Joirrnal of Neurochemisfry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium- and Calmodulin-Dependent Kinase I1 Severn B. Churn, William C . Taft, *Melvin S. Billingsley, tBanumathi Sankaran, and Robert J. DeLorenzo Departments of Neurology and Pharmacology, Medical College of Virginia, Richmond, Virginia; *Department of Biochemistry, Pennsylvania State University, State College, Pennsylvania; and tDepartment of Biochemistry, University of Kentucky, Lexington, Kentucky, U.S.A.

Abstract: The activity of multifunctional calcium/calmodulin-dependent protein kinase I1 (CaM kinase 11) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase I1 was purified to > 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase I1 from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase I1 was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase I1 activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase I1 was not different from control enzyme in affinity for calmodulin, Caz+,Mg2+,or exogenously added substrate or rate of autophosphorylation. CaM kinase I1 isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3)directed toward the @ subunit of the enzyme. In addition, ischemia

caused a significant decrease in affinity of CaM kinase I1 for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase 11, the covalent-binding ATP analog 8-azidoaden~sine-S-[cu-~~P]triphosphate was used. Covalent binding of 25 p M azido-ATP was decreased 40.4 & 12.3%in ischemic CaM kinase I1 when compared with control enzyme (n = 5; p < 0.0 1 by paired Student’s t test). Thus, CaM kinase I1 levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase I1 and that results in an apparent decrease in enzymatic activity. Key Words: Phosphatase-Phosphorlation-Purification -Stroke-ATP-Binding site. Churn S. B. et al. Global forebrain ischemia induces a posttranslational modification of multifunctional calcium- and calmodulin-dependent kinase 11. J. Neurochem. 59, 1221-1232 (1992).

In recent years, it has been shown that loss of calcium homeostasis may be an important mechanism of ischemic brain damage (Siesjo, 1981, 1988; Raichle, 1982). Neurons maintain extremely low intracellular levels of calcium and utilize transient increases in intracellularcalcium levels as a second mes-

senger system. Therefore, prolonged exposure to high levels of intracellular calcium may have irreversible consequences(Farber,198 1 ;Simon et al., 1984; Sakamot0 et al., 1986; Desphande et al., 1987). The calcium hypothesis of ischemia-induced cell death suggests that cessation of blood flow to the brain induces

Received September 27, 199 1; final revised manuscript received March 10, 1992; accepted March 23, 1992. Address correspondence and reprint requests to Dr. S. B. Churn at Department of Neurology, Medical College o f Virginia, Richmond, VA 23298, U.S.A. The present address of Dr. W. C. Taft is Division of Neurosurgery, University of Texas Health Science Center, Houston, TX, U.S.A. Abbreviations used: azido-ATP, 8-azido-adenosine-5’-triphos-

phate; CaM kinase 11, multifunctional calcium/calmodulindependent protein kinase 11; IgG, immunoglobulin G, MAP-2, microtubule-associated protein 2; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N’-bis(2ethanesulfonicacid); PMSF, phenylmethylsulfonyl fluoride; PRP-C, phosphatase mixture of phosphatase 1 and phosphatase 2A; SDS, sodium dodecyl sulfate; TPBS, phosphate-buffered saline containing 0.05%(vol/vol) Tween-20.

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S, B. CHURN ET AI,.

a prolonged increase in intracellular calcium concentration (Siesjo, 1981). The prolonged exposure to high intracellular calcium levels initiates a series of intracellular events that culminate in cell death. To elucidate possible mechanisms by which prolonged exposure to excess calcium results in cell injury, it is important to study calcium-regulated intracellular systems. Protein phosphorylation is a major calcium-dependent effector system that controls many neuronal calcium-regulated processes (see, e.g., Hemmings et al., 1989). These processes include neurotransmitter synthesis and release (DeLorenzo and Freedman, 1978; Joh et al., 1978; DeLorenzo et al., 1979; El Mestikaway et al., 1983; Albert ct al., 1984; Pocotte and Holz, 1986; Nichols et al., 1990), synaptic vesicle mobilization (DeLorenzo et al., 1979; Llinas et al., 1985; Chou and Rebhun, 1986; Hemm i n g et al., 1989), cytoskeletaldynamics (Jameson et al., 1980; Burke and DeLorenzo, 1982), and regulatics cf icn conductances (Sakakibara ei al., 1986). Because of the physiological importance of calciumdependent protein phosphorylation, our laboratory has been studying the role of alterations of calciumdependent phosphorylation elicited by ischemia. Calcium-dependent protein phosphorylation was initially shown to be very labile postmortcm and is especially scnsitive to decapitation ischemia (Burke and DeLorenzo, 1982;Goldenring et al., 1983; Wasterlain and Powell, 1986). Therefore, alteration of calciumdependent phosphorylation by ischemia may play a central role in the cascade of events culminating in delayed ncuronal cell death. Ischemia has been shown to decrease calcium/calmodulin-dependent protein kinase I1 (CaM kinase 11) activity in several models, including transient global forebrain ischemia in the gerbil (Taft et al., 1988; Churn et al., 1990a,b), rabbit spinal cord (Zivin et al., 1990), and decapitation ischemia (Goldenring et al., 1983; Wasterlain and Powell, 1986). Transient forebrain ischemia results in -50% inhibition of CaM kinase 11 activity in homogenates from hippocampus (Taft et al., 1988; Churn et al., 1991) and cortex (Churn et al., 199I). Ischemia has been shown to alter almost every aspect of neuronal metabolism, including ion homeostasis, ATP levels, and membrane potential (Siesjo, 198l, 1988). However, these important physiological parameters return to preischemic levels on recirculation. Unlike most metabolic indices, the decrease in CaM kinase I1 activity after ischemia is an early (within 10 s) and long-lastingphenomenon that precedes the development of delayed cell death (Taft et al., 1988; Churn et al., 1990b). Thus, inhibition occurs too early to be explained by a switch in isozyme coding-i.e., coding for a nonenzymatic form of CaM kinase 11. Furthermore, quantification of enzyme levels in gerbil forebrain homogenates by biotinylated-calrnodulin binding shows that the observed ischemia-induced decrease in CaM kinase I1 activity is not due to large-scale proteolysis of the enJ. Neurochem., Vol. 59, No. 4, 1992

zyme (Churn et al., 1990a; Zivh et al., 1990). Thus, the ischemia-induced inhibition of CaM kinase I1 would best be explained by a posttranslational modification of the enzyme that results in loss of enzymatic activity. A posttranslational modification of CaM kinase IT that results in subsequent inhibition of the enzyme can be determined by isolation and characterization of enzyme from ischemic animals. In this report, CaM kinase I1 was isolated from control and ischemic animals and rigorously compared. The results are consistent with the hypothesis that ischemia induces a selective posttranslational modification of CaM kinase I1 that leads to decreased activity of the enzyme. MATERIALS AND METHODS Materials All reagents and salts used were reagent grade except as follows. ~T-~*P]ATP (5-iO Ci/mmolj and formula 963 scintillation fluid were purchased from New England Nuclear (Wilmington, DE, U.S.A.). 8-Azido-adeno~ine-5'-[y-~~P]triphosphate (azido-[y-"PIATP) and -[~~-~~P]triphosphate (azid~-[cy-'~P]ATP)were a generous gift of Boyd E. Haley, Ph.D. (Department of Biochemistry, University of Kentucky, Louisville, ICY, U.S.A.). Acrylamidc and bisacrylamide were purchased from Kodak Chemical Co. (Rochester, NY, U.S.A.). Calmodulin and calmodulin-Sepharose were purchased from Pharmacia (Piscataway, NJ, U.S.A.). Halothane was purchased from Halocarbon Laboratories (North Augusta, GA, U.S.A.). All anesthetic gases were purchased from Airco (Richmond, VA, U.S.A.). NHS-D-biotin was purchased from Calbiochem (San Diego, CA, U.S.A.).

Gerbil ischemia model All animal use procedures werc in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and werc approved by the local Animal Care and Use Committee. Male Mongolian gerbils (Mcriones unguiculatus) weighing 50-70 g were anesthetized under 2.5% halothanc. 60%nitrous oxide, and 40% oxygen for 5 min in a preequilibrated, temperaturc-controlled exposure tank. Animals were placed on a heated surgical table with anesthesia maintained by placing the animal's head in an anesthesiadosing cap. Temperature was monitored continuously during surgery via a lubricated rectal probe (YSI. Yellow Springs, OH, U.S.A.). The ventral neck area was soaked with Operanol (Red-Products, Pritchard, WV, U.S.A.) and shaved using a safety razor. The neck was again washed with Operanol, and the gerbil was covered with a surgery blanket. An incision was made by lifting the skin above the sternum and making two or three clean cuts rostrally up the neck. Chest muscles were separated from the trachea by a stripping motion parallel to the trachea to expose the carotid arteries. Using 90" hemostats, the carotid arteries were hooked, and a 2-inch strip of sterile 5-0silk suture was looped under each artery. To induce ischemia, the carotid arteries were raised by lifting the sutures, and Heifitz aneurysm clips were attached. Cessation of blood flow was confirmed visually with the aid of Zeiss prism Joupes at a magnification of 3.5. After 5 min of occlusion, the aneurysm clips were removed, and recirculation was confirmed visually. The 5-0 suture was cut

ISCllEMIC CaM KINASE II and removed with care so as not to damage the artery. The skin was folded over, and the wound was sutured with 5-0 silk. Surgery, ischemia, and wound closure required 15-20 min. Animals were allowed to recover in specialized recovery cages with food and water available, ad libitum. Recovery from anesthesia required 5-10 min, during which time animals were observed for seizure activity and general behavior.

Quantification of kinase activity Protein samples and column fractions were normalized for protein concentrations and assayed for kinase activity under standard conditions, unless otherwise specified (Goldennng et al., 1983; Taft et al., 1988; Churn et al., 1990a,b). Standard phosphorylation reaction solutions contained 10 mM piperazine-N,N’-bis(2-ethanesulfonicacid) (PIPES; pH 7.4), 10 m M MgCl,, and 7 pA4 [y-32P]ATP. For maximal CaM kinase I1 activity, 5 pMCaC1, and 1 pg of calmodulin were included. Reactions were initiated with addition of calcium, continued for 1 min, and then terminated by addition of 5% sodium dodecyl sulfate (SDS) stop solution. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) and stained for protein resolution with Coomassie Brilliant Blue or sensitive silver stain as previously described (Goldenring et al., 1983). Stained gels were dried and exposed to x-ray film (XRP-I; Kodak) for autoradiography. The autoradiograph was then used as a template for excising radioactive phosphoproteins for quantification in a liquid scintillation spectrometer (model LS 2800; Beckman) with a counting efficiency of 80%. In addition, the autoradiograph was scanned with a microdensitometer (Ras- 1000; LOATS Systems) (Taft et al., 1988; Chum et al., 1990~).

CaM kinase I1 purification Tissue preparation. Parallel CaM kinase I1 purification was performed for control and ischemic gerbils at 4°C by standard proccdures (Goldenring et al., 1983). Gerbils were killed by decapitation 2 h after ischemia. Forebrains were removed and placed in ice-cold homogenization buffer [ 100 mM PIPES (pH 6.9), 1 mMEDTA, 2 mM EGTA, and 0.3 mM phenylmethylsulfonyl fluoride (PMSF); 3 ml per brain]. Brains homogenized by 13 upand-down strokes of a Teflon pestle. Owing to the postmortem lability of CaM kinase I1 activity (Burke and Debrenzo, 1982; Goldenring et al., 1983), each brain was rcmoved and homogenized within 20 s after decapitation. Homogenates (10-1 5 brains per preparation) were pooled and centrifuged at 100,000 g at 4°C (41,000 rpm; 70-Ti rotor; Beckman). The supernatant (“cytosol”) was decanted and used for column chromatography. The entire purification process, from decapitation to elution from the Sephacryl sieving column, was performed within 16 h. Phosphocellulose column chromatogruphv. Phosphocellulose resin (Cellex-P; Bio-Rad 0.5 g per brain) was hydrated by a series of acid (0.25 M HC1) and base (0.25 M NaOH) washes and equilibrated with column buffer [30 mM PIPES (pH 6.9) and 0.3 mM PMSF]. Cytosol was loaded onto precquilibrated phosphoccllulosc resin (3.5X 8.5cm column) and washed with column buffer until the void protein eluant reached baseline (optical density at 280 nm). The column was then washed with 100 mM NaCl in column buffer until the wash eluant reached baseline. CaM kinase I1 activity was then eluted with 500 mM NaCl in column buffer.

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Cnlmodiilin-Sepharose column chromntogruphy. Phosphocellulose eluant was adjusted to 15 m M 3-(N-morpho1ino)propanesulfonic acid (MOPS), I75 mM NaCI, and 0.5 mM CaCI,. The diluted eluant was loaded onto calmodu1inSepharosc resin (3.5- X 3-cm column: Pharrnacia) preequilibrated with calmodulin column buffer [ 15 mM MOPS ( ~ € 7.1). 1 175 mMNaC1, and 0.5 mMCaCl,]. After loading of the phosphocellulose eluant, the column was washed with buffer until baseline (optical density at 280 nm) was obtained. CaM kinase I1 activity was eluted by replacing the column buffer with 15 mMMOPS, 175 mM NaCl, and 1 mM EGTA. Sephucryl column chromatography. The EGTA eluant was immediately concentrated 10-fold by filtration (YM- 10 membrane; Amicon, Danvers, MA, U.S.A.). Concentrated EGTA eluant was loaded onto Sephacryl S-300 superfine resin (2.5- X 100-cm column) equilibrated with 200 mM NaC1 and I 5 mM MOPS (pH 7.1) and allowed to enter the column under gravity. Once loaded, the head volume was replaced, and the column was pumped at a flow rate of 30 ml/h. Eluting protein was collected into glass test tubes, 2 ml per tube. Fractions containing peak CaM kinase I1 activity were pooled and used for biochemical assays.

Phosphatase reactions To determine the prephosphorylated state of isolated CaM kinase 11, the enzyme was treated with phosphatases shown to be active against forebrain CaM kinase I1 (Shields et al., 1985; Lou and Schulman, 1989). CaM kinase I1 from control and ischemic samples was incubated with a phosphatase mixture of phosphatase 1 and phosphatase 2A (PRP-C) for 20 min at 30°C in buffer containing 10 mM PIPES (pH 7.4), 10 mMMgCI,, 0.5 mMdithiothreito1, and 1 pg of calmodulin. Phosphatase reactions were terminated by incubating samples in 0.62 pM okadaic acid (BioMol, Plymouth Meeting, PA, U.S.A.)for 1 min at 30°C (Bialojan and Takai. 1988). Standard reactions of CaM kinase 11 were performed except that the reaction temperature was 30°C. CaM kinase I1 reactions were terminated by addition ofstop solution, proteins were resolved, and phosphate incorporation was quantified as previously described. Phcsphatase purifirztion Phosphatases active against CaM kinase I1 were purified by modifications of the techniques of Resink et al. (1983) and Ingebritsen et al. (1 983). Male Sprague-Dawley rats were decapitated, and forebrains were removed and homogenized in icecold buffer A [20 mM Tris (pH 7.0), 0.1% (vol/vol) P-mercaptoethanol, 2.0 mM EGTA, and 0.3 mM PMSF]. Homogenization was performed by 10 up-anddown strokes with a Teflon pestle. Forebrain homogenates were centrifuged at 6,000 g a t 4°C for 30 minutes. The supernatant was collected, and phosphatase activity was precipitated with addition of saturated ammonium sulfate to a concentration of 55% (wt/vol) at 4°C. The pellet was resuspended into buffer A and adjusted to 80% ethanol at room temperature. The suspension was immediately centrifuged at 6,000 g for 5 min at 4°C. The pellet was resuspended in buffer A and centrifuged at 10,OOO g for 15 min at 4°C. The resultant pellet was resuspended in buffer A and centrifuged, and the supernatants from both centrifugation assays were pooled. Pooled supernatants were adjusted to 10%glycerol and loaded onto a diethylaminoethyl-cellulose column equilibrated in buffer A plus 10% glycerol. Diethylaminoethyl-cellulose was washed in series with buffer A and

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0.08 M NaCl in buffer A. Phosphatase activity was eluted with 0.2 M NaCl in buffer A. The eluted proteins contained PRP-C (Ingebritsen et al., 1983; Resink et al., 1983; Cohen, 1989).

Calmodulin overlay Derivatization of calmodulin. Calmodulin (Pharmacia) was dialyzed against reaction buffer containing 0.1 M sodium phosphate, pH 7.4. Dialyzed calmoddin was adjusted to 1 mM CaCl, and reacted with 0.285 pLM NHS-D-biotin solution for 2 h at 4°C. Derivatized calmodulin was dialyzed against 100 volumes ofdialysis buffer at 4°C. Quantification of derivatization was performed by multiplying biotinylated absorbance minus starting, underivatized calmodulin absorbance against an extinction coefficient(2.3E4) at 260 nm (Kincaid et al., 1988). Derivatized calmodulin was adjusted to 20% (vol/vol) glycerol and stored at -80°C. Detection of culmodulin-binding proteins. Proteins were resolved by standard SDS-PAGE and western-blotted onto nitrocellulose (Bio-Rad). Strips of nitrocellulose were incubated in blocking solution containing 50 mMTris HCI (pH 7.5), !50mUNaC!, 1 mMCaCI,, O.!%'oan:ifoaziA(Sigms, St. Louis, MO, U.S.A.) and 5% nonfat dry milk (Carnation, Los Angeles, CA, U.S.A.).Strips were incubated with 10-25 pg/ml of derivatized calmodulin in blocking solution for 60 minutes. Excess calmodulin was removed by three washings in buffer containing 50 m M Tris-HC1 (pH 7 . 3 , 150 mM NaC1, and I mMCaCI,. Calmodulin-binding proteins were stained with an alkaline phosphatase staining kit (Vector, Burlingame, CA, U.S.A.). To demonstrate the calcium dependence of calmodulin binding, parallel strips were assayed in the presence of 5 m M EGTA. Quantification of staining was estimated by computer-assisted densitometry (Ras- 1000; LOATS Systems).

Immunodetection of CaM kinase I1 To study further CaM kinase I1 isolated from ischemic animals, immunoreactivity with an antibody directed against CaM kinase I1 was performed. A monoclonal immunoglobulin G3 (IgG3) antibody directed against the B s u b unit of rat CaM kinase I1 was developed in our laboratory. In brief, micc (Mus musculus,BALB/c; Charles River) were immunized with two injections (2 1 days apart) of purified rat CaM lunase II,0.2 ml per intraperitoneal injection (100 pg per injection). Thirty days following the second injection, the mice were killed by cervical dislocation, and a spleen cell suspension was made. The spleenocytes were fused with a nonsecreting mouse myeloma cell line (P3X63AG8.653). Monoclonal-producing cells were obtained from antibody-secreting polyclonal hybridomas by limited dilution methods (Kennett, 1979). Subtyping of monoclonal antibodies directed against CaM kinase I1 was performed by enzyme-linked immunosorbent assay (Kennett, 1979). Specificityof the antibody was tested by western blot analysis. Proteins were resolved on SDS-PAGE under standard conditions and blotted onto nitrocellulose. Nitrocellulose was immersed in phosphate-buffered saline (10 mMsodium phosphate and 0.9% NaCl, pH 7.5) containing 0.05% (vol/ vol) Tween-20 (TPBS; Sigma). The nitrocellulose strip was incubated in TPBS containing anti-CaM kinase I1 antibody (IC3-3D6, monoclonal) for 1 h. Unbound antibody was removed by three washings in TPBS. The blot was incubated in a solution of biotinylated secondary antibody in TPBS. Excess secondary antibody was removed by three washings with PBS (no Tween-20). Labeled proteins were J. Neurochem., Vol. 59. No. 4, 1992

AL.

reacted with an alkaline phosphatase staining kit (Vector). Development of bands was stopped by two changes of water. Developed strips were allowed to air-dry, and quantification was performed by computer-assisted densitometry (Ras-1OOO;LOATS Systems). Equal blotting of protein was confirmed by protein staining using a highly sensitive gold staining of parallel strips of nitrocellulose (Janssen, Beerse, Belgium). Strips of nitrocellulose were incubated in phosphate-buffered saline as above supplemented with 0.3% Tween-20. Washed membranes were briefly rinsed in water and stained with AuroDye (Bio-Rad) until band formation reached saturation. Stained strips were stored in distilled water.

Azido-ATP binding assay Azido-ATP binding was performed essentially under standard reaction conditions for CaM kinase 11. CaM kinase I1 was incubated in buffer containing 10 mM PIPES (pH 7.4), 10 pM MgCI,, 1 pg of calmoddin, and 5 CaCl,. The reaction was initiated by addition of 25 ptM azido-[a''PIATP. The reaction mixture was mixed for 15 s and exposed to UV light for 45 s. The reaction was terminated by addition of stop solution, and proteins were resolved by SDS-PAGE on 10% gels. Gels were dried and exposed to x-ray film at -70°C for autoradiography. X-ray patterns were used as a template for excision of ATP-binding protein. Radioactive bands were excised and counted for radioactivity in a scintillation spectrometer.

RESULTS Purification and characterization of control and ischemic CaM kinase I1 To study the ischcmia-induced modifications of CaM kinase 11, simultaneous, parallel purification of control and ischemic kinase was performed. An established method of purifying CaM kinasc I1 by ion exchange, calmodulin-affinity chromatography, and molecular-weight sieving to purify CaM kinase I1 from forebrain homogenates to > 1,000-fold was used as previously described (Goldenring et al., 1983), with minor modifications. Careful quantification of specific activity was conducted at each step of purification to confirm that CaM kinase 11 properties during purification were not altered by ischemia (Table 1). To minimizc loss of enzyme activity during the purification, all of the column chromatography procedures were done consecutively and without freezing the enzyme betwcen procedures. All purification steps were conducted on unfrozen samples, within 16 h of death. Cdlmodulin binding and percent recovery calculations were performed within 24 h of isolation and without freezing ofthe samples. In addition, the lyophilization step previously performed to concentrate the enzyme before molecular-weight sieving (Goldenring et al., 1983) was replaced by Amicon filtration to preserve activity. To rule out the possibility that ischemia influenced the partitioning of CaM kinase I1 in a purification step or that ischemia differentially affected CaM kinase I1 in individual regions, e.g., membrane versus cytosol, control and ischemic CaM kinase I1 activity in each subcellular fraction and purification step was care-

ISCHEMK CaM KINASE II TABLE 1. Percent recovery of protein and activity during CaM kinase II purification from control

and ischemic gerbils Control

Ischemia

Ilomogenate Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

715.5 498.6 0.697 100 1

602. I 216.8 0.360 100

Cytosol Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

297.4 386.6 1.300 77.5 1.865

258.0 167.7 0.650 77.4 1.805 50.00

Phosphocellulose void Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

249.8 247.3 0.990 49.6 1.42 1

234.6 108.8 0.464 50.2 1.288 53.15

Phosphocellulose 500 Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

54. I 186.1 3.439 37.3 4.935

50.0 89.0 1.778 41.0 4.939 48.29

CaM-EGTA Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

0.460 133.9 291.1 26.8 417.7

0.290 45.4 156.6 20.9 434.8 46.22

0.0404 49.9 1,235.1 10.0 1,772.5

0.0233 12.5 536.5 7.65 1,489.9 56.56

Poolcd Sephacryl S-300 Total protein Total activity Specific activity Percent recovery Fold purification Percent inhibition

1

48.33

Gerbils were subjected to 5 min of bilatcral carotid artery occlusion, and homogenates were obtained 2 h postischemia (see Materials and Methods). Data presented are from a representativepurification. Protein content is expressed in milligrams; activity is expressed in picomoles per minute. Specific activity is expressed as phosphate incorporation into the a subunit ofCaM kinase I1 (picomoles per milligram per minute).

fully evaluated (DeRobertis and DeLores, 1969). Ischemia resulted in inhibition of CaM kinase I1 activity in nuclear, synaptosomal, microsomal, and cytoplasmic fractions studied (data not shown). In addition, CaM kinase I1 activity from crude membrane pellets of material from high-speed centrifugation (see Materials and Methods) was inhibited to the same extent as soluble (cytosolic)enzyme activity. Therefore, the sol-

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uble fraction from ischemic gerbils was representative of the ischemia-induced inhibition and was used for further purification. The results presented in Table 1 demonstrate that CaM kinase I1 was purified in equivalent amounts through each step ofthe purification process. The ischemia-induced inhibition of CaM kinase I1 activity was present to the same extent in forebrain cytosol as was observed for homogenates. The results demonstrate that total homogenate and cytosolic CaM kinase I1 species are affected to a comparable extent. Because the effect of ischemia on CaM kinase I1 was observed in the cytosolic fraction to the same extent as was observed for total CaM kinase 11, cytosol was used as a source for crude CaM kinase I1 for purification. CaM kinase I1 was isolated from ischemic animals in levels equivalcnt to that of control animals, based on protein staining and calmodulin-binding data (Table 1). CaM kinase I1 activity was determined following each step of the purification process as described previously (Goldenring et al., 1983).During each step of purification the percent recoveries of protein and activity were equivalent in both ischemic and control preparations (Table 1). Percent recovery of CaM kinase I1 was calculated by total phosphate incorporation into the a subunit (see Materials and Methods). Using this method, a purification of > 1,000-fold was obtained, which is in agreement with previous reports using this purification paradigm (Goldenring et al., 1983). In addition, although Table 1 represents a typical purification, similar recovery values were obtained in other isolation experiments, indicating that this value is representative of this technique for the purification of gerbil kinase. However, total recovery of enzyme is less than reported by other laboratories (Kuret and Schulman, 1984; Miller and Kennedy, 1985; Kwiatkowsky and King, 1989). Total recovery ofenzyme ranges from 0.1 (Goldenring et al., 1983)to i .O% (Erondu and Kennedy, 1355). The lower calculated recovery value of CaM kinase I1 in Table 1 may represent a difference in measuring enzyme activity (autophosphorylation vs. substrate phosphorylation) or some differences in the method of purification. In addition, the lower recovery of CaM kinase I1 from gerbil brain may represent a lower level of protein expression compared with rat brain. The phosphocellulose eluant was tested for calmodulin-binding affinity, and no significant difference was observed. Because ischemic CaM kinase I1 had the same calmodulin-binding affinity as control CaM kinase 11, the calmodulin-affinity resin was used for both ischemic and control CaM kinase 11. The phosphocellulose column eluant was adjusted for salt concentration and passed over calmodulin-Sepharose resin to enhance purification. No significant kinase activity voided from the calmodulin-Sepharose column in the presence of calcium. The EGTA eluant for both control and ischemic preparations was >400-fold purified from homogenate and was free from major conJ. Neurochem., Vol. 59, No. 4, 1992

S. B. CHURN ET AL.

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'

0 0

- ---A

7

'

.*

to an equivalent level through each step and was present after the enzyme had been purified > 1,000-fold. Thus, the results observed in the purified enzyme are not an artifact of isolation of a subpopulation of CaM kinase 11. Therefore, purified CaM kinase I1 can be studied to try and determine what ischemia-induced posttranslational modifications have occurred that result in inhibition of the enzyme.

'-

10 20 30 40 50 60 70 80 90

B "

Tube I

FIG. 1. Elution profile of protein and CaM kinase II activity off a Sephacryl S-300 column from (A) control and (6)ischemicgerbils. Calmodulin-enriched CaM kinase II from control and ischemic gerbils was concentrated by Amicon filtration and chromatographed on Sephacrylresin. Protein content (solid line) was monitored at 280 nm. The elution pattern of protein into fractions greater than tube 95 are not shown to emphasize the profile of relevant proteinelution. The data shown represent a partialrecovery of the total protein loaded onto the column. Activity (broken line) was quantified under standard reactions (see Materials and Methods) and is expressed as a percentage of maximal control kinase activity.

taminating bands with molecular masses of 50 or 60 kDa. Sephacryl molecular-weight-sieving resin was used as the final purification step. The protein and CaM kinase I1 activity elution profiles from the Sephacryl column are shown in Fig. 1. The profiles from control and ischemic preparations are similar in pattern, and the majority of CaM kinase I1 activity eluted in equivalent fractions. Sample tubes from under the CaM kinase 11 peak were pooled for both control and ischemic preparations and used for subsequent studies to compare the properties of the purified enzymes. Samples were normalized for protein content and reacted and resolved on SDS-PAGE (Fig. 2). Silver staining patterns showed balanced protein staining patterns for both the a and 0 subunits between control and ischemic lanes. However, activity was dccreased -56%, which corresponds with a 48% decrease in the starting homogenate activity (Table 1). Thus, when protein content was balanced for the staining of the a and fl subunits of CaM kinase 11, the decrease in activity was still observed. Total protein and percent recovery values for control enzyme and ischemic CaM kinase I1 were not different in any fraction studied. However, the ischemia-induced inhibition of CaM kinase I1 was prescnt J. Neurochem.. Vol. 59, No. 4, 1992

Calmodulin binding is not affected by ischemia CaM kinase I1 preparations from control and ischemic samples were subjected to western blotting and further evaluated by biotinylated-calmodulin overlay and immunoreactivity. Gold staining of blotted protein and examination of the blotted gel by silver staining showed no differences in blotting properties of CaM kinase I1 from control and ischemic animals (Fig. 2). Examination of blotted proteins by the biotinylated-calmodulin overlay technique showed equivalent calmodu!in binding in purified CaM kinase I1 samples (Fig. 2). The observed calmodulin binding for purified CaM kinase I1 agrees with equivalent calmodulin binding was observed in crude homogenates from ischemic gerbils (Churn et al., 19904. Equivalent calmodulin binding provides additional evidence that the ischemia-induced inhibition of CaM kinase 11 activity is not due to significant loss of the enzyme subunits. Ischemia alters immunoreactivity of CaM kinase I1 To study further CaM kinase I1 isolated from ischemic animals, immunoreactivity with an antibody di-

6050-

I

C

PROTEIN

I

C

AUTORADIOGRAPH

I

C

CALMODULIN

I

C

ANTIBODY

FIG. 2. Photograph of pooled Sephacryl S-300 eluant resolved on SDS-PAGE and subjected to western analysis. Protein from purified control (C) and ischemic (I) Sephacryl fractions were pooled. normalized for protein, reacted under standard conditions, and resolved by SDS-PAGE on 8.5% gels. CaM kinase II activity, as measured by autophosphorylation, is inhibited in samples from ischemic gerbils when enriched >l,OOO-foId. Percent inhibition is equivalent to that observed in starting homogenate material. Purified CaM kinase II was subjected to western analysis and tested for biotinylated-calmodulinand antibody binding. No differences were observed in total calmodulin binding, demonstrating equivalent transfer of protein between control and ischemic samples. lmmunoreactivity of ischemic CaM kinase II was decreased, demonstrating that the decreased immunoreactivity observed in the starting homogenate material remains after the enzyme is purified. The (Y (50-kDa) and p (60-kDa) subunits are denoted by arrowheads.

ISCIIEMIC' CaM KINASE 11 rected against CaM kinase I1 was performed. A monoclonal IgG3 antibody directed against the p subunit of rat CaM kinase I1 was developed in our laboratory as described in Materials and Methods. In a cytosolic fraction, the antibody reacts with a doublet of -60 kDa (data not shown). Different isoforms of the p subunit exist and have been termcd p and p' (Miller and Kennedy, 1985). Thus, the monoclonal antibody reacts with the p subunits of CaM kinase I1 but does not differentiate between the two major isoforms of the subunits. The observed immunostaincd bands are similar to those seen in the purified CaM kinase 11. The data demonstrate that the monoclonal IgG3 reacts specifically with the j3 subunits of CaM kinase 11. The effect of ischemia on the immunoreactivky of CaM kinase I1 was studied. Immunoreactivity of ischemic CaM kinase I1 with a monoclonal IgG3 antibody raised against the p subunit of CaM kinase 11 was substantially decreased (Fig. 2). CaM kinase 11immunoreactivity was decreased in starting homogenate samples and in purified CaM kinase 11 preparations from ischemic animals when compared with control samples. Western analysis of crude and purified CaM kinase I1 fractions suggests that ischemia induces a modification in the CaM kinase I1 molecule that results in inhibition of enzymatic activity and decreased immunoreactivity of the enzyme. However, when quantified by protein staining on SDS-PAGE and biotinylated-calmodulin overlay, no differences were observed. Thus, CaM kinase I1 level was equivalent in ischemic fractions when quantified by protein staining, percent recovery, and calmodulin binding but was decreased when measured by activity and immunoreactivity. Inhibition of CaM kinase I1 activity is not due to an inhibitor Protein kinase C activity is also decreased in response to ischemia, and the decrease in activity is due to an ischemia-released inhibitor of the enzyme (Zivin et al., 1990). However, the inhibition of CaM kinase I1 activity caused by ischemia was not due to an inhibitor that can be separated away by purification of the enzyme (Table 1). Furthermore, studies to test for the presence of an inhibitor of CaM kinase I1 showed additive activities, Ischemic CaM kinase reacted alone displayed activity of 456 fmol/band/min, and control CaM kinase 11 displayed activity of 1,543 fmol/band/min. Combined ischemic plus control CaM kinase I1 displayed activity of 2,086 fmol/band/ min, which was additive. Thus, the presence of an inhibitor of CaM kinase activity that was released in response to ischemia was not likely. In addition, the ischemia-induced inhibition of CaM kinase I1 cannot be reversed by incubation in Cleland's reagent and therefore was not due to simple disulfide linkage between the enzyme and other sulfhydrylcontaining peptides (Davies and Delsignore, 1987).

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Inhibition of CaM kinase I1 is not due to autophosphorylation Autophosphorylation in certain circumstances has been shown to lead to inhibition of CaM kinase I1 activity (Shields et al., 1984; Lai ct al., 1986). However, the autophosphorylation-inducedinhibition of CaM kinase I1 activity is reversible on incubation of CaM kinase I1 in the presence of phosphatases. Phosphatases 1 and 2A have been shown to be responsible for almost all phosphatase activity against CaM kinase I1 and can reverse the autophosphorylation of the kinase (Shields et al., 1985; Lou and Schulman, 1989).To determine whether ischemia induces extensive autophosphorylation of CaM kinase I1 that results in subsequent inhibition of the enzyme, control and ischemic enzymes were preincubated in the presence of phosphatases I and 2A. The activity of the phosphatase preparation (PRP-C) was shown by the ability to remove radioactive phosphates enzymatically added to CaM kinase I1 via autophosphorylation (Fig. 3A).

0'

20

€4

40

80

1

Time

No Treatment

PRP-C Treated

FIG. 3. Phosphatase treatment of CaM kinase II isolated from gerbil forebrain. A CaM kinase II was phosphorylated in vitro in the presence of 7 pM [y-32P]ATPunder standard conditions. Reactions were terminated with addition of 20 pA4 EGTA buffer (0) or EGTA buffer plus PRP-C (0). Aliquots were removed at the indicated time points, proteins were resolved on SDS-PAGE, and phosphate incorporation into the (Y subunit of CaM kinase II was quantified as described in Materials and Methods. Addition of PRP-C results in significant removal of labeled phosphates, demonstrating that the isolated phosphatases recognize phosphe amino acids within the CaM kinase II holoenzyme. B Effect of phosphatase pretreatment on ischemic and control CaM kinase II activity. Control (0)and ischemic (m) preparations were pretreated with PRP-C for 20 min at 30°C. Phosphatase reaction was terminated with addition of okadaic acid and CaM kinase II reacted as described in Materials and Methods. Ischemic CaM 1.4% of control activity before PRP-C kinase II displayed 42 treatment and 43 f 1.1% of control activity following PRP-C pretreatment. Data are mean SEM (bars) values.

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Howcver, the ischemia-induced inhibition of CaM kinase I1 was not reversed by phosphatase pretreatment (Fig. 3B). Ischemic CaM kinase I1 activity was reduced 58% before phosphatase treatment (control, 1,129.0 f 20.0 fmolhand; ischcmic, 474.2 k 16.0 fmol/band) and remained reduced 57% following phosphatase treatment (control, 1,799.3 +. 53.1 fmol/ band; ischemic, 773.6 19.6 fmol/band). Therefore, the inhibition observed was not likely to be due to an autophosphorylation effect of CaM kinase 11, nor was it due to phosphorylation of CaM kinase I1 at a site accessible to the above phosphatases.

*

Kinetics of control and ischemic CaM kinase I1 Saturation curves were generated for control and ischemic CaM kinase I1 for calmodulin, ATP, and substrate affinities and reaction kinetics under standard reaction conditions (see Materials and Methods), except that the parameter under study was varied (Fig. 4). The ischemia-induced decrease in CaM kinase I1 activity could not be reversed by increasing the concentration of any parameter studied, including Ca”, Mg2+, calmodulin, exogenously added substrate, or ATP. CaM kinase I1 activity was maximal at 5 m M Ca2+ for both control and ischemic enzyme. The K,,, for Mg2+was similar for control and ischemic CaM kinase I1 and was 10 mM. The affinity for calmodulin was not significantly different between control and ischemic CaM kinase I1 (Fig. 4A). The estimated K, for control CaM kinase I1 was -24.05 +. 2.00 nM, and ischemic CaM kinase I1 showed a similar affinity for calmodulin, with a K, value of 21.75 f 1.31 nM. Studies with exogenously added microtubule-associated protein 2 (MAP-2), a known substrate for CaM kinase I1 (Goldenring et al., 1983; Kuret and Schulman, 1984), revealed that ischemia did not alter substrate recognition by CaM kinase I1 (Fig. 4C). Control CaM kinase I1 showed an apparent K, for MAP-2 of 2.66 5 0.14 pM. Ischemic CaM kinase 11 exhibited a similar affinity for MAP-2 (2.26 f 0.24 p M ) . The reaction rate of control and ischemic CaM kinase I1 was studied in the presencc or absence of calcium (Fig. 4B). In the absence of calcium, no significant incorporation of labeled phosphate was observed in either the control or the ischemic sample. In the presence of calcium, the rate of autophosphorylation was not significantly different between control and ischemic enzyme. The k,,2 of the reaction rate was 3 1.87 f 0.70 s for control and 28.67 f 3.18 s for ischemic enzyme.

-

ATP binding properties of CaM kinase I1 To investigate whether ischemia alters ATP rcaction kinetics, standard phosphorylation reactions were performed, except the level of ATP was vaned (Fig. 4D). The previously reported affinity of ATP for rat CaM kinase 11, when measured by autophosphorylation, was -7 p M . Control gerbil CaM kinase I1 affinity was lower, with an apparent K, of 2.42 f 0.84 J. Neurochem., Vol. 59, No. 4, 1992

pM. Ischemia induced a significant alteration in affinity for ATP by CaM kinase 11. Ischemic CaM kinase I1 cxhibitcd an apparent K,,, for ATP of 8.52 f 1.50 p M . This difference was significant (Fig. 4D; n = 5 , p < 0.005 by paircd Student’s t test). To characterize further the change in ATP affinity, covalent ATP binding studies were performcd. Equivalent protein samples, as determined by the method of Bradford ( 1976)and silver staining of the a subunit of CaM kinase 11, were resolved on SDS-PAGE. Ischemia rcsulted in an overall decrease in ATP binding to all proteins in thc ischemic fraction (Fig. 5A). Azido-ATP binding to the a subunit of CaM kinase I1 was quantified to determine whether ischemia altered ATP binding by CaM kinase 11. Ischemia resulted in a decrease of a-labeled azido-ATP binding compared with control enzyme (Fig. 5B). Control CaM kinase I1 bound an average of 3,253.4 ? 598 cpm, whereas ischemic cnzyme bound 1,940.8 i 401.2 Cpiii (1-1 - 5 , p < 0.01 b y paired Student’s I test). Initial studies with 1C3-3D6 and control globulin demonstrate that IC3-3D6 immunoreactivity interfered with normal ATP binding by CaM kinase I1 at saturating levels of antibody. Azido-ATP binding studies were performed for control enzyme as described in Materials and Methods, except that lC33D6 was included in the reaction mixture. Parallel reactions with a nonspecific mouse y-globulin (Sigma) were used as a negative control for the monoclonal antibody. The results suggest that the epitope for 1C3-3D6 recognition may be near the ATP binding site.

DISCUSSION Ischemia has been shown to decrease CaM kinase I1 activity in several models, including transient global forebrain ischemia in the gerbil (Taft et al., 1988; Churn et al., 1990u,b); spinal cord ischemia in the rabbit (Zivin et al., 1990), and decapitation ischemia (Goldenring et al., 1983; Wasterlain and Powell, 1986). Previous reports have demonstrated that the ischemia-induced inhibition of CaM kinase I1 activity is an early and long-lasting event in the gerbil model of forebrain ischemia (Taft el al., 1988; Churn et al., 1990b). In addition, ischemia does not result in loss of CaM kinase I1 enzymc subunits as measured by biotinylated-calmodulin binding in forebrain homogenates (Churn et al., 1990~).Thus, ischemia results in inhibition of CaM kinase I1 activity without significant loss of total enzyme level and at a point that cannot be accounted for by turnover of the enzyme. In this report, we developed a protocol to purify CaM kinase I1 to > I ,000-fold from both control and ischemic gerbil forebrain. When purified, the ischemiainduced inhibition of CaM kinase I1 activity remained, suggesting the possibility that ischemia induced a posttranslational modification of the enzyme. Direct evidence for a posttranslational modi-

ISCHEMIC CaM KINASE 11

1229

A

11111)

'-1

I

C

w-I q FIG. 4. Saturation curves of (A) calmodulin, (B) rate of reaction, (C) substrate, and (D) ATP for purified control and ischemic CaM kinase II. CaM kinase II purified from control (0)or ischemic(0)gerbils was reacted under standard conditions, except for varying concentrations of the parameter under study (curves shown are representativeof three or more experiments). Phosphate incorporation into the (Y (50-kDa) subunit was quantified as described in Materials and Methods. Increasing the concentration of any parameter did not overcome the inhibitory effect of 5 min of bilateral carotid occlusion. A: Five minutes of bilateral carotid artery occlusion did not affect the affinity for calmodulin in ischemic CaM kinase II (n - 4; paired Student's t test). Ischemic CaM kinase II displayed an apparent K,,, for calmodulin of 21.75 -t1.31 nM; control CaM kinase II displayed an apparent K,,, of 24.05 I 2.00 nM. B: Half-maximalactivity was not different between control (31.87 f 0.70 s) and ischemic (28.67 f 3.18 s) CaM kinase II (n - 3; paired Student's t test). C: Five minutes of bilateral carotid artery occlusion did not affect the affinity for MAP-2 in ischemic CaM kinase II (n -- 3; paired Student's t test). Ischemic CaM kinase II displayed an apparent K, for MAP-2 of 2.26 0.24 pM; control CaM kinase II displayed an apparent K,,, of 2.66 f 0.14 pM. D: Bilateral carotid occlusion did alter 'the affinity of CaM kinase II for ATP. Ischemic CaM kinase II displayed an apparent K, for ATP of 8.52 ~f 1.50 pM, whereas control CaM kinase II displayed an apparent affinity for ATP of 2.42 f 0.84p M (n = 5; p < 0.005 by paired Student's t test).

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A

C

I

B

‘. 7-

Control

Ischemic

FIG. 5. Five minutes of global forebrain ischemia results in decreased azido-ATP labeling of CaM kinase ll. A Azido-ATP covalent binding to purified control and ischemic CaM kinase II. Cah4 kinase II purified from control (C) or ischemic (I) gerbils was reacted with 25 phl a z i d ~ [ a - ~ P ] A T P Samples . were exposed to UV light to initiate covalent binding of the azido group of the labeled probe with a free amino group on CaM kinase II. Ischemic CaM kinase II binding to azido-ATP was decreased 40.4 f 12.3% when compared with control CaM kinase II. The 01 (50-kDa) and p (60-kDa) subunits are denoted by arrowheads. B: Bar graph of the effects of 5 min of bilateral carotid occlusion on azido-ATP binding to CaM kinase II.CaM kinase II purified from control (0)or ischemic (m) W l s was reacted with 25 p M azid~+a-~P]ATP. Samples were exposed to UV light to initiate covalent binding of the azldo group of the labeled probe with a free amino group on CaM kinase II. Incorporation of label was quantified as described in Materials and Methods and expressed as a mean SEM (bars) percentage of control binding. CaM kinase II from control animals bound an average of 3,253.4 k 598.0 cpm, whereas Ischemic CaM kinase II bound an average of 1,940 k 401.2 cpm (n - 5; p < 0.01 by paired Student’s t test).

*

fication of CaM kinase I1 was shown in experiments demonstrating decreased ATP binding affinity and decreased immunoreactivity with a monoclonal antibody directed against the P subunit of the enzyme. Furthermore, the posttranslational modification of CaM kinase I1 is selective, in that many parameters are not affected by ischemia. Ischemia did not affect calmodulin-binding affinity, substrate recognition, or phosphorylation kinetics. The results suggest that there is a selective posttranslational modification in CaM kinase I1 that results in significant inhibition of CaM kinase I1 activity and affects immunoreactivity and ATP binding without affecting calmodulin binding. Ischemia induced an alteration in immunoreactivity of CaM kinase I1 with a monoclonal antibody

J . Neurochem.. Vol. 59, No. 4, 1992

directed against the j3 subunit of the enzyme. The decrease observed in the purified enzyme was also observed in the starting homogenate material, suggesting that the decreased immunoreactivity was not due to artifactual isolation of a subpopulation of the enzyme. We have previously shown that the ischemia-induced inhibition of CaM kinase II in forebrain homogcnates was not due to proteolysis of the enzyme (Chum et al., 1990a,b). Thus, ischemia induced a specific alteration in the enzyme that resulted in decreased antibody recognition, even though the enzyme is present. When balanced for protein staining in the purified fractions, the decreased immunoreactivity is still present. It is possible that ischemia altered the ability of CaM kinase I1 to transfer onto nitrocellulose. However, balanced transfer of CaM kinase I1 from control and ischemic samples onto the nitrocellulose membrane was demonstrated by equivalent calmodulin binding. At present, it is not known whether the Atered imrnunorextivity is dge to an alteration of the specific epitope of antibody recognition or due to an ischemia-induced masking of the epitope. CaM kinase I1 contains many sulfhydryl groups within its structure (Schulman, 1988), and therefore disulfide “bridges” may form in response to ischemia that mask the epitope recognized by the antibody (Davics, 1987). However, because the decreased immunoreactivity is not reversible by incubation in Cleland‘s reagent or SDS, it is unlikely that simple disulfide bonding is responsible for masking of the epitope. Kinetic studies of CaM kinase I1 revealed that many biochemical parameters are not affected by ischemia. Ischemia does not induce a change in calmodulin-binding affinity. This was fortunate because it allowed for the use of biotinylated-calmodulin overlay to quantify enzyme levels. It also made possible the use of calmodulin-Sepharose affinity resin for the punfication of the enzyme. There was also no change in substrate recognition or reaction rate of the enzyme. Thus, the alteration of the CaM kinase I1 holoenzyme induced by ischemia may be specific. Experiments designed to study the affinity of CaM kinase I1 for ATP were performed to determine whether ischemia altered recognition for ATP. Reported K , values for ATP using various substrates have been published for rat CaM kinase I1 (Goldenring et al., 1983; Kuret and Schulman, 1985; Miller and Kennedy, 1985; Kwiatkowsky and King, 1989). Apparent K,,, values for ATP vary according to the substrate used to study the kinetic parameters of the enzyme. Reported affinities for ATP range from 7 pM [autophosphorylation (Goldenring et al., 1983)] to 22 pLM for casein phosphorylation (Kwiatkowsky and King, 1989). Saturation experiments for gerbil CaM kinase 11, using the enzyme as substrate and analyzed with the aid of PharmCalc software, resulted in an apparent affinityof CaM kinase 11 for ATP of -2 pM.

ISCHEMIC CaM KINASE 11 This affinity is lower than previously reported values for the rat (Kuret and Schulman, 1984; Miller and Kennedy, 1985; Kwiatkowsky and King, 1989)but is in agreement with the rat data of Goldenring et al. (1983). The lower apparent K , value may represent a difference in substrate used to measure enzyme kinetics (autophosphorylation vs. substrate phosphorylation) or stability of the enzyme with different purification methods or may be a subtle interspecies difference. In gerbils subjected to 5 min of forebrain ischemia, a significant decrease in ATP affinity was observed. The physiological relevance of such a specific change in affinity was studied using the covalent-binding azido-ATP compound. Ischemic kinase exhibited a 40.4 f 12.3% decrease in ability to bind azido-ATP under conditions for maximal activity. The decrease in binding was seen with an ATP concentration 10fold greater than the apparent K , observed for control enzyme. Thus, the ischemia-induced effect on CaM kinase 11 activity may bc located at or near the ATP recognition domain. Oxygen radical-mediated damage has been shown to cause alterations in physical properties of proteins (Davies, 1987;Davies and Delsignore, 1987),and forebrain ischemia results in increased production of free radicals (Demopoulos et al., 1982). Therefore, free radical-mediated damage to CaM kinase I1 may occur, resulting in decreased enzymatic activity. However, some evidence argues against a free radical-mediated effect. First, the ischemia-induced inhibition of CaM kinase I1 activity appears to be selective to the ATP-binding domain. It would be expected that a free radical-mediated effect would be more random, causing a change in greater than one biochemical parameter. Also, if dimerization or fragmentation were to occur, as would be expected for radical-mediated damage, extra peptide bands would be observed on SDS-PAGE. If low-molecular-weight products were produced, unbalanced protein staining would result owing to inaccurate protein quantification by the Bradford or Lowry method. When the protein concentration is balanced by the level of subunit staining, the inhibition is still observed. Thus, although still possible, it is unlikely that oxygen radical-mediated damage is responsible for theischemia-inducedinhibition of CaM kinase I1 activity observed. Because the alteration of CaM kinase I1 is an early and long-lasting event (Taft et al., 1988; Churn et al., 1990b)that remains after purification of the enzyme, ischemia appears to induce a posttranslational modification of the enzyme. The site of this modification appears to be located near or within the ATP-binding domain. Future studies will focus on ischemia-induced modifications within the ATP-binding domain, using the covalent-binding ATP analog azido[3ZP]ATP.In addition, ischemia induces a change in immunoreactivity of CaM kinase I1 with a monoclo-

1231

nal antibody raised against the subunit of the enzyme. Therefore, studies involving mapping of the epitope of immunoreactivity with the monoclonal antibody will also be performed. Acknowledgment: This work was supported by Jacob Javits award R01-,”\IS23350and Epilepsy Program Project (POI-NS25630) from the National Institute of Neurological Disorders and Stroke to R. J. DeLorenzo, postdoctoral grant HM7537 from the National Institutes of Health to S. R. Churn, and the Sophie and Nathan Gumenick Neuroscience and Alzheimer Research Fund. The authors are grateful to Dr. Douglas Coulter for reading the manuscript and to Robert E. Blah for performing the gerbil surgeries.

REFERENCES Albert K. A., Helmer-Matyjek E., Nairn A. C., Muller T. H., Haycock J. W., Greene L. A,, Goldstein M., and Greengard P. (1984) Calcium/phospholipiddependentprotein kinases (protein kinase C) phosphorylates and activates tyrosine hydroxylase. Proc. Natl. Acad. Sci. USA 81, 7713-7717. Bennett M. K. and Kennedy M. B. (1987) Deduced primary structure of the beta subunit of brain type I1 calcium/calmodulindependent protein kinase determined by molecular cloning. Proc. Null. Acud. Sci. USA 84, 1194-1798. Bertram E. H., Lothman E. W., and Lenn N. J. (1990) The hippocampus in experimental epilepsy: a morphometric analysis. Ann. Neurol. 2 1 , 4 3 4 8 , Bialojan C. and Takai A. (1988) Inhibitory effect of a mannesponge toxin, okadaic acid, on protein phosphatases. Biochern. J. 256,283-290. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the pnnciple of proteindye binding. Anal. Biochem. 72,248-254. Burke B. E. and DeLorenLo R. J. (1982) Calcium and calmodulindependent phosphorylation of endogenous synaptic vesicle tubulin by a vaicle-bound calmodulin kinase system. J. Neure chem. 38, 1205-12 18. Chou Y.-H. and Rebhun L. 1. (1 986) Purification and characten7~tion of a sea urchin egg calcium-calmodulindelependentkinase with myosin light chain phosphorylating activity. J. Biol. Chem. 261,5389-5395. Chum S. R., Taft W. C., Billingsley M. L., Blair R. E., and DeI& renzo I?. J. ( 1 9 9 0 ~Temperature ) modulation of ischemia-induced neuronal cell death and ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase 11 in gerbils. Stroke 21, 17 15-172 1 . Churn S. B., Taft W. C., and DcLorenzo R. J. ( I 990b) Effects of ischemia on multifunctional calcium/calmodulin-delependent protein kinase I1 in the gerbil. Stroke 21 (Suppl. HI), 111-112111-1 16.

Churn S. B.. Tali W. C., and DeLorenzo R. J. (1991) Ischemia-induced modifications of calcium/calmodulin kinasc 11. (Abstr. 134) liuns Am. SOC.Neurochem. 22,7. Cohcn P. (1989) The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58,453-508. Davies K. J. A. (1 987) Protein damage and degradation by oxygen radicals: I. General aspects. J. Biol. Chem. 262,9895-990 1 . Davies K. J. A. and Delsignore M. E. (1987) Protein damage and degradation by oxygen radicals: 111. Modification of secondary and tertiary structure. J. Biol. Chern. 262, 9908-99 13. DeLorenzo R. J. and Freedman S. D. (1978) Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles. Biochem. Biophys. Res. Cornmiin. 80, 183-1 92. Dehrenzo R. J., Freedman S. D., Yohe W. B., and Maurer S. C. (1979) Stimulation of Ca2’-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation

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Global forebrain ischemia induces a posttranslational modification of multifunctional calcium- and calmodulin-dependent kinase II.

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient gl...
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