Proc. Natl. Acad. Sci. USA Vol. 76, No. 9. pi). 4687-4690, September 1979 Neuirobiology Regulation of the state of phosphorylation of specific neuronal proteins in mouse brain by in vivo administration of anesthetic and convulsant agents (protein phosphorylation/protein I/phosphoprotein) ULF STRdMBOM*, JAVIER FORNt, ANNETTE C. DOLPHIN, AND PAUL GREENCARD Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Contributed by Paul Greengard, June 4, 1979

ABSTRACT The effect of drug treatment in vivo on the A. O. Geiszler of Abbott); ethyl ether (Mallinckrodt); chloral state of phosphorylation of two specific neuronal proteins, hydrate (Fisher); and Ly-32PIATP (ICN Chemical and Radio- proteins Ta and Tb, has been studied in mouse brain. For this isotope). Bovine heart protein kinase used in initial experiments purpose, animals were killed by immersion into liquid nitrogen, and proteins Ta and Tb were extracted by a procedure designed was purified through step 3 of the procedure of Rubin et J. (9) to prevent alterations in their state of phosphorylation. Several and then subjected to one further step of purification by chro- anesthetic agents (pentobarbital, chloral hydrate, and urethane) matography on DEAE-cellulose (DE 52, Whatman). For sub- each caused a decrease in the state of phosphorylation of these sequent experiments, the catalytic subunit of bovine heart proteins. Conversely, the convulsant agents pentylenetetrazol protein kinase was purified according to Beavo et al. (10). Bo- and picrotoxin each caused an increase in the state of phos- vine heart protein phosphatase was purified through step 4 of phorylation of these proteins. Neither the anesthetic nor the convulsant agents affected the total amount of these proteins. the procedure of Chou et al. (11). Drugs to be injected were The results are compatible with a role for proteins Ia and Tb in dissolved or diluted in 0.9% sodium chloride and given intra- neuronal function. peritoneally in a volume of 0.4 ml/20 g of body weight. Male CD, mice (Charles River Breeding Laboratories), When subeellular fractions of brain enriched in synaptic weighing 25-30 g, were immersed in liquid nitrogen after membranes are incubated with L[y-32P]ATP, there is a phos- various treatments in vivo. Control animals were included in phorylation of many proteins catalyzed by endogenous protein each experiment. The frozen brain, without the cerebellum and kinase (for review, see ref. 1). However, the phosphorylation part of the lower brain stem, was removed. All subsequent of only a few of these proteins is markedly stimulated by the procedures were carried out at 40C unless otherwise stated. addition of adenosine 3',5'-cyclic monophosphate (cAMP) (2, Determination of Dephosphoprotein I. The brain was 3). Two of the most prominent of these proteins, protein Ia and homogenized in 5 ml of 5 mM Zn(OAc)2 with a motor-driven protein Tb, collectively referred to as protein I, have apparent Teflon pestle. [The use of Zn(OAc)2 was designed to prevent molecular weights of 86,000 and 80,000, respectively. Recent alteration of the state of phosphorylation of protein I during its studies have indicated that protein I can also be phosphorylated isolation (8). l A 1-ml portion of this concentrated homogenate by a Ca2+-dependent protein kinase present in nerve terminals was further diluted into 5 ml of 5 mM Zn(OAc)2 and used for (4, 5). Protein I is found only in nervous tissue (2, 6, 7) and ap- analysis of dephosphoprotein I. pears to be associated specifically with presynaptic vesicles, Determination of Total Protein I. In some experiments, synaptic membranes, and submembranous material in the both dephosphoprotein I and total protein I levels were mea- postsynaptic neuron (experiments carried out in collaboration sured. In these experiments, the frozen brain was split into the with the laboratories of P. Siekevitz and F. E. Bloom). two cerebral hemispheres, which were then weighed. One The development of a method for the selective extraction of hemisphere was homogenized in 5 ml of "standard buffer" protein I from nervous tissue (2) and a recent modification of 1containing 5 mM Hepes (p1l 7.4), 2 mMl ethylene glycol- this method designed to allow extraction of protein I without bis(,3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), alteration of its state of phosphorylation (8) permit a study of 2 mM EDTA, and I 00 units of the protease inhibitor trasylol the state of phosphorylation of protein I in intact tissue. By these per ml]. The other hemisphere was homogenized in 5 ml of 5 techniques, the state of phosphorylation of protein I in rat brain mM Zn(OAc)2, and an aliquot (100 Al) was immediately further slices has recently been found to be altered by incubation in diluted into 5 ml of 5 mM Zn(OAc)2 and used for analysis of vitro with depolarizing agents or with cAMP (8). In the present dephosphoprotein I. investigation, we have studied the effect of in vivo treatment For analysis of total protein I, an aliquot (100 Al) of the ho- wvith anesthetic and convulsant agents, two classes of drugs with mogenate in standard buffer was incubated for 1.3 min at 30'C profound and opposite effects on brain function, on the state with 2 microunits ( 1) of the heart phosphatase in a final vol- of phosphorylation of protein I and on the total concentration ume of 120 Mul in order to convert phosphoprotein I present in of protein I in mouse brain. the extract to dephosphoprotein I; the reaction was stopped by MATERIALS AND METHODS dilution with 5 ml of 5 mM Zn(OAc)2. With [32Plprotein I as substrate, this procedure was found to dephosphorylate 90- Materials. Drugs and chemicals were obtained as follows: 100% of protein I in the homogenate, while resulting in little Trasylol (Mobay, New York, NY); urethane, strychnine sulfate, or no of protein I (unpublished results). As a control, picrotoxin, and pentylenetetrazol (Sigma); pentobarbital sodium proteolysis solution and halothane (Abbott); trimethadione (donated by Abbreviations: cAMP, adenosine 3'.5'-cyclic monophosphate; Na- DodSO4, sodium dodecvl sulfate. The publication costs of this article were defrayed in part by page * Present address: Department of Pharmacology, University of charge payment. This article must therefore be hereby marked "ad- Goteborg. Sweden. vertisement " in accordance with 18 U. S. C. §I1734 solely to indicate t Present address: Department of Pharmacology, University of Bar- this fact. celona, Faculty of Medicine, Barcelona, Spain. 4687 Downloaded by guest on September 26, 2021 4688 Neurobiology: Strombom et al. Proc. Natl. Acad. Sci. USA 76 (1979)

a second aliquot (100 gl1) from the homogenate in standard Table 1. Dephosphoprotein I in mouse brain after in vivo buffer was immediately diluted into Zn(OAc)2 and used for the treatment with some central nervous system depressant and analysis of dephosphoprotein I. No difference was found in the convulsant agents level of dephosphoprotein I between the hemisphere homog- Significance enized in Zn(OAc)2 and the hemisphere homogenized in of standard buffer with immediate dilution of an aliquot into Dephospho- difference Zn(OAc)2, indicating that homogenization in standard buffer proteiti I, from had no effect on the subsequent measurement of protein I. Dose, % of control Analysis of Protein I. The brain homogenates in Zn(OAc)2 Agent mg/kg control (P value) were centrifuged at 1000 X g for 10 min. Protein I was ex- Saline (control) 100 i 3 (24) tracted from the resultant pellet by means of an acid extraction procedure described in detail elsewhere (2, 8). In brief, the Anesthetics pellet was resuspended in 10 mM citric acid and rehomogenized Pentobarbital 75 124 ± 3 (19) <0.01 by hand. An aliquot (1 ml) was adjusted to pH 3.0 and centri- Urethane 2000 120 ± 3 (15) <0.01 fuged at 23,500 X g for 15 min. The supernatant was adjusted Chloral hydrate 400 117 i 3 (15) <0.01 to pH 6 with 0.2 M Na2HPO4 and centrifuged at 4000 X g for Ether Inhaled 102 I 5 (11) NS Halothane Inhaled 95 + 6 (5) NS 15 min. The supernatant was used for the assay of protein I. The amount of dephosphoprotein I present in each extract Convulsants was assayed by determining the amount of 32p incorporated Pentylenetetrazol 100 78 + 3 (15) <0.01 into protein I in the presence of an excess of [y-32P]ATP and Picrotoxin 10 75 + 1 (4) <0.05 added protein kinase. The standard assay mixture (final volume, Strychnine 5 95 + 7 (5) NS 100 ,ul) contained 50 mM Hepes buffer (pH 7.4), 10 mM MgCl2, Anticonvulsant 1 mM EDTA, 0.4 ,g of catalytic subunit of cAMP-dependent Trimethadione 400 105 + 4 (6) NS in an amount protein kinase (or, initial experiments, equivalent Combinations of partially purified cAMP-dependent protein kinase plus 10 Pentobarbital 75 MM cAMP), and 30 Mul of the extract. The presence of EDTA in + pentylenetetrazol 100 108 + 6* (8) NS the final assay mixture was necessary in order to chelate the Trimethadione 400 Zn2+ carried over from the original homogenate which other- + pentylenetetrazol 100 90 ± 5t (6) NS wise would have inhibited the activity of the exogenous protein kinase. The phosphorylation reaction was initiated by the ad- Except for the inhalation anesthetics, agents were given intra- peritoneally. Animals were killed by immersion into liquid nitrogen dition of 100 pmol of ['y-32P]ATP (3-6 X 107 cpm/nmol) in a 15 min after administration of the injectable anesthetics, after 10 min volume of 10 ,l. Incubation was carried out at 300C for 20 min. of exposure to anesthetic concentrations of the volatile anesthetics, The reaction was terminated by the addition of 50 Ml of a 1-2 min (during convulsions) after injection of the convulsants when "NaDodSO4-stop solution" containing 9% sodium dodecyl given alone, 2 min after pentylenetetrazol when given in combination, sulfate (NaDodSO4), 6% 2-mercaptoethanol, 15% glycerol, and and 10 min after injection oftrimethadione. Dephosphoprotein I levels 0.01% bromophenol blue dye in 186 mM Tris.HCl (pH 6.7). of whole brain were measured. Results were calculated as percent of the mean value obtained in saline-treated control animals included Samples (80 Mul) were subjected to NaDodSO4/polyacrylamide in each experiment. Data represent the means +SEM of individual gel electrophoresis as described in detail elsewhere (12), except determinations on the number of animals indicated in parentheses. that the final concentration of acrylamide in the separating gel In separate experiments, dephosphoprotein I levels ofsaline-injected was 8%. The gels were stained for protein with 0.025% Coom- mice were found not to differ from those of noninjected mice (data assie blue, destained, and dried. The protein I band was located not shown). Statistical significance of differences from controls were by autoradiography (6) and cut out from the gel, and the calculated by means of Student's t-test. NS, not significant. * Significantly different from pentobarbital alone (P < 0.05) and amount of 32p was measured by liquid scintillation spectrom- pentylenetetrazol alone (P < 0.01). etry. Under the conditions used, maximal phosphorylation of t Significantly different from pentylenetetrazol alone (P < 0.05). protein I was achieved. The incorporation of radioactive phosphate into dephosphoprotein I was proportional to the injection of various doses of pentobarbital on the amount of amount of dephosphoprotein I present in the assay mixture dephosphoprotein I in brain is shown in Fig. 1. The two lowest (data not shown). doses used produced little or no drowsiness, but slight ataxia; The possibility of in vitro effects of the various drugs used 37.5 mg/kg produced considerable drowsiness and a slight in this study was investigated by their addition to brain ho- decrease in susceptibility to pain; 75 mg/kg produced complete mogenates from control mice at concentrations greater than anesthesia. The level of dephosphoprotein I increased over the those expected in the brain after their in vivo administration. same dose range. No alteration in phosphorylation of protein I was found with The effect of pentobarbital on the amount of dephospho- the following drug concentrations added to the initial homog- protein I was studied as a function of time after administration enate: pentobarbital (40,Mg/ml), pentylenetetrazol (50,Mg/ml), of the anesthetic agent (Fig. 2). The level of dephosphoprotein urethane (1,000 Mg/ml), chloral hydrate (200,Mg/ml), and pi- I reached a peak level between 5 and 20 min after adminis- crotoxin (10,Mg/ml). tration, returning to control levels about 1 hr after drug ad- ministration. Similarly, the anesthetic effect of pentobarbital RESULTS reached a maximum in 10-20 min and had largely disappeared Effect of Anesthetic Agents on Dephosphoprotein I. The within 1 hr. effects of in vivo administration of various anesthetic agents In contrast to the increase in dephosphoprotein I observed on the amount of dephosphoprotein I in mouse brain are shown in animals treated with pentobarbital, chloral hydrate, or ure- in Table 1. When mice were injected intraperitoneally with thane, no such increase was obtained in mice inhaling ether or pentobarbital, urethane, or chloral hydrate in doses sufficient halothane for 10 min in amounts causing complete anes- to cause complete anesthesia, as judged by absence of pain re- thesia. flexes, the amount of dephosphoprotein I was significantly in- Effect of Convulsant Agents on Dephosphoprotein I. The creased above that found in saline-treated mice. The effect of effects of in vivo administration of various convulsant agents Downloaded by guest on September 26, 2021 Neurobiology: Str6mbom et al. Proc. Nati. Acad. Sci. USA 76 (1979) 4689 enetetrazol (100 mg/kg) is shown in Table 2. A statistically 140 significant decrease in dephosphoprotein I relative to controls z was found only when mice were killed during convulsions. In c ° 120 addition, the effect of various doses of pentylenetetrazol on the u level of dephosphoprotein I was examined. In contrast to the 0 effect on dephosphoprotein I observed with a convulsant (100 :.. 100 mg/kg) dose of pentylenetetrazol, lower (25 and 50 mg/kg) C 4a) doses of pentylenetetrazol, which failed to produce convulsions, 0 80 also failed to decrease the amount of dephosphoprotein I (data not shown). 0Q. Pentobarbital (75 mg/kg) given 15 min before pentylene- 0. tetrazol (100mg/kg) was able to antagonize both its convulsant action and its effect on protein I phosphorylation (Table 1). ,, I Animals given both pentobarbital and pentylenetetrazol dis- 0 9.4 18.8 37.5 75 played considerable drowsiness but intact pain reflexes. The Pentobarbital, mg/kg anti-epileptic drug, trimethadione, is a specific antagonist of FIG. 1. Effect of various doses of pentobarbital on the concen- the type of convulsions produced by pentylenetetrazol tration of dephosphoprotein I in mouse brain. Animals were killed (16). 15 min after intraperitoneal administration of the indicated dose of Trimethadione, when given in a dose of 400 mg/kg 10 min pentobarbital. Data represent the means + SEM of individual de- before pentylenetetrazol (100 mg/kg), antagonized both the terminations on three to four experimental animals, and are expressed convulsant action and the biochemical effect (Table 1) of as percent of dephosphoprotein I in saline-injected control animals. pentylenetetrazol. When injected alone, trimethadione (400 Statistical significance of differences from control values were de- mg/kg) had no readily observable behavioral effects and did termined by means of Student's t-test. *, P < 0.05; t, P < 0.01. not alter the state of phosphorylation of protein I compared to control animals (Table 1). This indicates that gross sedation is on the amount of dephosphoprotein I are also included in Table not a prerequisite for inhibition of the biochemical effect of 1. pentylenetetrazol. In a few experiments, a lower dose of tri- Pentylenetetrazol and picrotoxin are thought to produce methadione (200 mg/kg) failed to antagonize either the con- convulsions by an action in the brain. Picrotoxin is thought to vulsant action or the biochemical effect of pentylenetetrazol act as an antagonist of the effects of 7y-aminobutyric acid at (data not shown). receptors for this inhibitory neurotransmitter (13); the mech- Measurements of Total Protein I. In one series of experi- anism of action of pentylenetetrazol is unknown, although there ments, the amounts of both dephosphoprotein I and total pro- is some evidence that it increases cellular K+ permeability (14). tein I were measured in the same samples. The amount of The convulsant action of strychnine is thought to be due to phosphoprotein I was calculated by difference, in brains from blockade of tonic presynaptic inhibition of neurons in the spinal control mice and from mice treated with pentobarbital or cord (15). Mice were injected intraperitoneally with pentyl- pentylenetetrazol (Table 3). The total brain content of protein enetetrazol, picrotoxin, or strychnine and killed at the onset of I was not altered by treatment with either drug; the mean value convulsions, which occurred 1-2 min after administration. for all animals was 1.4 ± 0.03 pmol/mg of tissue weight (n = Pentylenetetrazol and picrotoxin decreased the amount of 26). Pentylenetetrazol (100 mg/kg) increased the amount of dephosphoprotein I relative to controls, but strychnine did not. protein I from 32.8 ± 1.9% in the phospho-form in control an- In some were experiments mice injected with the convulsant imals to 45.1 ± 2.2% during convulsions. Pentobarbital (75 agents, in the doses indicated in Table 1, for purposes of ob- mg/kg) reduced the amount of protein I in the phospho-form servation. In these mice, pentylenetetrazol was almost never from 33.1 + 2.7% in control animals to 21.2 + 3.3% during fatal, picrotoxin produced fatal convulsions in most animals anesthesia. about 10 min after administration, whereas strychnine caused Comparison of Procedures for Tissue Fixation. Because death a few seconds after the onset of convulsions in all ani- many metabolic changes occur rapidly during the killing of mals. animals (see ref. 17), it seemed possible that the level of de- The amount of protein I in the dephospho form in mice be- phosphoprotein I measured in control animals, as well as the fore, during, and after initial convulsions induced by pentyl- changes in this level induced by drug treatment in vivo, might be critically dependent on the mode of killing and tissue fixa- p 140- tion. In a series of experiments, the levels of a dephosphoprotein 0 t "' 120- Table 2. I in mouse in 1aR Dephosphoprotein brain after vivo treatment with pentylenetetrazol c 100- Condition % of control Q. 80 Control 100 + 5 (6) 0Q. I1 Pentylenetetrazol-treated 0.Q. (a) Before convulsion 88 + 8 (5) a) I I I L ,, 0 025 10 20 40 60 120 (b) During convulsion 74 + 3* (4) min after pentobarbital (c) After convulsion 83 ± 6 (5) FIG. 2. Dephosphoprotein I in mouse brain as a function of time Mice were killed by immersion into liquid nitrogen, after admin- after intraperitoneal injection of pentobarbital. Data represent the istration of pentylenetetrazol (100 mg/kg): (a) before any behavioral means SEM of individual determinations on four to eight experi- signs of the onset of convulsions were evident, (b) during the clonic mental animals treated with pentobarbital (75 mg/kg), and are ex- phase of the first convulsion, or (c) after recovery from the first con- pressed as percent of dephosphoprotein I in noninjected control an- vulsion. Results were calculated as described in the legend to Table imals. Statistical significance of differences from control values were 1. determined by means of Student's t-test. *, P < 0.05; t, P < 0.01. * P < 0.05 compared to control saline-treated group. Downloaded by guest on September 26, 2021 4690 Neurobiology: Strombom et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 3. Dephosphoprotein I, total protein I, and calculated phosphoprotein I levels in brains of control mice and of mice treated with pentobarbital or pentylenetetrazol Phospho- Dephospho- Total protein I n protein I protein I (calculated) pmol/mg of tissue weight Control 5 0.91 i 0.06 1.36 + 0.06 0.45 ± 0.03 Pentobarbital 5 1.11 + 0.08* 1.41 + 0.06 0.30 ± 0.04* Control 8 0.95 + 0.04 1.42 + 0.05 0.47 + 0.03 Pentylenetetrazol 8 0.82 ± 0.04* 1.48 ± 0.08 0.67 ± 0.07t Mice were given pentobarbital (75 mg/kg), pentylenetetrazol (100 mg/kg), or saline (control) intra- peritoneally and killed, as described in the legend to Table 1. Dephosphoprotein I levels were determined by measuring the amount of 32p incorporated into protein I in extracts prepared from tissue homogenized in 5 mM Zn(OAc)2 and in extracts prepared from tissue homogenized in standard buffer with immediate dilution into Zn(OAc)2. The results obtained by the two procedures showed no significant difference. Therefore, in each experiment the data obtained by the two procedures were pooled and used as the level of dephosphoprotein I for calculation of the amount of protein I in the phosphorylated form in vivo. Total protein I was determined after pretreatment of the homogenate in standard buffer with a partially purified phosphatase. Each value represents the mean i SEM for n experiments, in each of which triplicate or quadruplicate aliquots of homogenate were taken through the extraction and assay procedure. * P < 0.05 compared to respective control values. t P < 0.01 compared to respective control values. I found after decapitation of mice and immediate immersion studies in brain slices and in intact brain, taken together, indi- of the head into liquid nitrogen were similar to those obtained cate that neuronal activity alters the state of phosphorylation in animals killed by direct immersion into liquid nitrogen, both of protein I and suggest that protein I plays an important role in control mice and in mice treated with either pentobarbital in neuronal function. (75 mg/kg) or pentylenetetrazol (100 mg/kg) (data not shown). Thus, the rapid freezing procedure used in the present study This work was supported by U.S. Public Health Service Grants may provide a reasonable assessment of drug-induced changes DA-01627, MH-17387, and NS-08440, and grants from the McKnight in protein I phosphorylation occurring in vivo. However, the Foundation and from Hoffmann-La Roche. possibility cannot be excluded that alterations occur in the state of phosphorylation of tissue proteins during freezing (18). 1. Greengard, P. (1978) Cyclic Nucleotides, Protein Phosphoryl- ation and Neuronal Function (Raven, New York). 2. Ueda, T. & Greengard, P. (1977) J. Biol. Chem. 252, 5155- DISCUSSION 5163. In a recent study, it was found that depolarization of neuronal 3. Kelly, P. T., Cotman, C. W. & Largen, M. (1979) J. Biol. Chem. membranes by veratridine or by high K+ caused a Ca2+-de- 254, 1564-1575. pendent increase in the state of phosphorylation of protein I in 4. Krueger, B. K., Forn, J. & Greengard, P. (1977) J. Biol. Chem. slices of rat cerebral cortex (8). Incubation of the slices with 252, 2764-2773. 5. Schulman, H. & Greengard, P. (1978) Proc. Natl. Acad. Sci. USA cAMP, 8-bromo-cAMP, N6-monobutyryl-cAMP, or a phos- 75,6295-6299. phodiesterase inhibitor, isobutylmethylxanthine, also increased 6. Ueda, T., Maeno, H. & Greengard, P. (1973) J. Biol. Chem. 248, the state of phosphorylation of protein I but in a Ca2 -inde- 8295-8305. pendent manner. In the present study, the convulsant agents 7. Sieghart, W., Forn, J., Schwarcz, R., Coyle, J. T. & Greengard, pentylenetetrazol and picrotoxin increased, and the depressant P. (1978) Brain Res. 156,345-350. agents pentobarbital, urethane, and chloral hydrate decreased, 8. Forn, J. & Greengard, P. (1978) Proc. Natl. Acad. Sci. USA 75, the state of phosphorylation of protein I in mouse brain in 5195-5199. vivo. 9. Rubin, C. S., Erlichman, J. & Rosen, 0. M. (1972) J. Biol. Chem. A significant difference between the measurements of brain 247,36-44. 10. Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1974) Methods En- protein I in slices and in whole animals was the amount of zymol. 38C, 299-309. protein I in the phosphorylated form under "control" condi- 11. Chou, C.-K., Alfano, J. & Rosen, 0. M. (1977) J. Biol. Chem. 252, tions. In the slices, the amount of protein I in the phosphorylated 2855-2859. form was almost undetectable (8). In contrast, in the present 12. Liu, A. Y.-C. & Greengard, P. (1976) Proc. Natl. Acad. Sci. USA studies on whole mouse brain, about one-third of protein I was 73,568-572. in the phosphorylated form. The reasons for this marked dif- 13. Galindo, A. (1969) Brain Res. 14, 763-768. ference are not clear. It could be that the "control" brain slices, 14. Gross, G. J. & Woodbury, D. M. (1972) J. Pharmacol. Exp. Ther. lacking the physiological activity characteristic of intact living 181,257-272. brain, had an artifactually low level of phosphoprotein I. 15. Goodman, L. S. & Gilman, A. (1976) The Pharmacological Basis it may be that excessive neuronal activity in the of Therapeutics (Macmillan, New York), Chap. 18. Conversely, 16. Everett, G. M. & Richards, R. K. (1944) J. Pharmacol. Exp. Ther. brain, associated with the procedure of freezing the whole 81, 402-407. animals, resulted in an artifactually high level of phosphopro- 17. McIlwain, H. & Rodnight, R. (1962) Practical Neurochemistry tein I. It would appear impossible, with the techniques currently (Churchill, London), Chap. 1. available, to determine the true state of phosphorylation of 18. Barany, K., Barany, M., Gillis, J. M. & Kushmerick, M. J. (1979) protein I in the brain in vivo. Nevertheless, the results of these J. Biol. Chem. 254,3617-3623. Downloaded by guest on September 26, 2021