Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.     

Phosphorylation of B-50 Protein by Calcium-Activated, Phospholipid-Dependent Protein Kinase and B-50 Protein Kinase

B-50 is a brain-specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B-50 protein. Only calcium-activated, phospholipid-dependent protein kinase (kinase C) and B-50 protein kinase were able to use B-50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B-50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B-50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B-50 protein kinase is a calcium-dependent, phospholipid-stimulated protein kinase of the same type as kinase C.     

Comparison of a 52-kDa Phosphoprotein from Synaptic Plasma Membranes Related to Long-Term Potentiation and the Major Coated Vesicle Phosphoprotein

In the in vitro hippocampal slice preparation a short tetanus induces long-term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52-kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52-kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52-kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: relative molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, phospho-amino acid content, and isoelectric point. Since coated vesicles are thought to be involved in receptor-mediated endocytosis and membrane recycling, our data suggest that LTP-correlated changes in 52-kDa phosphorylation may reflect increased coated vesicle activity.     

Neurone-Specific Enolase and Creatine Phosphokinase Are Protein Components of Rat Brain Synaptic Plasma Membranes

Abstract: Neuron-specific enolase and creatine phosphokinase were found, by 2-dimensional gel analysis, in rat brain synaptic plasma membranes (SPM). The identity of these enzymes was confirmed by comigration with purified rat brain NSE and CPK and by peptide analysis. The specific enzymatic activities of enolase and creatine phosphokinase, as well as of pyruvate kinase, also present on the membranes, were comparable to those in the homogenates when these three enzymes were fully activated. In the SPM all three enzymes, particularly enolase, were partially cryptic in that enzymatic activities were very low unless the membranes were treated with Triton X-100. They were resistant to both low-salt and high-salt extraction and to trypsin, except when Triton X-100 was present. These results suggest that the enzymes are tightly bound protein components of the membrane and that they may constitute an assembly capable of generating ATP.     

A Radioimmunoassay for the Phosphoprotein B-50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

Resolution of Rat Brain Synaptic Phosphoprotein B-50 into Multiple Forms by Two-Dimensional Electrophoresis: Evidence for Multisite Phosphorylation

Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5-5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.     

A Protein Modulator Stimulates C Kinase-Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

Okadaic Acid-Induced Inhibition of B-50 Dephosphorylation by Presynaptic Membrane-Associated Protein Phosphatases

The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Evidence for the Binding of Calmodulin to Endogenous B-50 (GAP-43) in Native Synaptosomal Plasma Membranes

The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.     

Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4-, 14–15-, and 24–25-month-old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.     

Decreased Phosphorylation of GAP-43/B-50 in Striatal Synaptic Plasma Membranes after Circling Motor Activity

The effects of spontaneous circling motor activity on the in vitro phosphorylation of the protein kinase C substrate GAP-43/B-50 was studied on striatal membranes of developing rats (30 days of age). At this time of postnatal development, permanent plastic changes in cholinergic and dopaminergic systems are produced by physiological motor activity. Exercised animals showed a significant reduction of 31% in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum respect to the ipsilateral side (P < 0.01), while control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of exercised animals showed a 33% reduction in the incorporation of ³²P-phosphate into GAP-43/B-50 30 minutes post-exercise (P < 0.01). This change in GAP-43/B-50 phosphorylation was correlated with the running speed developed by the animals (r:0.8986, P = 0.015). GAP-43/B-50 immunoblots revealed no changes in the amount of this protein in any group. Moreover, a significant variation of 25% (P < 0.05) in the PKC activity was seen between both exercised striata. Interhemispheric differences were not found in control animals. We conclude that endogenous phosphorylation of this protein is also altered by motor activity in the same period that permanent changes in striatal neuroreceptors are triggered after motor training.     

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attachment of the Synapse-Specific Phosphoprotein Protein I to the Synaptic Membrane: A Possible Role of the Collagenase-Sensitive Region of Protein I

The purified synapse-specific phosphoprotein Protein I was previously shown to be degraded by a bacterial collagenase, through a series of intermediates, to a collagenase-resistant fragment of molecular weight about 48,000 containing a phosphorylated serine residue. In this study, a purified synaptic membrane fraction containing Protein I was treated with Cl. histolyticum collagenase; membrane-bound and membrane-free proteins were then phosphorylated using [gamma-32P]ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. It was observed that Protein I bound to the synaptic membrane was susceptible to the collagenase and degraded to fragments of molecular weights about 68,000, 62,000, and 48,000; the 68,000 fragment remained bound to the membrane whereas the 62,000 and 48,000 fragments were dissociated from the membrane. These observations suggest that the peptide moiety of mol. wt. 6000, present in the 68,000 fragment but absent from the 62,000 fragment, may play a crucial role in anchoring Protein I to the synaptic membrane.     

Synaptic Protein Tyrosine Kinase: Partial Characterization and Identification of Endogenous Substrates

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.     

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

具异型胞蓝细菌 Anabaena sp.PCC 7120质膜和类囊体膜的分离纯化 李斌 徐冬一 赵进东*     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.