoethanol. The homogenate, pH 6.3, was filtered through several layers of cheesecloth and centrifuged at 15,000 x g for 10 min. The supernatant was filtered through glass wool and divided into two equal portions for the purification of phosphorylase a or b. The extract con- tained 17 units of activity per g, wet weight, of thorax and had a specific activity of 0.65 unit per mg of protein (Table I).

Approximately 10% of the total phosphorylase in the extract, at this stage, was in the a form. To convert all of the phos- phorylase activity in the extract to phosphorylase b, EDTA (pH 7), to a final concentration of 5 InM, was added. For the complete conversion to phosphorylase a, additions were made to the extract to give the following concentrations: 10 mm NaF, 1 InM ATP, and 1 mM MgClz. Although a slow conversion to phosphorylase a usually occurred when NaF alone was added, the addition of ATP and MgClz allowed a complete conversion within the 1st hour of incubation (Fig. 1). The complete con- version to phosphorylase b was always rapid following the addition of EDTA. The phosphorylase activity of the extract was the same regardless of the form (a or b) of the enzyme. As shown below, phosphorylase b was inactive in the absence of added AMP but was maximally activated by 2 mM AMP. Con- version of an aliquot of phosphorylase b to a by the ATP-de- pendent insect flight muscle phosphorylase kinase resulted in the same activity as was produced by AMP activation of phos- phorylase b. As described later in this paper, flight muscle phosphorylase a was stimulated by very low levels of AMP.

TABLE I

Purijkation of flight muscle phosphorylase

The values shown represent a typical purification of either phos- phorylase a or b from 150 g of flight muscle. Both forms of the enzyme behaved identically in the purification scheme and the activity per unit of enzyme protein was the same for the a and b forms under the conditions of assay. The values for the DEAE-

cellulose eluate are expressed for comparative purposes as only one-third this amount of enzyme was actually chromatographed at one time.

Fraction Activity

- I

“2’ 15,000 X g supernatant 3.5 Supernatant after acid

precipitation. 3.5

30-50y0 (NH&SO, pre- cipitate. 85.0

Heat-treated (NHJ$Oh precipitate. 85.0

DEAE-cellulose eluate

(concentrated). 60.0 -

units

2660

2660

2600

2600

1500

-I- 1

-

Protein Specific activit:

- w/ml

5.4

3.0

19.8

13.0

5.0 -

7%

4066

605

390 6.50

125 12.00

units/ w

0.65

4.30

R.5 WWY

%

100

100

95

95

56

HOURS OF INCUBATION AT 37°C

FIG. 1. Conversion of phosphorylase to the a or b form. The crude enzyme extract was a 15,000 X g supernatant from flight muscle, prepared as described in the text. The concentrations of reactants were 5 mM NaF, 1 mM ATP, 1 mM MgC12, or 5 mM EDTA, where indicated. p, conversion of phosphorylase b to a; - - -, conversion of phosphorylase a to b.

The comparison of the two enzymic activities represented the AMP-stimulated rates for both enzymes.

Each phosphorylase was then treated identically in subsequent purification steps. The extracts were chilled in an ice bath and the pH was adjusted to 4.8 by dropwise addition of 1.0 N

acetic acid. The preparations were centrifuged at 15,000 x g for 10 min, the supernatants were filtered through glass wool, and the pH was immediately readjusted to 7.0 with solid KHCOa. The extracts could not be allowed to remain at pH 4.8 for pro- longed periods because of the instability of phosphorylase at this pH. A large amount of protein was precipitated during this step, resulting in a 2-fold increase in specific activity with 100% recovery of phosphorylase activity (Table I).

The supernatant was fractionated by the dropwise addition of 0.43 volume of a room temperature-saturated solution of (NH&SO4 which had been adjusted to pH 7.0 with concen-

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2930 Insect Flight Muscle Phosphorylase Vol. 245, No. 11

trated NHIOH and filtered just before use. This 30% saturated solution was kept at 3” for 18 hours or longer. The precipitate was centrifuged and discarded. The (NH&SO4 concentration was then increased to 50% saturation by the addition of 0.57 volume of saturated solution. The enzyme preparation was again kept at 3” for 18 hours or longer to allow complete pre- cipitation. The 30 to 50% precipitate was harvested by cen- trifugation and redissolved in a minimal volume of buffer con- taining 1 mM Tris, 1 mM EDTA, 5 mu mercaptoethanol, and 5 mnir NaF, pH 8.0.

The clear enzymic solution was adjusted to pH 8.5 with 1.0 M

unneutralized Tris. The preparation was incubated at 41” for 1 hour, chilled to O”, and centrifuged to remove denatured pro- tein. The pH was then readjusted to 8.0 with 1.0 N acetic acid. The enzymic solution, containing 6.5 units per mg of protein, was stored at -20” and smaller batches were subjected to further purification as required. The specific activity slowly decreased during storage, but could be restored by repeating the heat treatment at pH 8.5 just prior to chromatography.

A lo-ml aliquot, containing 130 mg of protein and 850 units of activity, was passed through a column, 2 x 45 cm, of Sephadex G-25 (coarse) which had been equilibrated with 1 mu Tris, 1 mM EDTA, 5 mM mercaptoethanol, and 5 mM NaF, pH 8.0, to remove traces of (NH&Sod. The Sephadex G-25 eluate, 30 ml, was concentrated by ultrafiltration and placed on a column, 2 x 23 cm, of DEAE-cellulose which had been equilibrated in the same buffer. The column was eluted with 100 ml of the starting buffer and then a gradient was begun by passing 0.1 M Tris, 1 mM EDTA, 5 mu mercaptoethanol, and 5 mu NaF, pH 6.0, into a mixing reservoir containing 250 ml of the starting buffer. The eluate was collected at a rate of 0.5 ml per min. The elution pattern of an equal mixture of phosphorylase a and b is shown in Fig. 2. The enzyme was concentrated by ultra- filtration, centrifuged, and stored at -20” for further studies. The specific activities of the final product range from 12 to 15 units per mg of protein (Table I).

Usually the enzyme remained entirely in the a or b form throughout the purification procedure. Partial reversion to the b form to the extent of a few per cent did occur in a few preparations of phosphorylase a. In these instances, the earlier

j : .---.--.-ml.\.

.?” it ; \Phosphorylose”b”

- 80 - ,600 1.

5 ‘A j i

s, 20 -- ,500 “>. i. c

z 2aom,oqP~ + 1.i ::

b

f !2 ~:-‘i , \J, ~~~~~6,01

0 I00 200 300

I 400 500

Begin

gradlent MLEFFLUENT

FIG. 2. DEAE-cellulose chromatography of a phosphorylase solution containing a mixture of 50% each of purified phosphoryl- ases a and b. The sample contained 64 mg of protein and 415 units total activity. Elution was carried out as described in the text. Recovery of activity was 65%.

eluted fractions of phosphorylase a, which were contaminated with the b form of the enzyme (Fig. 2), were discarded.

Stability-In contrast to muscle phosphorylases from other species (19), the flight muscle enzyme was rapidly inactivated by storage at 3”. It was quite stable, however, when stored frozen at concentrations of 5 to 10 mg per ml in buffer containing 1 mM EDTA, 5 m&r mercaptoethanol, 5 mM NaF, and 50 mM Tris, pH 7.0. Under these conditions, the enzyme lost ap- proximately 5% of its total activity per month.

Homogeneity-Sedimentation velocity measurements on flight muscle phosphorylase b showed that the enzyme migrated as a single symmetrical boundary with an s20 value of 7.4. The sedimentation pattern for flight muscle phosphorylase a was identical with that of flight muscle phosphorylase b. The ~20 value of 7.4 was obtained whether the enzyme was purified in the a form or converted from b to a with phosphorylase b kinase and ATP just before sedimentation analyses. It is interesting to note1 that, although blowfly flight muscle phosphorylase b kinase converted flight muscle phosphorylase b to a without affecting the aso value, the insect kinase converted rabbit muscle phosphorylase b to the a form with a corresponding increase in the sedimentation coefficient from 8.4 to 13.5.

Electrophoresis of preparations on polyacrylamide gel showed that purified flight muscle phosphorylase b formed a single sharp band when stained for either protein or enzyme activity, also indicating a high degree of homogeneity.

Molecular Weight-Estimations of the molecular weight of purified flight muscle phosphorylase b were obtained by several methods. Plots of the data for the distribution of enzyme (0.1 mg of protein per ml) at equilibrium (16), at 10,000 rpm for 48 hours at lo”, in 0.1 M P-glycerophosphate (pH 7.1) in Hz0 or DZO were linear, again pointing to the homogeneity of the enzyme preparation. From the difference in the slopes in Hz0 and DzO buffers, the partial specific volume was calculated to be 0.735 ml per g. This compares to a value of 0.733 calculated from the amino acid composition by the method of McMeekin et al. (17). The results of several sedimentation equilibrium analyses, performed at temperatures ranging from 0” to 20”, indicated a molecular weight of approximately 97,000.

An estimation of molecular weight was also made, in crude preparations, by gel filtration (Fig. 3). Values of approximately 100,000 were obtained for the molecular weights of both flight muscle phosphorylase b and a. The diffusion coefficient, deter- mined by this method, was 6.3 x lo-’ cm2 per sec. The molec- ular weight calculated from this value and an sZo value of 7.4 was about 115,000.

In earlier preliminary experiments sedimentation coefficients of crude phosphorylase preparations were determined by sucrose density gradient centrifugation. By this technique an S value of approximately 8 was obtained for both the a and b forms of flight muscle phosphorylase, in reasonable agreement with the more precise measurements performed with homogeneous preparations by analytical ultracentrifugation.

Treatment with p-Chloromercuribenzoute-Rabbit muscle phos- phorylases a and b, when treated with low concentrations of CMB,2 dissociate into their monomeric subunits of molecular weight 90,000 (21). In view of the low molecular weight of the flight muscle phosphorylases, the effect of CMB on the insect

1 C. C. Childress and B. Sacktor, unpublished observations. 2 The abbreviation used is: CMB, p-chloromercuribenzoate.

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Issue of June 10, 1970 C. C. Childress and B. Sacktor 2931

- 256 $

- 218

75 m 10 30 100 300 1000

MOLECULAR WEIGHT x lo3

FIG. 3. Gel filtration of flight muscle phosphorylase and several standard proteins on a column, 1.5 X 90 cm, of Sepharose 6B. The standard proteins shown are: 1, cytochrome c; 2, chymotryp- sin; 3, bovine serum albumin; 4, flight muscle phosphorylase a or 6; 6, alcohol dehydrogenase; 6, rabbit muscle phosphorylase b; 7, pyruvic kinase; 8, rabbit muscle phosphorylase a. V. and BO represent elution and void volumes, respectively.

enzyme was determined. As a control, rabbit muscle phos- phorylase b was treated with 0.1 mu CMB; the sedimentation coefficient decreased from 8.4 to approximately 5, indicating a dissociation into smaller molecular species. Concentrations of CMB greater than 1 mu precipitated the enzyme. In contrast, when flight muscle phosphorylase ij was treated with CMB, no change in the sedimentation coefficient (7.4) was found, even at CMB concentrations as high as 10 mM.

Pyridoxal Phosphate Content-The pyridoxal phosphate con- tent of flight muscle phosphorylase was not measured directly because of the limited availability of pure enzyme. However, treatment of the enzyme from the blowfly with a solution con- taining imidazole, citrate, and I-cysteine, which removes pyri- doxal phosphate from the rabbit muscle enzyme (22), caused a rate of inactivation linear with time of exposure. The rate of inactivation was increased by higher temperatures or ATP, and was decreased by AMP. Following the complete inactiva- tion of the enzyme, the protein was passed through Sephadex G-25 to remove the imidazole-citrate-cysteine reagent. The enzyme could then be reactivated, in part, by the addition of pyridoxal phosphate. These results, although not definitive, suggest that flight muscle phosphorylase contains pyridoxal phosphate as a prosthetic group.

Amino Acid Composition-Table II shows an amino acid analysis of flight muscle phosphorylase b. In general, its com- position was similar to those of the phosphorylases of human, rabbit, and frog muscle (23, 24). However, striking differences between the flight muscle and the vertebrate muscle enzymes were found in the contents3 of half-cystine, 60 as compared to 9 to 12; arginine, 35 as compared to 57 to 70; and lysine, 66 as compared to 46 to 54, respectively.

pH Optima-The activity of flight muscle phosphorylases a and b at several pH values is shown in Fig. 4. Phosphorylase a had a somewhat sharper optimum at around pH 7.0 than did the b form of the enzyme.

Kinetic Properties of Phosphorylase a-It was found previously (3) that flight muscle phosphorylase a had a lower affinity for

a Values are expressed in per cent relative to the values of as- partate which were arbitrarily set at 100. Therefore, the differ- ences in amino acid compositions for flight muscle and the three vertebrate muscle enzymes which are found by this method of calculation are not due to differences in molecular weight.

TABLE II

Amino acid composition of flight muscle phosphorylase Calculated on the basis of a molecular weight of 100,000. The

values in parentheses give the amino acid compositions expressed in percentiles relative to the value of aspartate which was set arbitrarily at 100.

Amino acid I

Composition

Aspartic acid. Threonine

Serine . Glutamic acid. Proline.

Alanine. Methionine . Isoleucine. .

Leucine.. Tyrosine. . . Valine

Glycine. Lysine . . Histidine . . . .

Arginine . . Phenylalanine. . Tryptophan.. Half-cystine

. .

.

. ......... ......... ......... .........

Tt?SidUeS/~Ol~GUl~

86 (100) 30 (35) 27 (32) 82 (95)

33 (38) 57 (66) 21 (24)

46 (53) 68 (79) 34 (40) 45 (52)

49 (57) 57 (66) 14 (16)

30 (35) 26 (32)

9 (10) 52 (60)

I / I I I

"s.0 6.5 7.0 7.5 8.0 8.5 PH

FIG. 4. Activity of flight muscle phosphorylases as a function of pH. Reactant concentrations were 80 mM Pi, 1 mM AMP, and 2.5 mM glycogen (as end groups).

native flight muscle glycogen than for the same substrate fol- lowing its treatment with 1 N KOH for 30 min at 100”. The apparent K, values were 0.09 and 0.29 mu (as end groups) for the alkali-treated and native glycogens, respectively, in the presence of saturating levels of Pi and AMP. Values for ap- parent V,,, with native substrate were approximately 50% those obtained with alkali-treated or commercial shellfish gly- cogens. In addition, the enzyme was found to have differing affinities for Pi, depending upon which glycogen was present as cosubstrate (3). At saturating levels of AMP and glycogen, the apparent K,,, values for Pi were 4.5 and 9.5 mM, respec- tively, for KOH-treated and native glycogens. In view of these significant differences, all kinetic experiments in the present paper contained as substrate nat.ive glycogen extracted from flight muscle.

The relationship between rate of phosphorylase a activity

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2932 Insect Flight Muscle Phosphorylme Vol. 245, No. 11

and concentration of substrate, at several fixed levels of cosub- strate and saturating levels of AMP, is illustrated in Fig. 5. Double reciprocal plots resulted in a series of straight lines that intersected in the fourth quadrant, indicative of a sequential type of mechanism (25) in which increasing levels of either substrate enhance the binding of the other. The values of apparent K, for Pi ranged from 47 mrvr at 0.15 mM glycogen to 7.3 mM at 1.7 mM glycogen. The values of apparent K, for glycogen ranged from 6.4 mM at 1.6 mM Pi to 0.72 mM at 49.3 !nM Pi.

Although not required for activity, low levels of AMP stimu- lated phosphorylase a 2- to S-fold at saturating levels of sub- strates and lo-fold or higher at very low substrate concentrations. A value of 0.59 PM for the apparent K, of AMP at saturating levels of both Pi (80 mM) and glycogen (1.8 mM) was obtained. Lowering the level of either substrate decreased the affinity of the enzyme for the activator; the apparent K, value of AMP was increased to 1.2 PM and 2.8 pM when glycogen or Pi was lowered to 0.18 InM and 3 mM, respectively.

AMP enhanced the affinity of phosphorylase a for both sub- strates. The apparent K, of glycogen was decreased to ap- proximately one-third by excess AMP (Fig. 6). The effect of

0 I 2 3 4 5 6 7

1 [Glycogeo] x 1t13M End Groups

FIG. 5. Reciprocals of initial

’ Molar m velocity of phosphorylase a ac-

tivity as a function of reciprocals of concentration of substrate, at several fixed levels of cosubstrate. The AMP concentration was 1.6 mM. The values of apparent K,,, for Pi were, respectively, 47.6,23.7,16.3,11.2, and 7.3 mM in order of the lowest to the highest glycogen concentration. The values of apparent K, for glycogen were, respectively, 6.4, 4.5, 2.6, 1.7, and 0.7 mM in order of the lowest to the highest Pi concentration.

0 200 400 600

1 [Giycogen]

x IO3 M End Groups &j Molar

FIQ. 6. Double reciprocal plots of initial velocity of phosphoryl- ase a as a function of glycogen or Pi concentration at several fixed levels of AMP. The Pi or glycogen concentration in the respec- tive experiments was 80 or 1.7 mM. The values of apparent K,,, for glycogen were, respectively, 0.88, 0.73, 0.58, 0.28, and 0.27 mM in order of the lowest to the highest AMP concentration. The values of apparent Km for Pi were, respectively, 100, 20, 9, and 9 IIIM in order of the lowest to the highest AMP concentration.

AMP on the apparent K, of Pi was much more marked, de- creasing from 100 mu at 0.3 pM AMP to 9 mM in the presence of 16 pM AMP. At low levels of AMP, near the apparent K, for activation, the kinetic relationships became nonlinear. Double reciprocal plots of rate and Pi concentration were concave up, typical of sigmoidally shaped substrate-velocity curves. The sigmoidal character of these substrate-velocity plots was even more pronounced at low levels of glycogen. The presence of saturating levels of glycogen partially reversed the nonlinearity, while saturating levels of AMP seemingly completely eliminated the sigmoidal shape of the curves.

The observations that at low levels of AMP the saturation curves for Pi became sigmoidal might be interpreted on the basis of the model of Monod, Wyman, and Changeux (26). High concentrations of Pi or low levels of Pi in the presence of AMP would effect a shift from a less to a more active state. Inter- action coefficients, 12, were calculated from the Hill equation (27), shown in Fig. 7. At high levels of AMP, a condition in which double reciprocal plots were linear (Fig. 6), the slope was approximately equal to unity. However, at low AMP con- centrations, n was 1.9.

Double reciprocal plots of velocity and glycogen concentra- tion, at low levels of AMP and Pi, were also nonlinear (Fig. 8). However, in this case the plots were concave down. This sug- gests that increasing the glycogen concentration in a situation

I I I 1.0 10.0 100.0

[Pd mM

FIG. 7. Hill equation plots showing the effect of AMP on the activit,y of phosphorylase a at various concentrations of Pi.

0 12 3 4 5 6 7

FIG. 8. Double reciprocal plots of initial velocity of phos- phorylase a as a function of glycogen concentration at several fixed levels of Pi and AMP.

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Issue of June 10, 1970 C. C. Childress and B. Sac&or 2933

in which both AMP and Pi are limited may favor a shift to a pathway or state having higher activity.

Kinetic Properties of Phosphorylase &In contrast to phos- phorylase a, double reciprocal plots of rate of phosphorylase b activity and substrate concentration were nonlinear at all levels of cosubstrate and AMP. However, the nonlinear character was much more pronounced at low levels of cosubstrate or AMP (Figs. 9 and 10). As in the case of phosphorylase a, increasing levels of one substrate enhanced the binding of the other to phosphorylase b (Fig. 9). Values for apparent K, for Pi ranged from 12 mM at 0.34 mM glycogen to 5 mM at 2 mM

glycogen. Values for apparent K, for glycogen ranged from 0.8 mM at 4.8 mM Pi t0 0.2 mM at 40.3 mM Pi.

Unlike the a form of the enzyme, phosphorylase b had an absolute requirement for AMP. Furthermore, levels of AMP loo-fold greater than those that stimulated phosphorylase a were required to activate the b form. Increasing amounts of AMP lowered the apparent K, values for both Pi and glycogen, as shown in Fig. 10. The apparent K, values for Pi ranged from 100 mM at the lowest to 5 InM at the highest level of AMP. The apparent K, values for glycogen ranged from 0.9 mM at the lowest to 0.2 mM at the highest level of AMP. Lineweaver- Burk plots of rate and glycogen concentration were concave down at low levels of AMP, as was seen with the a form of the enzyme. However, in the case of the b form, high levels of Pi did not eliminate the nonlinear pattern.

Double reciprocal plots of velocity and AMP concentration (Fig. 11) showed that increasing levels of either substrate lowered the apparent K, value for AMP. The values for AMP de- creased from 2 mM to 0.2 mM as the Pi concentration increased

0001 oo= 11 I II 0 1 2 3 4 5 6 0 200 400 600

I -x IO3 M End Groups [W]

h Molar

FIG. 9. Double reciprocal plots of initial velocity of phosphoryl- ase b as a function of Pi or glycogen concentration at several fixed levels of cosubstrate. The AMP concentration was 0.96 mM.

1.00

0 25

1 x lo3 M End GWQS Fwl

FIG. 10. Double reciprocal plots of initial velocity of phos- phorylase b as a function of substrate concentration at several levels of AMP. The concentrations of the fixed cosubstrate were 2 InM glycogen and 40.3 mM Pi in the two respective experiments.

from 4.8 mM to 40.3 InM. The apparent K, for AMP ranged from 0.5 mM to 0.2 mM as the glycogen concentration increased from 0.34 mM to 2.0 mivr.

Inhibition of Phosphorylase 6 by ATP-ATP was found to be a potent inhibitor of phosphorylase b but not of phosphorylase a (Fig. 12). The Ki value was approximately 2 mM. At high AMP concentrations the inhibition by ATP was less, perhaps suggesting competitive inhibition with respect to AMP, as had been previously reported for mammalian skeletal muscle phos- phorylase (23, 28). This view is strengthened by the observa- tions, shown in Fig. 13, that ATP increased the apparent K,

FIG. 11. Double reciprocal plots of initial velocity of phos- phorylase b as a function of AMP concentration at several levels of substrates. The cosubstrate concentrations were fixed at 2 mM glycogen and 40.3 mM Pi in the two experiments, respectively.

FIG. 12. Double reciprocal plots showing the effect of ATP on the initial activity of phosphorylase b as a function of AMP con- centration. Substrate concentrations were 16 mM Pi and 2 mM glycogen.

2.5 1’” 5-

x IO3 M End Groups

FIG. 13. Double reciprocal plots showing the effect of ATP on the initial activity of phosphorylase b as a function of substrate concentration. The concentration of AMP was 0.32 mM. The concentration of the cosubstrate was 2 mM glycogen or 16 mM Pi in the two experiments, respectively.

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2934 Insect Flight Muscle Phosphorylase Vol. 245, No. 11

values for both substrates, as if AMP had been displaced from its binding site on the enzyme.

Examination of the effect of ATP on phosphorylase a was complicated by the finding that commercial ATP preparations were contaminated with trace quantities of AMP. Since the a form of the enzyme was activated by AMP concentrations as low as 0.5 PM, it was impossible to add ATP at the levels found in viva (8) without simultaneously adding saturating amounts of AMP. In order to circumvent this difficulty, the ATP used

in these experiments was treated with adenylic acid deaminase to convert contaminating AMP to IMP. When this was done, no effect of ATP upon phosphorylase a could be detected.

Several other nucleoside triphosphates were tested for in- hibitory effects on both the b and a forms of phosphorylase, Fig. 14 shows that all of the nucleotides except UTP were strongly inhibitory to phosphorylase b. ATP was the most potent inhibitor, however. None of the nucleotides inhibited phos- phorylase a.

Several other metabolites, including arginine phosphate, glucose-6-P, and trehalose, at the concentrations found in flight muscle (2, S), were without effect on both phosphorylase b and a.

Phosphorylase Activities under Ximuluted Conditions of Rest and Flight-The kinetic studies on the effects of substrates, activator, and inhibitor on flight muscle phosphorylases, as described above, plus previous findings (2, 8) on the concen- trations of these metabolites in the muscle during rest and flight, permitted estimates of the phosphorylase activities under sim- ulated conditions in viva. As shown in Table III, the level of glycogen in the muscle would be sufficient to saturate the en- zymes, except after 10 min or more of flight when the muscle glycogen reserve was nearly depleted (2). The concentration of Pi in the muscle at rest was 7.0 mu, increasing to 7.5 mM during flight. On the other hand, the concentration of AMP

in the muscle was increased sharply from 0.1 mu to 0.3 mM in the rest to flight transition. However, examination of the apparent K, values for Pi and AMP revealed that each ligand had a marked effect upon the affinity of the enzyme for the other in these concentration ranges. In the case of phosphorylase b, the apparent K, for Pi at 0.1 mu AMP was about 100 mu, very much above the level found in the flight muscle, in viva. The apparent K, of phosphorylase b for AMP at 8 mM Pi was

0 32 64 96 [AMP] mM

FIG. 14. The effect of nucleoside triphosphates on the initial velocity of phosphorylase b as a function of AMP concentration. Substrate concentrations were 40 mM Pi and 1.7 mM glycogen. The concentration of inhibitor was 16 mM.

about 1.0 mM, about lo-fold that in the muscle, at rest. These observations, alone, would indicate that the activity of the b form was limited to only a trace of its potential activity at sub- strate saturation. The strong inhibition of ATP at the low concentration of AMP would further decrease the activity of

phosphorylase 6 to practically zero under these conditions. In contrast, for phosphorylase a, the concentration of AMP

TABLE III

Comparison of metabolite levels and apparent K, values for jlight

muscle phosphorylases under conditions in vivo

Metabolite levels, measured previously (2, S), are reported as micromoles per g, wet weight, of thorax. These are probably

minimum values for the metabolite concentrations in the muscle. If one assumes that the flight muscle represents half of the wet weight of the thorax and the chitinous exoskeleton accounts for

the other half of the thoracic weight but contributes nothing to the metabolite content, maximal values double those given are obtained. The true values are in between the two extremes but

closer to those reported in the table. The differences between use of the minimal or maximal values in calculating the potential activity at simulated conditions are essentially insignificant.

-

I

Metabolite Level

during flight in vim

Apparent Km w&lea

Potential activity” at simulated conditions of

Rest 1 Flight

Glycogen. Pi

AMP...... ATP. _...,

?nM

6-9 6 --) 0.7P

7.0 7.5 0.1 0.3 7.0 6.5

b (r b a 6 a I I I

?nM %

0.6 1.7 100 8

21(.ii)i 0.001 /nil/ 5O/nili 50

a The apparent K,,, values were determined from the kinetic

data with the concentrations of cosubstrate and activator, as found in vivo, at rest.

b Activities are expressed as the percentage of the potential phosphorylase activity with saturating conditions of substrate and AMP, without ATP.

c The level of glycogen in the muscle in vivo decreased steadily during flight to a value of 0.75 mM after 10 min of flight. It re-

mained steady at this exhausted level throughout the remainder of the flight (2). A glycogen concentration of 1% corresponds to

5.9 mM end groups.

TABLE IV

Relative amounts of phosphorylases a and 6 in vivo at rest and during sight

The figures shown represent mean values and their standard

deviations. The number of extracts, each consisting of five thoraces, is shown in parentheses.

state Phosphorylase in the cz form

% total Resting-unmounted

Mounted-rested. Flown 5 sec. Flown 15 sec.

Flown 30 sec. . Flown 60 sec. Flown 10 min..

17.8 f 3.6 (17)

34.3 z!r 8.5 (27) 63.9 f 12.3 (16) 69.1 f 13.4 (16) 72.2 f 12.3 (10)

71.5 f 14.2 (10) 72.1 f 13.1 (10)

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Issue of June 10, 1970 C. C. Childress and B. Xacktor 2935

in the muscle was loo-fold the apparent K, for the nucleotide (Table III). In addition, ATP did not inhibit the a form of the enzyme. The apparent K m of phosphorylase u for Pi, with simulated conditions in U~UO, was 8 mrvr, approximating the concentration found in the muscle. Thus, about 50% of the potential activity of phosphorylase a would be found and gly-

cogenolysis by phosphorylase a would be moderately dependent on small changes in the level of Pi.

Based on the specific activity for flight muscle phosphorylase of 9.6 units per g, wet weight, of thorax, when assayed with native glycogen at 30” (3), and the knowledge that the potential activity of phosphorylase was the same whether in the a or the b form, it can be predicted from the kinetic data that 50% of the total phosphorylase must be in the a form in order to account for the rate of glycogen breakdown that occurs during flight, 2.4 pmoles of glycosyl residues per min per g, wet weight, of thorax (2).

Phosphoryluse a and b in Flight Muscle during Flight-Reliable determinations of the relative amounts of phosphorylases a and b in the flight muscle in viva proved to be difficult unless the procedures, described above, were adhered to strictly. As shown in Table IV, “control” flies had a phosphorylase a content of 18%. The procedure of mounting the f ly for flight, in itself, agitated the fly sufficiently to raise the level of the a form of the enzyme to over 50% of the total. For this reason, all flies were mounted and rested for 2 hours before flight was initiated, al- though they continued to struggle throughout this period. By this time, however, the percentage of total phosphorylase in the a form decreased to 34%. Initiation of flight induced an im- mediate increase in the relative amount of the enzyme in the a form. The level of phosphorylase a reached a maximum of about 70% at 15 set of flight and remained constant during flight. The total amount of phosphorylase (a +- b) did not change. This level of phosphorylase a was more than adequate to account for the observed rate of glycogenolysis during flight. From the kinetic data obtained in vitro, a 70% level of phos- phorylase a would catalyze a rate of glycogen breakdown of 4.3 pmoles of glycosyl residue per min per g, wet weight, as com- pared to a rate of 2.4 pmoles actually measured during flight (2). If, in “control” flies, a value of 18% of the total phos- phorylase in the a form was too high because of technical diffi- culties in extracting the phosphorylases in their state in situ,

and if these factors increased the amount of phosphorylase a in the flown fly to the same extent, the agreement between the predicted rate of glycogenolysis from the studies in vitro and that in the flying insect would be even more exact.

DISCUSSION

The present study established that the activity of the phos- phorylases in insect flight muscle was sufficient to account for the exceptional rate of glycogenolysis in the muscle during flight (2). Approximately 1.5% of the total muscle protein was estimated to be phosphorylase.

The molecular weight of phosphorylase b was estimated to be about 100,000, determinations, by several techniques, ranging between 97,000 and 115,000. Conversion of the flight muscle enzyme from the b to a form did not change the sedimentation coefficient (7.4), indicating that the molecular weights of the two enzymes are the same. Flight muscle phosphorylase b was not dissociated into subunits by dilute CMB. Other findings also suggested that phosphorylase from insect flight

muscle is unique. Differences in amino acid composition be- tween the insect and vertebrate muscle enzymes were par- ticularly striking.

Because of the dependence of kinetic constants upon the levels of cosubstrate and AMP, it is difficult to compare precisely the results of the present study with those reported previously for phosphorylases from other species. Nevertheless, a general qualitative comparison with other studies can be made. In the present study, nonlinear Lineweaver-Burk plots were found with phosphorylase a at low levels of cosubstrate and AMP. This phenomenon has not been observed with phosphorylase a from other sources, although it is well known with the b form of the enzyme (29).

The apparent K, for Pi was somewhat higher for flight muscle phosphorylase a than for the same enzyme from frog muscle (23), rabbit muscle (30), or brain (29). An even greater differ- ence was found in the apparent K, values for glycogen. Insect flight muscle phosphorylase a had a much higher apparent K, for native glycogen (3), which was used in the kinetic studies reported here, than for alkali-treated glycogen, which was used in most of the studies reported previously. However, even when alkali-extracted glycogen was tested with the flight muscle enzyme, the apparent K, was nearly IO-fold higher than the corresponding value for phosphorylase a of rabbit muscle (30) or brain (29). The enzyme from frog muscle (23) had an ap- parent K, for glycogen very nearly the same as the value ob- tained for the flight muscle enzyme.

The responses of phosphorylase a of flight muscle, frog muscle, rabbit muscle, and brain to the allosteric modifier AMP were similar. With all, AMP decreased the apparent K, values with only a small change in apparent V,,,. At saturating levels of glycogen and Pi, AMP stimulated the rabbit or frog muscle enzyme by 20 to 30%. The flight muscle phosphorylase a was stimulated 2- to 3-fold under these same conditions. However, the response of phosphorylase a from all tissues was modified by substrates. At low levels of substrates, the degree of stimu- lation was considerably increased. It was also evident that substrate or activator could substitute for each other, at least to some extent, since at low concentrations of one substrate the effect of AMP on the affinity of the enzyme for the cosubstrate was much greater than at high concentrations of substrate.

The strong inhibition of phosphorylase b by ATP and the high apparent K, for Pi at low levels of AMP appear to be the major factors regulating glycogenolysis in insect flight muscle. The high concentration of ATP and the low concentration of AMP in the muscle would severely limit the activity of phos- phorylase b in viva. On the other hand, phosphorylase a, un- affected by ATP, and with a lower apparent K, for Pi at a saturating level of AMP, would retain 50% of its potential activity under these conditions. Accordingly, the activity of phosphorylase b would be far too low to account for the rate of glycogenolysis during flight. The activity of phosphorylase a would be adequate, however, if at least 50% of the total enzyme was in the a form. Actual measurements of the relative amounts of phosphorylase a and b at rest and during flight showed that the conversion of the b to the a form was of this order of mag- nitude. Thus, in the blowfly flight muscle the mechanism for controlling the intense rate of glycogenolysis concomitant with the initiation of flight is the conversion of phosphorylase b to phosphorylase a. The factors that regulate the activity of

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2936 Insect Flight Muscle Phosphorylase Vol. 245, No. 11

flight muscle phosphorylase b kinase are currently being ex-

amined.

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Charles C. Childress and Bertram SacktorAND PROPERTIES OF PHOSPHORYLASES IN VITRO AND IN VIVO

Regulation of Glycogen Metabolism in Insect Flight Muscle: PURIFICATION

1970, 245:2927-2936.J. Biol. Chem. 

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