Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

Transcription

1 J Cmp Physil B (1993) 163: Jurnal f Cmparative ~~..m,., SyslrnJc, and Envl[n- Physilgy 9 Springer-Verlag 1993 B w.,., Physilgy Purificatin and characterizatin f glycgen phsphrylase A and B frm the freeze-aviding gall mth larvae Epiblema scudderiana C.P. Hlden, K.B. Strey Departments f Bilgy and Chemistry, Carletn University, Ottawa, Ontari, Canada K1S 5B6 Accepted: 2 July 1993 Abstract. The active a and inactive b frms f glycgen phsphrylase frm cld-hardy larvae f the gall mth, Epiblema scudderiana, were purified using DEAE + in exchange and 3'-5'-AMP-agarse affinity chrmatgraphy. Maximum activities fr glycgen phsphrylases a and b were 6.3_+0.74 and 2.7 _ gml glucse-l- P- rain- t. g wet weight- 1, respectively, in - 4 ~ mated larvae. Final specific activities f the purified enzymes were 396 and 82 units.mg prtein-i, respectively. Bth enzymes were dimers with native mlecular weights f _18000 fr glycgen phsphrylase a and _ fr glycgen phsphrylase b; the subunit mlecular weight f bth frms was 87000_ Bth enzymes shwed ph ptima f 7.5 at 22 ~ and a break in the Arrhenius relatinship with a tw- t furfld increase in activatin energy belw 10 ~ Michaelis cnstant values fr glycgen at 22~ were 0.12 _ rag. ml- t fr glycgen phsphrylase a and 0.87 _ rag. ml- 1 fr glycgen phsphrylase b; the Michaelis cnstant fr inrganic phsphate was 6.5_+0.07mm1.1-1 fr glycgen phsphrylase a and 23.6 retl.1 1 fr glycgen phsphrylase b. Glycgen phsphrylase b was activated by adensine mnphsphate with a K, f _ mm Michaelis cnstant and K, values decreased by tw- t fivefld at 5 ~ cmpared with 22 ~ Glycerl had a psitive effect n the Michaelis cnstant fr glycgen fr glycgen phsphrylase a at intermediate cncentratins (0.5 ml-1-t) but was inhibitry t bth enzyme frms at high cncentratins (2 tl. 1-1). Glycerl prductin as a cryprtectant in E. scudderiana larvae is facilitated by the lw temperature-simulated glycgen phsphrylase b Abbreviatins: Ea, activatin energy; GPa, glycgen phsphrylase a; GPb, glycgen phsphrylase b; h, Hill cefficient; Is, cncentratin f inhibitr that reduces enzymes velcity by 50%; Ka, cncentratin f activatr that prduces half-maximal activatin f enzyme activity; K m, Miehaelis-Menten substrate affinity cnstant; MW, mlecular weight; PEG, plyethylene glycl; Pi, inrganic phsphate; SDS PAGE, sdium ddecyl sulphate plyacrylamide gel electrphresis; Vmax, enzyme maximal velcity Crrespndence t: K.B. Strey t glycgen phsphrylase a cnversin and by psitive effects f lw temperature n the kinetic prperties f glycgen phsphrylase a. Enzyme shut-dwn when plyl synthesis is cmplete appears t be aided by strng inhibitry effects f glycerl and KC1 n glycgen phsphrylase b. Key wrds: Cryprtectant synthesis - Insect cld hardiness - Glycerl metablism - Regulatin f glycgenlysis - Gall mth, Epibtema Intrductin Larvae f the gall mth Epiblema scudderiana Clemens (Lepidptera: Olethreutidae) use the freeze-avidance strategy f cld hardiness t verwinter inside galls n gldenrd stems. During autumn cld-hardening the larvae accumulate glycerl as a cryprtectant in quantities as high as 2 ml.1-1 r mre in bdy fluids, abut 19% f ttal bdy mass, and this helps t push the supercling pint f the larvae t -38 ~ (Rickards et al. 1987). The bisynthesis f glycerl utilizes as its substrate the large reserves f glycgen accumulated by the larvae during summer feeding (Rickards et al. 1987). Cryprtectant synthesis takes place in the fat bdy f cldhardy insects (Hayakawa and Chin 1981; Shimada 1982; Yi et al. 1987; Strey and Strey 1991) and is primarily regulated via cntrl ver the activity f the enzyme glycgen phsphrylase (Ziegler etal. 1979; Hayakawa 1985). Glycgen phsphrylase exists in tw frms, an active phsphrylated a frm (GPa) and an inactive (r subactive) dephsphrylated b frm (GPb). Intercnversin f GPa and GPb via phsphrylase kinase and phsphrylase phsphatase is the majr mechanism fr changing the activity state f the enzyme, and these enzymes are themselves part f a phsphrylatin cascade that is regulated by the actins f prte~n kinases and

2 500 C.P. Hlden, K.B. Strey: Insect glycgen phsphrytase secnd messengers (Newgard et al. 1989; Meinke and Edstrum 1991). In cld-hardy insects the activities f GP kinase and GP phsphatase are differentially sensitive t temperature change with the result that lw temperature expsure (0-5 ~ causes a rapid net increase in GPa activity and thereby initiates glycgenlysis t fuel cryprtectant synthesis (Hayakawa 1985). In E. scudderiana the percentage f ttal phsphrylase in the a frm remains high thrughut the perid f active glycerl bisynthesis and then falls t lw levels nce maximal cryprtectant levels have been reached (Churchill and Strey 1989a). In additin t the increase in enzyme maximal activity brught abut by the GPb t GPa cnversin at lw temperature, the kinetic r regulatry prperties f E. scudderiana glycgen phsphrylase may be specifically adapted t prmte high rates f glycgenlysis at lw temperatures and enzyme functin may be sensitive t the extremely high cncentratins f glycerl that build up as the end prduct f glycgenlysis. In the present study GPa and GPb frm E. scudderiana larvae are purified, then characterized accrding t physical and kinetic prperties. Temperature, ph, glycerl, and salt effects n the enzymes are analyzed t prduce a full picture f phsphrylase cntrl during plyl bisynthesis. Materials and methds Animals and chemicals. Spindle-shaped gldenrd galls cntaining larvae f E. seudderiana were cllected in the autumn f Galls were acclimated fr 3 weeks in a labratry incubatr at either 15 r -4 ~ These temperatures were chsen because cryprtectant bisynthesis by the larvae des nt ccur at 15 ~ but glycerl builds up rapidly at -4 ~ (Rickards et al. 1987). After acclimatin, galls were quickly pened and the larvae were remved, frzen in liquid N2 and then stred at -80 ~ until use. Bichemicals were purchased frm Behringer-Mannheim Crp., Mntreal, P.Q., Canada, r Sigma Chemical Cmpany St. Luis M., USA. Sephacryl S-300 was frm Pharmacia Fine Chemicals, Uppsala, Sweden. Prtein cncentratins were determined by the methd f Bradfrd (1976) using the Bi-Rad Labratries prepared reagent and a standard f bvine gamma glbulin. Assay fphsphrylase a and b. Phsphrylase was assayed essentially as described by Mrishima and Uen (1990) using a Gilfrd 260 spectrphtmeter with a water-jacketed cell hlder fr cntrl f cuvette temperature and mnitring NADPH prductin at 340 nm. Optimal assay cnditins fr GPa were 50 mml.1-1 KH2PO4 buffer (ph adjusted t 7.2 at 22 ~ 4 mg.ml-1 yster glycgen, 15 mml.1 1 MgSO4, 5 Ixml-1 -~ glucse 1,6-bisphsphate, and 0.2 retl-1-1 NADP + with 1 U glucse-6-phsphate dehydrgenase and I U phsphglucmutase in a ttal vlume f I ml. Fr ttal phsphrylase activity r the assay f purified GPb, the assay further cntained 1 mml.l-t AMP; GPb activity in a mixture f the tw frms was determined as ttal activity minus GPa. One unit f phsphrylase activity is defined as the amunt f enzyme that utilizes 1 gml NADP +" min-~ at 22 ~ Optimal yield f phsphrylase a r b frms. Prir t enzyme purificatin, tests were carried ut t determine the animal surce (15 r -4 ~ larvae) and the hmgenizatin buffer that wuld prduce the highest yield f either GPa r GPb. Samples f frzen larvae (apprx. 0.2 g, generally five larvae) were hmgenized 1:5 w/v using a Plytrn hmgenizer. One f fur ice-cld >~ 2.0 ' k B O.B "~ ~) 0 I Fractin Number Fig. l. DEAE-Sephadex clumn chrmatgraphy f E. scudderiana GPa and GPb. 9 GPa; 9 GPb. Velcity is in units-ml 1-mg prtein -~ with Vm,x values f 1.98 and 1.64 U.ml-~'mg prtein -~ fr GPa and GPb, respectively. Data are means f n = 3 determinatins n separate preparatins f enzyme; SEM bars are within the size f symbls used buffers was used; all cntained 15 mml-1 1 imidazle-hc1 and 15 mml-1 ~ 2-mercaptethanl and were adjusted t ph 7.2 at 22 ~ (Buffer D). Buffer A was designed t inhibit bth prtein kinases and prtein phsphatases and cntained Buffer D plus 50 mml-t 1 NaF, 5 mml-i 1 EDTA and 5 mml-1-1 EGTA. Buffer B prmted enzyme phsphrylatin and cntained Buffer D plus 50mm1"1-1 NaF, lmml'l-1 Mg2+ and 2mml.l-1 ATP. Buffer C prmted enzyme dephsphrylatin and cntained Buffer D plus 2 mml.l- ~ Mg 2+. Hmgenates were nt centrifuged but were allwed t settle fr 1 h n ice fllwed by assay f enzyme activity in the supernatant. Purificatin f glycgen phsphrylase a and b. Optimizatin tests (abve) shwed that highest GPa activity culd be btained frm -4 ~ larvae hmgenized in buffer B. Fr purificatin frzen larvae (apprx. 1 g r 20 larvae) were hmgenized 1:5 w/v in buffer B and the hmgenate was allwed t settle n ice fr 1 h. The hmgenate was then centrifuged at g in an Eppendrf micrcentrifuge fr 5 rain. The pellet was discarded and the supernatant was fractinated using plyethylene glycl 8000 (PEG). Slid PEG was added t the supernatant t a final cncentratin f 2% w/v, mixed gently with a Lab Quake test tube inverter fr 20 min at 22 ~ and then centrifuged at g fr 10 rain in a Srvall RC-5B refrigerated centrifuge at 5 ~ The pellet was discarded and further PEG was added t the supernatant t adjust the cncentratin t 6% w/v. After mixing and centrifuging as abve, the pellet cntaining GPa was resuspended in 0.5 ml buffer D and then dialyzed fr 2 h against 5 mml. 1-1 imidazle buffer, ph 7.2 (buffer E). The enzyme was then applied t a clumn (7 cm 1.5 cm) f DEAE-Sephadex equilibrated in buffer E. The clumn was washed with 30 ml f buffer E and then the enzyme was elnted with a linear gradient f 0-1 ml-1-1 KC1 in 40 ml f buffer E; 1 ml fractins were cllected and assayed fr enzyme activity. This clumn readily separates GPa and GPb activity (Fig. 1); fractins with high GPa activity and minimal r n GBb activity were pled, placed in dialysis tubing, and cncentrated against slid sucrse t a final vlume f apprximately 2 ml. The preparatin was then dialyzed against 1 1 buffer E fr 2 h. The enzyme was then applied t a clumn (2 cmx 1 cm) f 3',5'-cyclic AMP agarse (Sigma) equilibrated in buffer E. The clumn was washed with 30 ml f buffer E and then enzyme was eluted with a linear gradient f ~25 mml.1-1 AMP in 40 ml

3 C.P. Hlden, K.B. Strey:Insect glycgen phsphrylase 501 buffer E; 1 ml fractins were cllected and assayed. The enzyme eluted in a single peak (with a maxima at 8.5 retl.1-1 AMP) and peak fractins were pled, cncentrated against slid sucrse t a final vlume f 4 ml, and then dialyzed fr 2 h against buffer E. After additin f glycerl (10% v/v) the purified enzyme was stable fr at least 2 weeks when stred at 4 ~ The purificatin scheme fr GBb fllwed the same steps except that the starting material was 15 ~ larvae, hmgenizatin f larvae was in buffer A, and peak fractins f GBb were saved frm the DEAE clumn. Enzyme kinetics. Substrate affinity cnstants (Kin) and Hill cefficients (h) were determined using Hill plts. K, values fr AMP fr GBb were calculated frm plts f enzyme activity (at subptimal glycgen and Pi cncentratins) versus increasing AMP cncentratins. I5 values (the cncentratin f inhibitr that reduced Vm,x by 50%) fr KC1 and glycerl were determined frm plts f activity versus [inhibitr]. Kinetics were assessed at bth 5 and 22 ~ Fr ph curves the buffer used was a mixture f 15 mml. 1-1 imidazle+20mml'1-1 KHzPO4; ph was adjusted t 7.2 at 22 ~ and ph at 5 ~ was calculated based n a ph unit increase per 1 ~ decrease in temperature fr imidazle. Data are means_+ SEM fr n = 3-5 separate preparatins f purified enzyme. Statistical testing used the Student's t-test. SDS-PAGE. SDS-PAGE was perfrmed n purified GPa and GBb samples as in Laemmli (1970) using a 10% w/v acrylamide gel. Subunit MW standards were rabbit muscle glycgen phsphrylase b (MW 97400), bvine serum albumin (66000), valbumin (45000), glyceraldehyde 3-phsphate dehydrgenase (36000), bvine erythrcyte carbnic anhydrase (29000), and bvine c~-lactalbumin (14200). Prtein was stained with 0.25% w/v Cmassie brilliant blue R. The subunit MW was determined frm a plt f Rf versus lg MW f the prtein standards. MW determinatin. The native MWs f GPa and GPb were determined by gel filtratin n Sephacryl S-300 (40 x 0.5 cm). The clumn was equilibrated and develped in 50retl.1 -x KH2PO 4 buffer cntaining 15retl.1 -a 2-mercaptethanl, 0.1% w/v NAN3, 10% v/v glycerl, ph 7.2. The clumn was calibrated with rabbit muscle phsphfructkinase (MW ), rabbit muscle aldlase (160000), rabbit liver fructse 1,6-bisphsphatase (140000), bvine bld hemglbin (64500), and bvine heart cytchrme c xidase (13370). MWs f GPa and GPb were determined frm a plt f Ka versus lg MW f the standards. Results Optimizatin f GPa and GPb yield Crude hmgenates f the larvae cntain bth phsphrylase kinase and phsphrylase phsphatase and by manipulating the cntents f the hmgenizatin buffer (+_ kinase inhibitrs, _ phsphatase inhibitrs) we aimed t ptimize the available starting material befre beginning purificatin f either GPa r GPb. Acclimatin state f the larvae als strngly influenced GPa and GPb cntents. Thus, when hmgenized in buffer A (cntaining bth kinase and phsphatase inhibitrs) the rati GPa: GPb was 9 : 91 fr 15 ~ larvae and 72:28 fr -4 ~ larvae, a result in line with the knwn effect f lw temperature in stimulating phsphrylase activatin and plyl synthesis in cldhardy insects (Ziegler etal. 1979; Hayakawa 1985; Churchill and Strey 1989a). Maximal activity f GPa in -4 ~ larvae was gml glucse- 1 -P-min - 1. g- 1 wet weight cmpared with 2.7 _ 0.87 U. g-1 fr GPb. Hmgenizatin and incubatin in buffer B (prmting enzyme phsphrylatin) increased the percentage f GPa t 68 and 81% fr 15 and -4~ larvae, respectively, whereas incubatin in buffer C (prmting dephsphrylatin) had the ppsite effect and %GPa fell t 7 and 47%, respectively. Final chices fr ptimizing the cntent f either the a r the b frm fr purificatin were -4 ~ larvae hmgenized in buffer B fr GPa and 15 ~ larvae in buffer A fr GPb (%GPb was slightly higher in buffer C but ttal units f activity was reduced cmpared t buffer A). By manipulating enzyme activity in the crude hmgenates prir t purificatin thrugh the use f endgenus kinases/ phsphatases we avided the need t treat with cmmercial kinases/phsphatases t achieve a high purity f the a r b frm, as has been dne fr mammalian phsphrylase (Fischer and Krebs 1959). Purificatin f glycgen phsphrylase a and b. Table 1 shws the purificatin f the a and b frms f glycgen phsphrylase frm E. scudderiana. The purificatin scheme fr each enzyme was essentially the same, differ- Table 1. Purificatin scheme fr Epiblema scudderiana glycgen phsphrylase Step Ttal activity Ttal prtein Yield Fld Specific activity (units) (mg) (%) Purificatin (U-mg- 1) Glycgen phsphrylase a Crude PEG, 2-6% DEAE-Sephadex AMP-Agarse Glycgen phsphrylase b ' Crude PEG, 2-6% DEAE-Sephadex AMP-Agarse

4 502 C.P. Hlden, K.B. Strey: Insect glycgen phsphrylase 6.5 ' I ' I ' I ' I ' I ' I ' I ' I ' I 2.0 i ' I ' I ' I i/i 6, ~: J f, I, I, I, I, I, I, I, I, I O g Peak Tube Number Fig. 2. Native mlecular weight f E. scudderiana GPa and OPb determined by Sephacryl S-300 gel filtratin. 9 GPa; 9 GPb; Standards (): 1 blue dextran, 2 phsphfructkinase, 3 aldlase, 4 fructse 1,6-bisphsphatase, 5 hemglbin, 6 cytchrme c xidase II > 0.8 A //---.-\,, " ph n I I \ i ing in the initial hmgenizatin buffer used t maximize a r b cntent and in which activity peak was saved frm the DEAE-Sephadex chrmatgraphy (Fig. 1). GPa was purified 1250-fld t a final specific activity f 396 U'mg- a prtein, whereas GPb was purified 328- fld t a final specific activity f 82 U. mg- 1. The specific activity f GPb was high cmpared with values fr the enzyme purified t hmgeneity frm ther surces (Mrishima and Sakurai 1985); hwever, SDS-PAGE indicated that the E. scudderiana GPb preparatin was nt cmpletely pure. SDS-PAGE als shwed a very minr additinal prtein band in the purified GPa preparatin. Mlecular weights The native MWs f GPa and GPb were _ and (bth n = 3), respectively (Fig. 2), values nt significantly different frm each ther (Student's t-test). SDS-PAGE shwed the same subunit MW f _ (n = 3) fr bth enzymes. "5 1.2 Ip > ,0 i 5.0, l, I r [ ph B Fig. 3A, B. Plt f activity versus ph fr E. scudderiana GPa at 22 ~ (A) and 5 ~ (B). 9 cntrl, n additins; 9 plus 0.5 m1.1-1 glycerl; 9 plus 2 tl-l-t glycerl. Reactin cnditins fr GPa assay are as in Materials and methds. Velcity is in units.ml 1. mg prtein- 1 with Vmax equal t 1.98 U'ml 1. mg prtein- 1. Data are means 4- SEM, n = 3 separate enzyme preparatins; SEM bars are cntained within the dimensins f the symbls used. All buffers were adjusted t specific ph values at 22 ~ the crrespnding ph values f these buffers when cled t 5 ~ were calculated assuming a ph unit increase per 1 ~ decrease in temperature fr imidazle buffer D Effect f ph n enzyme activity Prfiles f GPa activity versus ph are shwn in Fig. 3 a fr assays at 22 ~ and Fig. 3 b fr assays at 5 ~ Optimal activity at 22 ~ was at ph 7.5, similar t the ptima f reprted fr phsphrylase frm ther animal surces (Fischer and Krebs 1959; Childress and Sacktr 1970; Mrishima and Sakurai 1985). Maximal activity fr all animal phsphrylases, including E. scudderiana, seems t be cnserved at ph values between 7.0 and 7.5. The additin f 0.5tl-1-1 glycerl t the assay did nt change the ptimum but enzyme velcity decreased as glycerl cncentratin increased. At 2 mll- 1 glycerl the enzyme shwed a brad ptimum between ph 6.5 and 8.0. At 5 ~ the ph ptimum f GPa drpped t 7.31 under bth cntrl cnditins (n glycerl) and in the presence f 0.5 ml. 1-1 glycerl in the assay (Fig. 3 b). In the presence f 2 ml- 1-1 glycerl, hwever, the ptimum was higher at ph Figure 4 shws ph versus activity prfiles fr GPb. At 22 ~ the ptimum was again ph 7.5; the additin f plyls had n effect n the ptimum, but sharply reduced enzyme maximal activity (Fig. 4A). Unlike the situatin fr GPa, hwever, the ph ptimum at 5 ~ rse t 7.81 in the presence r absence f glycerl (Fig. 4B).

5 i C.P. Hlden, K.B. Strey:Insect glycgen phsphrylase I I I /\ 2.0 x 1.5 u E ~ O O J , A ph 1/TxlO -3 (~ I i L i I l [ I I i >, 1.2 /0 ~kl U 0 r 0.8 -J B ph Fig. 4A, B. Plt f activity versus ph fr E. scudderiana GPb at 22 ~ (A) and 5 ~ (B). 9 cntrl, n additins; 9 plus 0.5 tl-1 -t glycerl; 9 plus 2 ml-l-1 glycerl. Reactin cnditins fr GPb assay are as in Materials and methds. Velcity is in units.ml- t. mg prtein ~ v~ith Vm,~ equal t 1.64 U.ml -a.rag prtein -~. Data are means_+ SEM, n=3 separate enzyme preparatins; SEM bars are cntained within the dimensins f the symbls used 0.0 i i r I I i B 1/Tx10-3 (~ Fig. 5A, B. Arrhenius plts fr E. scudderiana glycgen phsphrylase a (A) and b (B). Assays were run under Vm,x substrate cnditins using phsphate buffer (in the presence f 1 mml AMP fr GPb) and in the absence (9 r presence (e) f 2 tl-l-1 glycerl. Velcity is in units-ml-1"rag prtein ~ with Vma~ equal t 1.98 and 1.64 U.ml t.mg prtein -t fr GPa and GPb, respectively. Data are means fr n=4 separate enzyme preparatins; SEM bars are cntained within the dimensins f the symbls used A rrhenius plts Figure 5 shws Arrhenius plts fr E. scudderiana glycgen phsphrylase in the presence and absence f 2 mll -1 glycerl. Fr bth GPa and GPb (Fig, 5A, B) the plts were discntinuus with a sharp change in slpe between 15 and 10 ~ in all cases. In the absence f glycerl Ea was 31_+l.5kJ.ml -~ ver the ~ range and increased significantly twfld t k J- tl-1 between 1 and 10 ~ (P<0.005). In the presence f 2 ml. 1-1 glycerl Ea was kj. tl- 1 between 15 and 35 ~ but increased furfld t 97 -t- 5.1 kj.tlver the range 1-10 ~ (P < 0.005). E, values in the presence and absence f glycerl were als significantly different fr each f the tw temperature ranges (P < 0.01). The additin f glycerl strngly reduced the activity f GPb but, as seen in the GPa plts, the Arrhenius plts fr GPb were again discntinuus with breaks between 15 and 10 *C (Fig. 5 b). In the absence f glycerl Ea was 37_+2.1 kj-m1-1 ver the range ~ and increased significantly twfld t kJ.ml -~ when temperature decreased t the range 1-10 ~ (P< 0.005). When glycerl was present Ea decreased within bth temperature ranges, being 28_4-1.1 kj-ml- 1 between 15 and 35 ~ and 1.5-fld higher at 56+_3.9 kj. tl -1 between 1 and 10 ~ (P<0.005).

6 504 C.P. Hlden, K.B. Strey: Insect glycgen phsphrylase Table 2. K~ values fr glycgen f E. scudderiana GPa and GPb: effects f variable csubstrate (phsphate) cncentratin, additin f glycerl, and temperature Cnditin Km Glycgen (mg-mi a) GPa GPb 22 ~ 50 mml.1-1 P~ (ptimal) _+0.034" 20mml'1-1 Pl " 10 retl.1 -~ Pi 0.172_ " 50 mml- 1-1 Pi q- 0.5 ml'l 1 glycerl r "'~ 2 tl-1 -t glycerl ~ 1.81 _+0.035a'~ 5 ~ 50 mml-1- t Pi (ptimal) 0.024_ b ~,b 50 mml.1- ~ Pi+ 0.5 ml.1 -~ glycerl q b'~ 0.117_ ~'b'c 2 tl-1 -~ glycerl 0.079_ b'r 0.086_ b'~ Data are means 4-SEM fr n = 3 5 separate enzyme preparatins. a Significantly different frm the crrespnding value fr GPa by the Student's t-test, P<0.005; b Significantly different frm the crrespnding value at 22 ~ P<0.001; c Significantly different frm the crrespnding value withut glycerl, P < "5 > ~ ~('" t i i i i i J _1... ~.... P A I, I ~ I, I, I, Glycgen (mg/ml) Fig. 6. Reactin velcity versus glycgen cncentratin fr GPb at 22 ~ at three different cncentratins f inrganic phsphate mml'1-1 Pi (ptimal); 9 20 mml.1-1 Pi; 9 10 retl-1 -t P~. All assays included 1 mml" 1-1 AMP. Velcity is in units, ml- t. mg prtein - i with Vm~x equal t 1.64 U" ml 1. mg prtein- i. Data are means_+ SEM fr n= 3-5 determinatins n separate enzyme preparatins; errr bars are enclsed within the symbls used Substrate affinities Table 2 shws Km values fr glycgen fr bth GPa and GPb. The relatinship between glycgen cncentratin and enzyme velcity was hyperblic (see Fig. 6 fr GPb) with h nt significantly different frm ~ in all cases. GPa shwed a substantially higher substrate affinity fr glycgen than did GPb; Km glycgen fr GPa was 7.3- and 8-fld lwer at 22 ~ and 5 ~ respectively, than the crrespnding value fr GPb (at ptimal phsphate Table 3. Km values fr inrganic phsphate fr GPa: effects f variable glycgen cncentratin and additin f glycerl [Glycgen] K~ Pi Hill (mml. 1-1) cefficient 22 ~ 0.5 rag-ml- t (ptimal) mg.ml -t mg.ml-t +0.5 tl.1 1 glycerl u mg-ml -~ +2 ml-i -~ glycerl b ~ 1 mg.m1-1 (ptimal) 3.74-t-0.14 ~ mg.ml -~+0.5 tl.1 t glycerl "'b mg.ml-~+2 tl.1 -~ glycerl 7.08_+0.17 ~'b 1.00 Data are means 4-SEM fr n = 3-5 separate enzyme preparatins. Km P~ at ptimal [glycgen] significantly different frm the crrespnding value at 22 ~ P<0.05; b Significantly different frm the crrespnding value withut glycerl, P < cncentratin). The cncentratin f the csubstrate, Pi, affected the glycgen Km fr bth enzyme frms with Km increasing as Pi cncentratin decreased. The effect was greater n GPb; at 10 mml'1-1 P~, Km glycgen increased 220% fr GPb cmpared with a 43% increase in Km fr GPa. Assay temperature als had a strng effect n Km glycgen: lw temperature (5 ~ sharply increased substrate affinity. GPa Km glycgen was 5-fld lwer at 5 ~ cmpared with 22 ~ (at ptimal Pl), and similarly 4.5-lwer fr GPb. Table 2 als shws the effects f glycerl n Km glycgen. During cryprtectant synthesis by the larvae, glycgenlysis must g frward under ever-increasing cncentratins f glycerl that reach at least 2000 laml'g wet weight-1 in fully cld-hardened larvae (Rickards et al. 1987; Churchill and Strey 1989 a). Glycerl effects n glycgen phsphrylase were, therefre, analysed at tw cncentratins: a cncentratin that is submaximal in viv (0.5 ml.1-1) and is characteristic f the phase f active glycerl bisynthesis, and a cncentratin that is maximal (2 ml. 1-1) in viv and characteristic f the cmpletin f cryprtectant synthesis. Glycerl had nvel effects n GPa Km fr glycgen; at 0.5 ml.1-1 glycerl Km decreased by abut 40% at bth temperatures, whereas at 2 ml. 1-t glycerl the ppsite effect was seen with Km increasing times cmpared with cntrl values. Fr GPb K~ glycgen increased abut twfld in the presence f glycerl at 22 ~ but the ppsite effect ccurred at 5 ~ with Km decreasing as glycerl increased. Table 3 shws GPa substrate affinity fr P~. The relatinship between velcity and Pi cncentratin was sigmidal and mre strngly s at 22 ~ cmpared with 5 ~ fr example, h was 2.3 and 1.3, respectively, at the tw temperatures under ptimal glycgen cncentratins. Enzyme affinity fr Pi (at 22 ~ was dependent n glycgen cncentratin, Km increasing as [glycgen] decreased. Km was als affected by temperature, decreasing by 40% at 5 ~ cmpared with 22 ~ under ptimal glycgen cncentratins. As fr Km glycgen, the Km

7 C.P. Hlden, K.B. Strey: Insect glycgen phsphrylase 505 Table 4. Km values fr inrganic phsphate fr GPb: effects f variable glycgen cncentratin and additin f glycerl [Glycgen] Km Pi Hill (mml.1 l) cefficient 22 ~ 4 rag-rt -1 (ptimal) mg-ml -~ mg.ml ml.1 -~ glycerl b mg-ml tl-1-1 glycerl b ~ 4 mg.ml -~ (ptimal) 14.5_+0.34 ~ 1.46 Data are means_+ SEM fr at least n = 3-5 separate enzyme preparatins. " Significantly different frm the crrespnding value at 22 ~ P<0.05; b Significantly different frm the crrespnding value withut glycerl, P < A cmbinatin f lw enzyme activity plus high glycerl inhibitin prevented analysis f glycerl effects n K~ at 5 ~ fr P~ was als affected by the additin f glycerl t the assay; Km increased with increasing [glycerl] at bth assay temperatures with Km values at 2 tl. 1 - a glycerl times greater than in the absence f added glycerl. Table 4 shws GPb affinity cnstants fr Pi. The b frm f the enzyme shwed a lwer affinity fr Pi than did the a frm, with GPb Km values abut furfld higher than the crrespnding values fr GPa at bth temperatures. Again, Km Pi was increased when ]glycgen] was decreased. Km Pi fr GPb was als dependent n temperature with affinity increasing at lw temperature; Km decreased by 40% at 5 ~ cmpared with 22 ~ The Hill cefficient was als lwer at 5 ~ than at 22 ~ The additin f glycerl increased Km fr P~ at 22 ~ threefld at 2 tl-1- ~ glycerl. Activatin by AMP AMP is a ptent activatr f GPb and increased the maximal activity f E. scudderiana GPb by abut 20- fld. /Ca fr AMP at 22 ~ was 176_+4 gml.l-1 (at 4 mg.ml glycgen -1' 50 mml.1 -~ P0. /Ca increased in the presence f glycerl t 338 _+ 5 gml-1- ~ at 0.5 ml. 1-1 glycerl and t 263 +_ 10 gml. 1- a at 2 tl. 1-1 glycerl. Lw temperature caused a significant increase in enzyme sensitivity t AMP, Ka drpping t 53 _+ 1 pml. 1-1 (P<0.005) at 5~ (at l mg.ml glycgen -1, 50 mml-1-1 Pi). Inhibitin by KCl and glycerl Table 5 shws that bth GPa and GPb were inhibited by KC1, GPb being mre strngly affected by the salt. Bth enzymes shwed an increase in inhibitin by KC1 (/s decreased significantly) at lw temperature; Is decreased by a factr f 2 fr GPa and 4.7 fr GPb at 5 ~ cmpared with 22 ~ KC1 inhibitin f GPa in- Table 5. Inhibitin f E. scudderiana GPa and GPb by KC1 Cnditin /s KC1 (mml. 1-1) GPa GPb 22 ~ N glycerl _+ 2 a +0.5 ml.1-1 glycerl 208 c +2 ml.1-1 glycerl ~ N glycerl b 38 1 "' b 0.5 ml" 1-1 glycerl 89 k 2 b'c - 2 ml.v 1 glycerl Data are means+_ SEM fr at least n = 3-5 separate enzyme preparatins. Assays were run with glycgen cncentratins f 0.5 mg. ml-1 fr GPa and 1 mg-ml-1 fr GPb. a Significantly different frm the crrespnding value fr GPa by the Student's t-test, P < 0.05; b Significantly different frm the crrespnding value at 22 ~ P< 0.005; c Significantly different frm the crrespnding value withut glycerl, P < A cmbinatin f lw enzyme activity plus high glycerl inhibitin prevented analysis f glycerl effects n Is at 5 ~ creased in the presence f glycerl, the 15 decreasing by 60% in the presence f 2 tl.l- 1 glycerl at 22 ~ and by 25% at 5 ~ Strng inhibitin by glycerl prevented analysis f glycerl effects n several prperties f GPb. Measurements fls values fr glycerl cnfirmed that the plyl was a much strnger inhibitr f GPb than f GPa. 15 values fr glycerl were 383_+18 mml-1-1 at 22 ~ but fell significantly t 61 _+2 retl.1-1 at 5 ~ (n=3-5 fr each; P<0.005; at 1 mg.ml glycgen -a, 50 mml. 1- a Pi)- Discussin The purificatin prcedure used fr E. scudderiana glycgen phsphrylase is a fast, tw-step methd that gives a high yield and a high purificatin factr and readily separates the a and b frms f the enzyme. Final specific activities fr E. scudderiana GPa and GPb were 396 and 82 U. mg- 1, respectively, much higher than specific activities previusly reprted fr the insect enzyme frm ther surces: blwfly flight muscle, 12 U- rag- ' and silkmth fat bdy, 47.3 U.mg -~ (Childress and Sacktr 1970; Mrishima and Sakurai 1985). The native ( kDa) and subunit (87kDa) MWs determined fr GPa and GPb indicated that bth frms f E. scudderiana glycgen phsphrylase are dimeric prteins; the slightly higher mean MW fr GPa (215 kda) can be attributed, at least in part, t the presence f cvalently-bund phsphate n the enzyme. The subnit MW f E. seudderiana phsphrylase is a little lwer than the kda reprted fr the enzyme frm mammalian and ther insect surces (Childress and Sacktr 1970; Titan et al. 1977; Dmbradi et al. 1986; Mrishima and Sakurai 1985; Van Marrewijk etal.

8 506 C.P. Hlden, K.B. Strey : Insect glycgen phsphrylase 1985). Early studies n glycgen phsphrylase prpsed that the cnversin f GPb t GPa invlved a dimer t tetramer transitin (Keller and Cri 1953), but mre recent studies have shwn that the mst active frm f GPa in the presence f glycgen is a dimer (Huang and Graves 1970). Similarly, active GPa frm E. scudderiana is a dimer, apparently with identical subunits, as has als been reprted fr the lcust fat bdy and the fruit fly enzymes (Dmbradi et al. 1986; Van Marrewijk et al. 1985). Hwever, the active frm f phsphrylase in sme ther insects (blwfly flight muscle, silkwrm fat bdy) appears t be a mnmer (Childress and Sacktr 1970; Mrishima and Sakurai 1985). The primary mde f phsphrylase regulatin in animals is the intercnversin between the active a and inactive b frms. A rapid increase in the amunt f the a frm has been well dcumented as a respnse t cld expsure in many species, including E. scudderiana, and initiates the glycgenlysis needed fr cryprtectant bisynthesis (Yamashita et al. 1975; Ziegler et al. 1979; Hayakawa and Chin 1982; Churchill and Strey 1989a; Strey and Strey 1991). Indeed, in the present study we fund 72% GPa in -4 ~ larvae cmpared with nly 9% GPa in 15 ~ animals. Our study f the kinetic prperties f E. scudderiana GPa and GPb, hwever, shw that a number f ther factrs, particularly temperature and glycerl effects n the kinetic prperties f the enzymes, can als have imprtant rles in regulating glycgenlysis. Bth GPa and GPb shwed a dependence f the Km fr ne substrate n the cncentratin f csubstrate; thus, K~ glycgen increases as [Pi] decreases (and vice versa). Such hetertrpic interactins have als been reprted fr ther insect phsphrylases (Childress and Sacktr 1970; Vaandrager etal. 1987). Km values fr bth substrates fr GPa are similar t values fr ther insect phsphrylases (Childress and Sacktr 1970; Mrishima and Sakurai 1985; Vaandrager et al. 1987; Mrishima and Uen 1990) and well within the physilgical cncentratin range f these cmpunds in viv. Km values fr GPa fr glycgen and Pi were much lwer than the crrespnding values fr GPb at bth temperatures, as is typical f phsphrylase frm all surces, and further indicates that GPa is the active frm. The [Pi] in the larvae, as measured by the methd f Atkinsn et al. (1973), was fund t be pml.g wet weight- 1 (C. Hlden, unpublished results) and this cmpares favrable with GPa Km values fr Pi f mml. 1-t at ptimal glycgen. Glycgen cntent f E. scudderiana larvae increases as high as 230 mg-g wet weight-1 in early autumn befre cryprtectant synthesis begins (Rickards et al. 1987), an amunt far in excess f the GPa Km values fr glycgen f mg. ml- 1 (at ptimal Pi) r the ptimal glycgen cncentratins f mg.ml-1. Thus, GPa activity appears unlikely t be limited by substrate availability in viv. With respect t GPb activity, larval glycgen cntent was als many-fld higher than the K~ glycgen values but the high Km P~ values may mean that the enzyme is limited by Pi availability. Indeed GPb is ften cnsidered t be functinally inactive in viv because f its lw activity in the absence f allsteric activatrs and the ften high, nn-physilgical cncentratins f activatrs needed t maximize activity (Childress and Sacktr 1970; Stalmans and Hers 1975; Newgard et al. 1989). AMP levels in unstressed E. scudderiana are abut gml. g wet weight - 1, cmpared with GPb Ka values fr AMP f mml.l-1 (Churchill and Strey 1989b, c), and this suggests that GPb is prbably subacrive in viv. Hwever, AMP levels rise sharply (frm 0.08 t 0.20 gml'g -1) in E. scudderiana in respnse t a lw temperature shck that stimulated glycerl synthesis (Churchill and Strey 1987b). Thus, it is pssible that in additin t the GPb t GPa cnversin that is stimulated by lw temperature, high AMP levels may als increase the activity f remaining GPb. The kinetic prperties f bth GPa and GPb frm E. scudderiana demnstrate that the enzyme is well designed fr lw-temperature functin and this wuld ptentiate the high rates f plyl synthesis that ccur within the range 5 t - 5 ~ in this and ther cld-hardy insects (Strey and Strey 1991). Bth temperature and temperature-mdulatr interactins n kinetic prperties facilitate lw temperature functin f the enzyme. Thus, GPa affinity fr bth substrates increased at 5 ~ cmpared with 22 ~ glycgen affinity fivefld and Pi affinity by nearly twfld. GPb shwed the same pattern and, furthermre, the Ka AMP drpped threefld at lw temperature. The Km fr glycgen f GPa als shwed a nvel respnse t the presence f glycerl. At intermediate glycerl levels (0.5 ml'1-1) the presence f the plyl increased enzyme affinity fr glycgen (Kin drpped by abut 40%) whereas when high glycerl (2 tl" 1-1) was present the effect was reversed and Km rse abut threefld cmpared with Km in the absence f glycerl (Table 2). During the perid f active glycerl accumulatin during the autumn cld-hardening it wuld be advantageus fr the accumulating glycerl t have a psitive feed-back effect n the rate f glycgenlysis via mdulatin f the Km fr glycgen. Hwever, at very high glycerl cncentratins the reverse effect, an inhibitin by high glycerl, culd be ne factr that slws phsphrylase activity and/r prmtes the recnversin f GPa t GPb when cryprtectant levels reach their maximum in viv, abut 2000 gml. g- 1 (Churchill and Strey 1987a). Intermediate levels f glycerl (0.5m1.1-1) als has n effect n the ph ptimum f the enzyme and relatively little effect n the maximum activity f GPa at either 22 r 5 ~ whereas high glycerl (2 ml.1-1) reduced V~ax, bradened the ph ptimum at 22 ~ and shifted the ptimum t a higher value at 5 ~ (Fig. 3). High glycerl als strngly depressed the maximum velcity f GPb at bth temperatures as shwn in the ph curves (Fig. 4) and the Arrhenius plt (Fig. 5) but did lwer the K~ glycgen at 5 ~ Indeed, with a GPb Is fr glycerl f 61 mml-1-1 at 5 ~ the very strng inhibitry effects f glycerl made it impssible t accurately assess varius kinetic parameters f GPb in the presence f glycerl at lw temperature. High salt (KC1) strngly inhibited E. scudderiana, gly-

9 c.p. Hlden, K.B. Strey: Insect glycgen phsphrylase 507 cgen phsphrylase althugh the a frm appeared t be less sensitive t KC1 than the b frm. The larvae lse abut 25% f their ttal bdy water during cld hardening (Rickards et al. 1987) s a reduced sensitivity f the a frm t high inic strength culd be advantageus in the intracellular milieu f the verwintering larvae. Analysis f Arrhenius plts f phsphrylase activity shwed a distinct break in the relatinship fr bth GPa and GPb and in the presence r absence f glycerl. The break ccurred between 10 and 15 ~ in all cases creating tw linear parts f the plt with tw- t furfld higher Kas at temperatures belw 10 ~ than at temperatures f i5-35 ~ Such discntinuities in the Arrhenius relatinship are nt unusual fr multimeric enzymes and indicate a temperature-induced change in cnfrmatin r subunit aggregatin state f the enzyme. Phsphfructkinase frm E. scudderiana and frm the cldhardy larvae f the gall fly Eursta slidaginis shws a break in the Arrhenius plt in the range ~ whereas E. scudderiana aldlase and fructse-l,6- bisphsphatase d nt (Strey 1982; C. Hlden, unpublished data). Calculated Ql values fr the interval 0-10 ~ were 3.3 fr GPa and 3.7 fr GPb in the absence f glycerl indicating a fairly rapid decrease in enzyme maximal velcity with decreasing temperature ver this range. This may seem t be at dds with the high rates f glycgenlysis and glycerl synthesis that ccur in the larvae in viv at lw temperatures. Hwever, effects f temperature n Vm,x may be utweighed by the lwtemperature-stimulated cnversin f GPb t GPa t prduce at least 70% GPa in viv during perids f active plyl synthesis (Churchill and Strey 1989 a; this study) and by the increased enzyme affinity fr bth substrates seen at lw temperatures. Acknwledgements. Thanks t J. M. Strey fr assistance in the preparatin f the manuscript. Supprted by an perating grant frm the Natural Sciences and Engineering Research Cuncil f Canada t K. B. S. References Atkinsn A, Gatenby AD, Lwe AG (1973) Phsphatase assay. Bichim Biphys Acta 320: Bradfrd MM (1976) A rapid and sensitive methd fr the quantificatin f micrgram quantities f prtein utilizing the principle f prtein-dye binding. Anal Bichem 12: Childress CC, Sacktr B (1970) Regulatin f glycgen metablism in insect flight muscle. J Bil Chem 245 : Churchill TA, Strey KB (1989 a) Seasnal variatin in the temperature-stimulated intercnversin f glycgen and glycerl pls in a freeze aviding mth larvae. Cry Lett 10: Churchill TA, Strey KB (1989 b) Regulatin f glycerl bisynthesis in a freeze aviding insect. J Cmp Physil B 159: Churchill TA, Strey KB (1989c) Metablic cnsequences f rapid cycles f temperature change fr freeze-aviding vs freeze-tlerant insects. J Insect Physil 35: Dmbradi V, Matk J, Kiss Z, Kiss L, Friedrich P, Bt G (1986) Structural and functinal prperties f Drsphila melangaster phsphrylase: cmparisn with the rabbit skeletal muscle enzyme. Cmp Bichem Physil 84B: Fischer EH, Krebs EG (1959) Muscle phsphrylase b. Methds Enzyml 49A : Hayakawa Y (1985) Activatin mechanism f insect fat bdy phsphrylase by cld. Insect Bichem 15 : Hayakawa Y, Chin H (1981) Temperature-dependent intercnversin between glycgen and trehatse in diapausing pupae f Philsamia cynthia ricini and pryeri. Insect Bichem 11:41-47 Hayakawa Y, Chin H (1982) Temperature-dependent activatin r inactivatin f glycgen phsphrylase and synthase f fat bdy f the silkwrm, Philsamia cynthia." the pssible mechanism f the temperature-dependent intercnversin between glycgen and trehalse. Insect Bichem 12: Huang CY, Graves DJ (1970) Crrelatin between subunit interactins and enzymatic activity f phsphrylase a. Methd fr determining equilibrium cnstants frm initial rate measurements. Bichemistry 9 : Keller P J, Cri GT (1953) Enzymatic cnversin f phsphrylase a t phsphrylase b. Bichem Biphys Acta 12: Krebs EG, Fischer EH (1955) The phsphrylase b t a cnverting enzyme f rabbit skeletal muscle. Bichim Biphys Acta 20: Laemmli UK (1970) Cleavage f structural prteins during the assembly f the head f bacteriphage T4. Nature 227: Marrewijk WJA van, Brck AThM van den, Beenakkers AMTh (1985) Glycgen phsphrylase in the fat bdy f Lcusta migratria. Insect Bichem 15: Meinke M, Edstrm R (1991) Muscle glycgenlysis: regulatin f the cyclic intercnversin f phsphrylase a and phsph 0- rylase b. J Bil Chem 266: Mrishima I, Sakurai S (1985) Purificatin and characterizatin f glycgen phsphrylase b frm fat bdy f the silkwrm, Bmbyx mri. Cmp Bichem Physil 81B: Mrishima I, Uen T (1990) Structural and kinetic prperties f glycgen phsphrylase a frm fat bdy f the silkwrm, Bmbyx mri. Cmp Bichem Physil 96B: Newgard CB, Hwang PK, Fletterick RJ (1989) The family f glycgen phsphrylases: structure and functin. Crit Rev Bichem Ml Bil 24: Rickards J, Kelleher MJ, Strey KB (1987) Strategies f freeze avidance in the lalwae f the gldenrd gall mth, Epiblema scudderiana: winter prfiles f a natural ppulatin. J Insect Physil 33 : Shimada K (1982) Glycerl accumulatin in develpmentally arrested pupae f Papili machan btained by brain remval. J Insect Physil 28 : Stalmans W, Hers H-G (1975) The stimulatin f liver phsphrylase b by AMP, fluride and sulfate. Eur J Bichem 54: Strey KB (1982) Phsphfructkinase frm the verwintering gall fly larva, Eursta slidaginis: cntrl f cryprtectant plyl synthesis. Insect Bichem 12: Strey KB, Strey JM (1991) Bichemistry f cryprtectants. In: Denlinger D, Lee RE (eds) Insects at lw temperature. Chapman and Hall, New Yrk, pp Titani K, Kide A, Ericssn LH, Kumar S, Wade R, Walsh KA, Neurath H, Fisher E (1977) Cmplete amin acid sequence f rabbit muscle glycgen phsphrylase. Prc Natl Acad Sci USA 74: Vaandrager SH, van Marrewijk WJA, Beenakkers AMTh (1987) Kinetic prperties f glycgen phsph0rylases a, ab, and b frm flight muscles f the lcust, Lcusta migratria. Insect Bichem 17 : Yamashita O, Suzuki K, Hasegawa K (1975) Glycgen phsphrylase activity in relatin t diapause initiatin in Bmbyx eggs. Insect Bichem 5: Yi S-X, Yin C-M, Nrdin JH (1987) The in vitr bisynthesis and secretin f glycerl by larval fat bdies f chilled Ostrinia nubilalis. J Insect Physil 33 : Ziegler R, Ashida M, Falln AM, Wimer LT, Silver Wyatt S, Wyatt GR (1979) Regulatin f glycgen phsphrylase in a fat bdy f Cecrpia silkmth pupae. J Cmp Physil 131 :