Synaptic Membrane Fractions Research Articles

Degradation of poly-phosphoinositides in brain subcellular membranes in response to decapitation insult

Although it has been well demonstrated that decapitation insult results in a rapid breakdown of the poly-phosphoinositides in brain, the subcellular site(s) for this event has not been examined in detail. Using rats that were injected intracerebrally with (32)Pi to label the brain membrane phosphoinositides, decapitation treatment (0.5, 1.5 and 3.5 min) resulted in a decrease in labeled phosphatidylinositol 4,5-bisphosphates and phosphatidylinositol 4-phosphates in almost all subcellular fractions except myelin. However, the fractions exhibiting the most changes were synaptic vesicles, synaptic plasma membranes and the non-synaptic plasma membranes. The rapid response of poly-phosphoinositides in synaptic vesicles towards decapitation insult demonstrated a role of these phospholipids in vesicular membrane function. Besides the decrease in labeled phosphatidylinositol 4,5-bisphosphates which is attributed mainly to the action of phospholipase C, decapitation insult also elicited a near parallel decrease in labeled phosphatidylinositol 4-phosphates, and this was accompanied by a concomitant increase in labeled phosphatidylinositol which was observed mainly in the synaptic vesicles and synaptic plasma membrane fractions. This latter event suggests that besides degradation of phosphatidylinositol 4,5-bisphosphates by phospholipase C, some phosphatidylinositol 4-phosphates may have been degraded through the phosphomonoesterase pathway.

Regulation of Ca2+/Calmodulin‐Dependent Protein Kinase II by Brain Gangliosides

Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.

Intracerebroventricular treatment of mice with pertussis toxin induces hyperalgesia and enhances 3H-nitrendipine binding to synaptic membranes: Similarity with morphine tolerance

The effect of intracerebroventricular treatment of mice with pertussis toxin (PTX) on pain perception and 3H-nitrendipine binding was examined to study a possible change in the GTP-binding proteins in morphine tolerant rodents. It was observed that both PTX treatment and chronic administration of morphine cause hyperalgesia in the acetic acid-induced writhing test. Analgesic effects brought by the acute administration of morphine or nifedipine, a calcium antagonist, were not affected by PTX treatment. In synaptic membrane fractions prepared from mice treated with PTX or morphine chronically, specific binding of 3H-nitrendipine was enhanced approximately 41.8% and 35.7%, respectively, without alteration in its affinity. Chronic administration of morphine followed by PTX treatment did not display further increases in 3H-nitrendipine binding. These results suggest that the PTX-sensitive GTP-binding proteins may not be involved in the manifestation of the analgesic effect of morphine in mice.

CGP 31358 binds to a site on the NMDA receptor that is coupled to both the transmitter recognition site and the channel domain

CGP 31358, a novel triazole, inhibited the binding of l-[ 3H]glutamate and [ 3H]MK-801 to the N- methyl- d-asparatate (NMDA) receptor complex in rat brain synaptic membrane fractions, and showed anticonvulsant activity in mice. It had no effect on the strychnine-insensitive binding of [ 3H]glycine. Saturation and Hill analyses indicated that CGP 31358 binds to a site on the NMDA receptor which is separate from, but coupled to, both the transmitter recognition site and the channel domain. Available data indicate that this site is distinct from those with which tricyclic antidepressants and ifenprodil interact. CGP 31358 is a new chemical entity with a novel mechanism of action at the NMDA receptor, and as such may form a tool for understanding the molecular pharmacology of this receptor-channel complex.

Ca++-ATPase in the central nervous system: an EM cytochemical study.

Ca++-ATPase plays an important role in regulation of the intracellular Ca++ concentration. Biochemical studies of brain have demonstrated that Ca++-ATPase co-purifies with synaptosomes, with synaptic plasma membrane and synaptic vesicle fractions. To better understand the role of this enzyme in normal brain function, we used an electron microscopic (EM) cytochemical method to determine the localization of Ca++-ATPase in rat brain. Reaction product occurred along cytoplasmic membranes. Specific areas of increased reaction product were seen at many but not all post-synaptic densities. Intracellular Ca++-ATPase reaction product was associated with all synaptic vesicles examined and with the Golgi and smooth endoplasmic reticulum (SER). Unlike the situation in peripheral nerve, Ca++-ATPase at the node of Ranvier in the CNS localized preferentially to the nodal axolemma. The localization of Ca++-ATPase at synaptic vesicles agrees with the biochemical evidence for its localization and with the cytochemical evidence for Ca++-ATPase sequestration in those vesicles. The restricted localization at postsynaptic densities suggests that it may be involved in extrusion of Ca++ at synapses where neurotransmitter release causes Ca++ influx.

Developmental changes in protein phosphorylation in chicken forebrain. II. Calmodulin stimulated phosphorylation

The development of calmodulin stimulated protein phosphorylation, with particular reference to calmodulin-stimulated protein kinase II (CMK II), was investigated in 3 subcellular fractions of chicken forebrain: cytosol (S3), crude synaptic plasma membranes (P2-M) and occluded cytosol (P2-S). Changes in the level of calmodulin-stimulated phosphorylation of endogenous proteins occurred over a protracted time course and were not complete until after day 52 post-hatching. By day 15 post-hatching, calmodulin-stimulated phosphoproteins characteristic of embryonic fractions had all disappeared and those characteristic of adult tissue were present but not necessarily at their mature levels. The levels of CMK II were estimated from the autophosphorylation of the α-subunit which was the only phosphoprotein present at 53,000 Da in the 3 fractions. Overall, calmodulin-stimulated phosphorylation and CMK II levels were low in embryonic brain and high in adult brain but two specific changes in CMK II were observed during development: (1) although CMK II concentrations increased in both membrane and cytosolic fractions until day 23 the kinase was predominantly cytoplasmic (approximately 75%) until day 23, after which it became increasingly membrane bound so that by day 52 post-hatching the majority of CMK II was present in the synaptic membrane fraction, and (2) the relative concentrations of the α- and β-subunits changed from an α:β-value of approximately 1:1 in the 19 day embryo to approximately 1:2 by 15 days post-hatch after which no further change was seen. The occurrence of major changes in the calmodulin stimulated protein phosphorylation system for up to 6–8 weeks after synapse formation is completed in the forebrain, provides further support for the existence of a synapse maturation phase of neuronal differentiation which is distinct from synapse formation. This phase involves only a specific subset of the developmental changes occurring in the calmodulin-stimulated phosphorylation system.

Developmental changes in protein phosphorylation in chicken forebrain. I. cAMP-stimulated phosphorylation

The net level of cyclic AMP-stimulated protein phosphorylation was investigated in cytosolic and membrane fractions from chicken forebrain between embryonic day 13 (E13) and 52 days post-hatching. Throughout this period the majority of the net level of cAMP-stimulated phosphorylation of endogenous proteins was in the cytosolic fractions. Between day −8 (E13) and adult, the net level of CAMP-stimulated phosphorylation of endogenous proteins in the cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions fell by 3 and 4 fold, respectively, when expressed per mg protein and rose by 5 and 10 fold, respectively, when expressed per fraction. The changes in specific activity were completed by 6–15 days post-hatching. The occluded cytosol (P2-S) fraction showed little change in the net level of cAMP-stimulated phosphorylation of endogenous proteins per mg protein. Major changes in phosphoprotein patterns involving both decreases and increases in phosphorylation occurred in all fractions from day −8 (E13) to day 6 post-hatch; thereafter the phosphoprotein bands and their relative intensities were unchanged. Three bands (P90 in S3; P41 and P31 in P2-M) contained major cAMP-stimulated phosphoproteins in embryonic brain but were absent after hatching. When cAMP-stimulated phosphorylation activity was measured in S3 and P-2M using an exogenous peptide substrate (Kemptide) there was no change in kinase activity per mg protein between day −8 (E13) and 30 days post-hatch. This suggests that the decrease in the net level of cAMP stimulated phosphorylation of endogenous proteins was due to the decrease in levels of endogenous phosphoproteins rather than protein kinase activity. These changes in cAMP-stimulated phosphoproteins during development correlated with the period of synapse formation in chicken forebrain.

Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent M r 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the M r of both glycoproteins by some 5–7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.

Transsynaptic impulse activity regulates postsynaptic density molecules in developing and adult rat superior cervical ganglion.

Ganglionic postsynaptic density protein (PSDp) was used to monitor the influence of transsynaptic impulse activity on synaptic structure in the developing and adult rat superior cervical sympathetic ganglion (SCG). Since transsynaptic activity is known to regulate ontogeny of postsynaptic transmitter enzymes, we initially studied the developing ganglion. Denervation in neonates prevented normal development, decreasing calmodulin binding to the ganglionic PSDp by 71% after 4 weeks. During this period, denervation elicited only a 42% decrease in total protein of the synaptic membrane fraction, suggesting that innervation regulates development of various synaptic components differentially. Effects of denervation were extremely rapid, resulting in a 44% decrease in calmodulin binding within 1 day, consistent with regulation by a signaling process such as impulse activity. The effect of impulse activity was examined more directly in adults by treatment with the agents reserpine or phenoxybenzamine, which elicit reflex increases in sympathetic transmission. Administration of reserpine resulted in a progressive 90% increase in calmodulin binding to the PSDp over 4 weeks. Phenoxybenzamine also elicited an increase, mimicking the effects of reserpine. Neither agent altered total protein of the synaptic membrane fraction, suggesting that impulse activity regulates specific synaptic components. Finally, ganglionic denervation in adults decreased PSDp binding within 12 hr, consistent with acute effects of impulse reduction. Our results suggest that transsynaptic impulse activity plays an important role in regulation of specific molecular components of the synapse.

Neurochemical characteristics of a postsynaptic density fraction isolated from adult canine hippocampus

Postsynaptic density and synaptic membrane fractions isolated from hippocampal tissue have been compared to those previously isolated from cerebellum and cerebral cortex. In all respects examined, the isolated hippocampal preparations are similar to the cerebral cortex fractions. The morphology of the postsynaptic density (PSD) preparation is the same and the protein composition is similar, but with higher concentration of the 51-kDa major protein and of calmodulin, and lower concentrations of actin, in the hippocampal PSD fraction. The binding characteristics for glutamate and GABA are also similar between the two fractions, but with higherB max andK D glutamate values and lowerB max and higherK D GABA values for the hippocampal PSD preparation. Both preparations contain GABA A and GABA B receptors. The PSD fraction contains, as does the cerebral cortex fraction, a calmodulin-dependent binding of the Ca 2+ channel antagonist, nitrendipine, as well as a cAMP-dependent and a Ca 2+/calmodulin-dependent protein kinase, with the same respective substrates. The value of the hippocampal fractions for studies on long-term potentiation and on kindling in the hippocampus is discussed.

Do vanadium ions exert any specific effect on brain protein phosphorylation.

It has been shown previously (1) that vanadate stimulates phosphorylation of the overall proteins from the synaptic membranes of rat cerebral cortex. The aim of the present experiments was to investigate whether the action of vanadate and also of vanadyl ions could exert any specific effect on endogenous phosphorylation of proteins from subcellular fractions of the rat brain cortex. Both vanadate and vanadyl ions stimulate phosphorylation of the overall proteins from synaptic membranes and to lesser extent from mitochondria. An attempt was made to estimate the contribution of inhibition of ATPase activity to nonspecific stimulation of phosphate labeling in the synaptic membrane fraction. A band of Mr approx. 37 kDa from synaptic membranes was particularly sensitive to vanadate. In mitochondria both vanadate and vanadyl caused a marked, concentration dependent inhibition of phosphorylation of a band corresponding to Mr approx. 34 kDa. The effect was confined exclusively to the mitochondrial fractions (total, perikaryal and two synaptic types). It was absent in all subcellular fractions tested, including the nuclear one. Phosphorylation of the mitochondrial 34 kDa band is not influenced by cyclic AMP, Ca-calmodulin, shift of pH from 6.6 to 8.1. Alkaline hydrolysis removed almost all phosphate-labeled bands of mitochondria, including that of 34 kDa.

Cerebral metabolism of plasma [14C]palmitate in awake, adult rat: subcellular localization.

Following intravenous injection of [U-14C]palmitate in awake adult rats, whole brain radioactivity reached a broad maximum between 15-60 min, then declined rapidly to reach a relatively stable level between 4 hr and 20 hr. At 44 hr total radioactivity was 57% of the 4 hr value (p less than 0.05). About 50% of palmitate which entered the brain from the blood was oxidized rapidly, producing 14C-labeled water-soluble components which later left the cytosol. Radioactivity in the cytosolic fraction peaked at 45 min and then declined, coincident with the decline in total brain radioactivity. Membrane fractions were rapidly labeled to levels which remained relatively stable from 1 to 44 hr. Increases in the relative distributions of radioactivity were seen between 1 and 4 hr for the microsomal and mitochondrial fractions, and beyond 4 hr for the synaptic and myelin membrane fractions (p less than 0.05). Radioactivity in membrane fractions was 80-90% lipid, 5-13% water-soluble components and 3-17% protein. The proportion of label in membrane-associated protein increased with time. Proportions of radioactivity in the combined membrane fractions increased from 65% to 76% to 80% at 4, 20 and 44 hr, respectively. The results show that plasma-derived palmitate enters oxidative and synthetic pathways to an equal extent, immediately after entry into the brain. At and after 4 hr, the radiolabel resides predominantly in stable membrane lipids and protein. Brain radioactivity at 4 hr can be used therefore, to examine incorporation of palmitate into lipids in vivo, in different experimental conditions.

The occurrence of microtubule-associated proteins 1 and 2 in a synaptic junction preparation from rat cerebrum

SJ proteins termed P1, P2, P3 and P4 were found to have molecular weights very close to MAP1 or MAP2. P1 and P2 reacted with an antibody to the combined antigens, MAP1a, MAP1b and MAP1c, but neither one of the peptide maps of P1, P2, nor P3 resembled either one of the maps of MAP1a plus MAP1b, MAP1c, MAP2a or MAP2b. Thus, proteins 1 and 2 were immunologically related to, but structurally different from, MAP1 subspecies. The protein P4 reacted with an antibody to combined MAP2a and MAP2b, and a peptide map of P4 resembled those of MAP2a and MAP2b. Thus, P4 is probably identical to MAP2. P1, P2, P3 and P4 appeared to be concentrated in SJ as compared to synaptosomes or a synaptic plasma membrane fraction, although the contents of these proteins, especially P1 and P4, were small. P2 (MAP1-related protein) was phosphorylated endogenously both in the presence and in the absence of CaC1 2 plus calmodulin, while the MAP2-related protein P4 in SJ was hardly phosphorylated by the endogenous kinases.

Regulation of molecular components of the synapse in the developing and adult rat superior cervical ganglion.

Rat superior cervical sympathetic ganglion was used to begin studying the regulation of molecular components of the synapse. Ganglionic postsynaptic densities (PSDs)exhibited a thin, disc-shaped profile electron microscopically, comparable to that described for brain. Moreover, the presumptive ganglionic PSD protein (PSDp) was phosphorylated in the presence of Ca2+ and calmodulin, bound 125I-labeled calmodulin, and exhibited a Mr of 51,000, all characteristic of the major PSD protein of brain. These initial studies indicated that ganglionic PSDp and the major PSD protein of brain are comparable, allowing us to study synaptic regulation in the well-defined superior cervical sympathetic ganglion. To obtain enough quantities of ganglionic PSDp, we used synaptic membrane fractions. During postnatal development, calmodulin binding to the ganglionic PSDp increased 411-fold per ganglion from birth to 60 days, whereas synaptic membrane protein increased only 4.5-fold. Consequently, different synaptic components apparently develop differently. Moreover, denervation of the superior cervical sympathetic ganglion in adult rats caused an 85% decrease in ganglionic PSDp-calmodulin binding, but denervation caused no change in synaptic membrane protein 2 weeks postoperatively. Our observations suggest that presynaptic innervation selectively regulates specific molecular components of the postsynaptic membrane structure.

G-proteins in Torpedo marmorata electric organ Differential distribution in pre- and post-synaptic membranes and synaptic vesicles

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the α-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the G o α-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.

Nerve terminal plasma membrane localization of Ca++-Mg++ ATPase

Depolarization of the frog neuromuscular junction results In an Influx of calcium from the extracellular space. The cell calcium concentration is increased several fold and release of neurotransmitter results. Following depolarization, a necessity exists for an outwardly directed calcium pump to return the intracellular Ca++ concentration to resting levels. A calcium pump such as this has been described In the erthrocyte plasma membrane as well as In synaptosomal plasma membrane from guinea pig, synaptic membrane fractions from rat cerebral cortex and the plasma membrane of mouse neuroblastoma X rat glioma hybrid cell line 108CC5. Jorgensen has shown a Ca+++Mg++-dependent ATPase in the sarcoplasmic reticulum of rat skeletal muscle using Indirect Immunoferritin labelling of ultra thin frozen sections.

Thyrotrophin releasing hormone degradation by rat synaptosomal peptidases: Production of the metabolite His-Pro

The dipeptide, His-Pro, is the major product of the degradation of TRH by rat synaptic membranes in vitro. A small amount of His-Pro is also formed from TRH by the synaptosomal soluble fraction. From inhibitor studies, the main route to His-Pro appears to involve removal of the pGlu residue by membrane-bound metal-dependent pyroglutamylaminopeptidase followed by deamidation. The deamidation step is not mediated by proline endopeptidase (EC3.4.21.26) nor dipeptidylpeptidase-IV (EC3.4.14.5) since it is insensitive to bacitracin and diprotin-A, and may therefore involve a novel membrane-bound TRH metabolizing enzyme. His-Pro is degraded rapidly by the soluble synaptosomal fraction, presumably by prolidase (EC3.4.13.9) and more slowly by the synaptic membrane fraction.

Identification and characterization of an N-methyl-D-aspartate-specific L-[3H]glutamate recognition site in synaptic plasma membranes.

Conditions have been developed for an L-[3H]glutamate binding assay in which 85-95% of the specific binding is to a site that corresponds to the N-methyl-D-aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L-[3H]glutamate in 50 mM Tris/acetate, pH 7.4, for 2-20 min at 2 degrees C results in binding with pharmacological characteristics of the electrophysiologically defined N-methyl-D-aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0-7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mM concentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to this N-methyl-D-aspartate site.

Brain spectrin, calpain and long-term changes in synaptic efficacy

This chapter discusses the possibility that proteolytic digestion of cytoskeletal proteins, in particular spectrin, is part of the mechanisms through which physiological activity elicits structural and chemical changes in brain synapses. Recent work from several laboratories has produced a description of the initial events that trigger the long-term potentiation (LTP) of synaptic responses that appears in hippocampus after brief episodes of high frequency electrical stimulation. A likely sequence is as follows: (1) suppression of IPSPs, (2) prolongation of EPSPs, (3) activation of N-methyl-D-aspartate (NMDA) receptors, (4) influx of calcium into target cells. After briefly describing the evidence for this triggering sequence, the review takes up the question of what types of calcium sensitive chemistries are available to synaptic region that could produce functional changes lasting for weeks (i.e., for LTP). It is argued that the partial degradation of spectrin by a calcium-activated protease (calpain) provides a mechanism of this type. Spectrin is a substrate for calpain and both it and a breakdown product comparable to that produced by calpain are found in postsynaptic densities. Moreover, there is substantial evidence that spectrin regulates the surface chemistry and morphology of cells and thus its partial degradation would be expected to produce pronounced and persistent modifications in synapses. To reinforce this point, the review discusses recent findings suggesting that calpain mediated proteolysis of spectrin and other cytoskeletal proteins produces substantial changes in the shape of blood-borne cells and the distribution of their surface receptors. This is followed by a summary of the evidence that morphological changes of a type expected from a calpain-spectrin interaction accompany LTP and that a specific biochemical correlate of LTP is reproduced in crude synaptic membrane fractions by stimulating calpain-induced breakdown of spectrin. The final section considers the possibility that excessive activation of this biochemical process is also responsible for some instances of neuropathology.

Similar physical and kinetic properties of rat brain synaptic membrane and cytosol phosphoinositide phospholipases C

Phosphoinositide phospholipase C (PLC) was extracted from the synaptic membrane fraction of rat brain by 1% sodium deoxycholate. The molecular weight and sedimentation coefficient of the membrane PLC were about 160,000 and 6.7 as estimated by gel filtration and sucrose density gradient centrifugation, respectively. These values of the membrane PLC were identical with those of the cytosol PLC of the same tissue. Moreover, the membrane PLC showed the substrate specificity for phosphatidylinositol, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate and the sensitivity to Ca 2+, sodium deoxycholate and N -ethylmaleimide similar to those of the cytosol PLC. These results indicate that the rat brain synaptic membrane PLC is indistinguishable from the cytosol PLC in physical and kinetic properties.