Rat Brain Particulate Fraction Research Articles

Characterization of 3H-AVP binding sites in particulate preparations of rat brain

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni>Co>Mg>Mn>no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays. Forty-six % of the specific 3H-AVP binding was concentrated in the synaptosomal fractions with only 17% and 7% in the myelin and mitochondrial fractions, respectively. Scatchard analysis of the high-affinity binding to the 12K hippocampal pellet gave a dissociation constant of 11 nM. This study shows that specific binding sites for AVP are present in rat brain and that the cellular location of binding of AVP in the hippocampus and septum differs from that in other brain regions.

Further characterization of the heat-stable factor in the α-hydroxylation and oxidation of lignoceric acid in brain: Effect of acidic amino acids and hexose-phosphates on brain fatty acid metabolism

Lignoceric acid and other very long-chain fatty acids are converted to α-hydroxy fatty acids and ceramide in brain. These fatty acids are also oxidized and produce glutamic acid and other water-soluble products. All of these metabolic conversions are catalyzed by a rat brain particulate fraction and require NADPH, heat-labile factor, and heat-stable factor. The heat-stable factor was prepared from calf cerebellum. Glucose 6-phosphate and N-acetylaspartic acid have previously been identified as active components of the heat-stable factor. We report in this manuscript that glutamic acid, glutamine, aspartic acid, and γ-aminobutyric acid as well as inorganic phosphate and adenosine nucleotides are also active components of the heat-stable factor. When the amino acids, glucose 6-phosphate, AMP, and phosphoric acid were combined, full activity of the heat-stable factor for the formation of cerebronate (by α-hydroxylation) and glutamate (presumably by β-oxidation) from lignoceric acid was recovered. The role of the acidic amino acids in the metabolic conversion of lignoceric acid in brain appears to be their conversion to the corresponding α-keto acids and then incorporation into the TCA cycle. Glucose 6-phosphate is also likely to be involved in the TCA cycle through the Emden-Meyerhof pathway. Inorganic phosphate and AMP seemingly are used to produce ATP. However, the addition of up to 20 m m ATP alone did not replace the heat-stable factor.

Purification and inhibition by neuropeptides of angiotensin-converting enzyme from rat brain.

Angiotensin-converting enzyme was solubilized with papain from a particulate fraction of rat brain and purified to apparent homogeneity by a procedure including DEAE-cellulose, hydroxylapatite, Sephadex G-200, Cys(Bzl)-Pro-Sepharose, and ricin-Sepharose chromatography. Bradykinin potentiators, SQ 14,225, and Arg-Pro-Pro strongly inhibited the activity of the purified enzyme, whereas Phe-Ala, phosphoramidon, and pentobarbital exerted little inhibitory effect on the activity. Among neuropeptides investigated, substance P, bradykinin, and Leu-enkephalin (Arg6) exerted strong inhibitory actions on the enzyme. Furthermore, the latter two peptides were shown to be good substrates for the enzyme. Thus, angiotensin-converting enzyme of rat brain is distinct from endogenous enkephalinase and may interact with various neuropeptides located in the brain.

Phorbol diester receptor copurifies with protein kinase C.

The phorbol diester receptor present in the particulate fraction of rat brain was solubilized by divalent ion chelation in the absence of detergents. The soluble receptor was partially purified by (NH4)2SO4 precipitation, DEAE-cellulose, and gel filtration chromatography. The purified receptor required exogenous phospholipid for activity and displayed a Kd of 7 nM for [3H]phorbol 12, 13-dibutyrate. Biologically active phorbol analogs inhibited binding, whereas inactive analogs did not. The Ca2+-dependent, phospholipid-sensitive protein kinase C copurified with the phorbol receptor. Purified protein kinase C was activated directly by phorbol 12-myristate 13-acetate in the presence of phospholipid.

ABSORPTION LOSS OF PEPTIDES TO THE PLASTIC TUBE IN RADIORECEPTOR ASSAY OF CALCITONIN AND β-ENDORPHIN : PROTECTION BY DETERGENTS

AbstractDuring development of the calcitonin binding assay using rat brain particulate fraction, it was noticed that 125I-salmon calcitonin-I (125I-SCT) was partly absorbed by the polypropylene tube surface in an irreversible manner. Absorption was partially prevented by cold SCT and complete prevention was achieved by bacitracin. Triton X-100, Brij 36T, and some Zwittergents in such concentration ranges which appeared not to disrupt the 125I-SCT binding ability of brain tissue. The. brain fraction itself exhibited a similar preventive effect. The a nti- absorbí ç g effect of Brij 36T was also observed in an opiate receptor binding assay for β-endorphin. These results led us to recommend that the binding assay for these peptides should be done only in the presence of an appropriate anti-absorbant.

Absorption loss of peptides to the plastic tube in radioreceptor assay of calcitonin and beta-endorphin: protection by detergents.

During development of the calcitonin binding assay using rat brain particulate fraction, it was noticed that 125l-salmon calcitonin-l (125-SCT) was partly absorbed by the polypropylene tube surface in an irreversible manner. Absorption was partially prevented by cold SCT and complete prevention was achieved by bacitracin. Triton X-100, Brij 36T, and some Zwittergents in such concentration ranges which appeared not to disrupt the 125l-SCT binding ability of brain tissue. The brain fraction itself exhibited a similar preventive effect. The anti-absorbing effect of Brij 36T was also observed in an opiate receptor binding assay for beta-endorphin. These results led us to recommend that the binding assay for these peptides should be done only in the presence of an appropriate anti-absorbant.

Specific binding of 125I-salmon calcitonin to rat brain: regional variation and calcitonin specificity.

Rat brain particulate fraction was found to contain binding sites for 125I-Salmon Calcitonin-I (125I-SCT). Maximum binding occurred in the physiological pH range of 7.25-7.5. The binding reaction proceeded in a temperature-dependent manner. Binding sites were broadly distributed among the various rat brain regions and considerable regional differences existed in the affinity and density as detected by Scatchard analysis. The highest affinity was recorded in the case of the hypothalamus and the lowest in the case of the cerebellum. The KD (nM) and Bmax (pmole/mg protein) estimated for the binding to four regions were as follows: hypothalamus: 1.4 and 0.19, midbrain, hippocampus plus striatum: 1.5 and 0.08, pons plus medulla oblongata: 3.0 and 0.15 and cerebellum: 8.3 and 0.20. Using a particulate fraction of rat brain void of cerebellum and cortices, a binding assay for calcitonins was developed. Binding of 125I-SCT was inhibited by unlabeled salmon, [Asu1,7]-eel and porcine calcitonins in a dose-dependent manner and the IC50s were 2.0, 8.0 and 30 nM, respectively. The IC50s were comparable to those estimated using a kidney particulate fraction. Human calcitonin, beta-endorphin and substance P were weak inhibitors of the binding. Other peptides, drugs and putative neurotransmitters tested (totally 23 substances) failed to inhibit the binding at concentrations of 1.0 microM. The physiological significance of brain binding sites for calcitonin, with the possibility that the brain may possess endogenous ligands for these sites are discussed.

Radioreceptor assay of methionine-enkephalin-like substance in human cerebrospinal fluid

A sensitive and simple radioreceptor assay system for measuring methionine(met)-enkephalin-like substance in human cerebrospinal fluid (CSF) was developed using a particulate fraction of rat brain as a receptor preparation and [ 3H]dihydromorphine as a radiolabeled ligand in the presence of 1 mM ethylenediamine tetraacetate (EDTA) and 2 mM magnesium acetate. Metenkephalin-like substance was purified from CSF by the combination of Sephadex G-10 and SP-Sephadex (H +) column chromatographies to be free of sodium and large molecular weight substance such as β-endorphin. The assays were carried out on samples obtained from normal subjects and patients with the disease of the brain or pituitary by lumbar or ventricular puncture. Mean level in samples obtained from normal subjects by lumbar puncture was 2.6 ± 1.0 pmoles/ml.

Specific binding of 3H-substance P to rat brain membranes.

The undecapeptide substance P is a putative neurotransmitter in the mammalian central nervous system (CNS), and may be associated with pain fibres in the spinal cord. Radiolabelled derivatives of other neuropeptides have been used to demonstrate specific interactions with receptor sites on brain membranes, and this approach has now been explored with substance P. We have now prepared [4-3H-Phe8]-substance P and we find that it binds reversibly to a saturable population of sites in rat brain particulate fractions. Scatchard analysis of concentration-dependent saturation of binding indicates a single population of non-interacting sites with a high affinity (Kd=0.38 nM) and a low density (Bmax=27.2 fmol per mg protein). Kinetic analyses indicate an apparent dissociation equilibrium constant of 0.46 nM. A variety of neurotransmitter amines and amino acids, and other peptides do not compete at the substance P sites, but structurally related peptides or shorter C-terminal fragments of substance P are active. The rank order of potency of these substance P-related peptides agrees with that reported for their effects in depolarizing spinal cord neurones. The regional distribution of the specific binding sites for 3H-substance P parallels that of substance P immunoreactivity, being high in the hypothalamus and low in the cerebellum and cerebral cortex. The characteristics of the 3H-substance P binding sites are consistent with those expected for substance P receptors.

Cholecystokinin receptors in the brain: characterization and distribution.

Specific cholecystokinin binding sites in particulate fractions of rat brain were measured with iodine 125-labeled Bolton-Hunter cholecystokinin, a cholecystokinin analog that has full biological activity. Binding was detected in brain regions known to contain immunoreactive cholecystokinin. Binding was saturable, reversible, of high affinity (dissociation constant, 1.7 x 10(-9) M), and was inhibited by cholecystokinin analogs but not by unrelated hormones.

Decreases in the binding of [ 3H]GABA and [ 3H]muscimol to a particulate fraction of rat brain produced by human sera; some speculations

1. 1. The effects of control human sera and sera from patients afflicted with certain neurological disorders on the binding of [ 3H]GABA and [ 3H]muscimol (both at 6.2 × 10 −9 M) were compared in a crude synaptic membrane fraction of rat brain. 2. 2. The presence of undiluted whole sera (representing about 22% of the total volume of final suspensions) decreased the binding of both [ 3H]GABA and [ 3H]muscimol. 3. 3. Sera from patients with epilepsy and encephalopathies decreased the binding of [ 3H]GABA, but not that of [ 3H]muscimol, to a greater extent than did control sera.

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.

Biological activity and enzymic degradation of substance P analogs: Implications for studies of the substance P receptor

Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.

Comparison of the “specific” binding of [ 3H]GABA and [ 3H]muscimol to a particulate fraction of rat brain

1. Over an identical concentration range (3.08 × 10 −9−3.08 × 10 −8 M), both [ 3H]GABA and [ 3H]muscimol were bound to a particulate fraction of rat brain by high-affinity processes in the absence of added Na +. 2. Components of these processes that were displaceable by excess unlabelled GABA had K B ⋍ 2 × 10 −7 M and B max ⋍ 1 pmol/mg protein for [ 3H]GABA and K B ⋍ 7 × 10 −8 M and B max ⋍ 1.9 pmol/mg protein for [ 3H]muscimol. 3. Pellet/supernatant distribution ratios and B max values indicated that more [ 3H]muscimol sites than [ 3H]GABA sites exist in rat brain. 4. [ 3H]Muscimol binding sites need not be the same as those that bind [ 3H]GABA to be sensitive to excess unlabelled GABA. 5. The concept of “specific” GABA binding is further qualified.

A novel synthesis of ceramide from lignoceric acid and sphingosine by rat brain preparation; the amide formation requires a pyridine nucleotide

The conversion of free lignoceric acid and sphingosine to lignoceroyl sphingosine (ceramide) by rat brain particulate fraction and two cytosolic factors, one heat-stable and the other heat-labile, requires pyridine nucleotide. This enzymatic reaction is apparently different from two previously published enzymic reactions, microsomal sphingosine:acyl CoA acyltransferase and the reverse reaction of lysosomal ceramidase. The reaction is strongly inhibited by common respiratory chain inhibitors, KCN, Antimycin A and sodium azide, this indicates the involvement of an electron-transfer system. From these observations it appears that the brain ceramide synthesis described above is catalyzed by an enzyme system which involves a mechanism for amide formation which has not been previously characterized.

Molecular resolution and reconstitution of the GPP(NH) and NAF sensitive adenylate cyclase system

When a special detergent-extraction procedure is applied to rat brain particulate fractions, the latter's adenylate cyclase activity becomes virtually unresponsive to NaF or Gpp (NH)p (guanylyl-5′-imidodiphosphate) despite the fact that under these conditions the enzyme does not appear to be removed (i.e., solubilized) from the membranes. Addition of exogenous fractions of detergent-solubilized membranes or of water-soluble samples of homogenates, obtained from various tissues, restores the stimulation of the enzyme by both Gpp(NH)p and NaF. These findings indicate that the stimulation caused by these agents is mediated by one or more regulatory component(s), and that these are molecular components physically distinct from the enzyme itself. The regulatory component(s) appear to be proteinaceous in nature and sensitive to SH-reactive reagents. The properties of the reconstituted system resemble those of the original particulate adenylate cyclase. This system may serve as a convenient tool for the study of the molecular properties of adenylate cyclase and of the basis of its regulatory control.

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of 125I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of 125I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K d of 1·10 −8 M. Nicotine and acetylcholine iodide at concentrations of 10 −5 M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10 −4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10 −4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain. Evidence for an activator deficient form

A cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain was partially purified after solubilization with a non-ionic detergent. Influence of divalent ions, of phosphodiesterase inhibitors and of the activator protein from different sources were tested. Determination of K m-values shows two enzymes with different values, one at 7.3 μM and the other at 15 mM. Two distinct activity peaks were determined after resolution by isoelectric focusing. It was concluded that this particulate enzyme is regulated in a way opposite to that of the soluble enzyme and is independent from calcium and the activator protein.

Selective control of calcium levels by naloxone

Acute treatment with morphine sulfate produces a selective loss of calcium from synaptosomal particulate fractions of rat brain. No changes in sodium, potassium or magnesium content were observed for myelin, synaptosomal particulate or mitochondrial fractions. Acute opiate treatment (90 min.) while causing calcium loss, produced no changes in regional brain content for sodium, potassium or magnesium. Naloxone, in the presence of morphine, reversed the calcium loss in both regional brain areas and synaptosomal particulate fractions. An hypothesis is offered that naloxone may bind to synaptosomal membranes protecting a morphine sensitive calcium pool, or may reverse the calcium loss seen after opiate agonist treatment.

Regulation of transsynaptically elicited increase of 3':5'-cyclic AMP by endogenous phosphodiesterase activator.

Experiments with cold exposure confirmed previous studies indicating that the endogenous protein acitvator of phosphodiesterase (PDEA) isolated by Cheung participates in the in vivo regulation of 3':5'-cyclic adenosine monophosphate (cAMP) in adrenal medulla. This activator of cAMP phosphodiesterase (PDE) (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) is present in the particulate as well as the soluble fractions of rat brain. It was found that a purified cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), in the presence of ATP and cAMP, stimulates 3-fold the release of PDEA from the particulate fraction of rat brain and adrenal medulla. The substrate for this phosphorylation could be either a membrane protein that binds PDEA or PDEA itself. In vivo evidence, however, obtained by injecting rats intraventricularly with [gamma-32P]ATP, indicates that the PDEA does not contain radioactive phosphate in its structure. Also, PDEA could not be phosphorylated by protein kinase in vitro. The following mechanism is postulated: when the intracellular content of cAMP increases it activates a protein kinase which phosphorylates a PDEA-binding membrane protein and releases PDEA. In turn this binds to activator-deficient high Km PDE and decreases its Km to facilitate the hydrolysis of the increased concentration of cAMP.