Long-chain acyl-CoA hydrolase from rat brain cytosol: purification, characterization, and immunohistochemical localization

To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL; one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF-treated macrophages. Treatment of human monocyte-derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated approximately 50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte-derived macrophages, which might have implications for foam cell formation.     

Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.     

Lipoxygenase activity in rat dermis and epidermis: partial purification and characterization

Lipoxygenase (LOX) activity in epidermis and dermis was distributed among microsomal and cytosolic fractions. The main products of polyunsaturated fatty acid metabolism were 12-hydroperoxy-cis-5,8,14, trans-10-eicosatetraenoic acid (12-HPETE), 15-hydroperoxy-cis-5,8,11, trans-13-eicosatetraenoic acid (15-HPETE) and 13-hydroxy-cis-9, trans-11-octadecadienoic acid (13-HOD). Enzyme activities were isolated from rat dermis and epidermis by ammonium sulphate precipitation, hydrophobic chromatography and gel filtration. In the dermis, activity was found at a molecular mass of 68 kDa, a pI of 4.6 and a Km of 50 microM. This activity was inhibited by known LOX inhibitors. The main reaction products indicated that this was 15-LOX. In the epidermis, activity was found in a fraction with a molecular mass of 68 kDa, a pI of 4.6 and a Km of 80 microM. Activity was inhibited by known LOX inhibitors whereas the reaction products indicated that this was 12-LOX. LOX activity in rat skin may involve one enzyme with dual regional specificities or may comprise two different enzymes.     

Proscillaridin A immunoreactivity: its purification, transport in blood by a specific binding protein and its correlation with blood pressure

A material crossreacting with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump was found in human blood plasma. The concentration of the material with a retention time similar to ouabain in a reversed phase HPLC correlated to systolic blood pressure and pulse pressure. Affinity purification of this compound from bovine adrenals resulted in the isolation of a compound with molecular mass of 600 Da that was not identical with ouabain. Consistent with the postulate that endogenous ouabain and proscillaridin A immunoreactivities may belong to a new class of cardiotonic steroid hormones, a protein of Mr = 60 kDa has been found in bovine serum by affinity-labeling with N-hydroxysuccimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate.     

Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.     

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

The pharmacological specificity of the binding of 125I-labeled alpha-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 alpha-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 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd 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 a partially purified nicotinic acetylcholine receptor protein.     

Human stress protein hsp70: overexpression in E coli, purification and characterization

The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.     

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [3H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). Kd values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). Kd values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. Bmax values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls. These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in Bmax values are consistent with a transient elevation in ACh concentrations after TBI.     

Quantitative autoradiography of halothane binding in rat brain

14C-halothane direct photoaffinity labeling was used to characterize the distribution of halothane binding in rat brain to test the hypothesis that anesthetics bind preferentially to a specific, heterogeneously distributed, receptor or channel. Slide-mounted sagittal rat brain sections were placed in gas-tight quartz cuvettes with 100 microM 14C-halothane in phosphate buffered saline with 0 to 7.5 mM unlabeled halothane, or unlabeled chloroform and isoflurane at 10 times the clinical EC50, and then exposed to UV light for 60 to 100 sec. Autoradiograms of nine brain regions (cortex, corpus callosum, hippocampal molecular and pyramidal layers, dentate molecular and granule cell layers, and cerebellar molecular, granular and white matter layers) were prepared and quantitated using Image 1.44. Total label incorporation was widespread, but exhibited subtle heterogeneity. There was significantly more total labeling in regions of high synaptic density than in regions containing primarily cell bodies or white matter. Most labeling (approximately 80%) was displaced by unlabeled halothane and can therefore be considered specific. Significantly more specific labeling was found in regions of high synaptic density. Isoflurane did not inhibit halothane photolabeling significantly, but chloroform inhibited it by approximately 50%. In conclusion, halothane photolabeling distribution in the mammalian brain is widespread, saturable and selective, but does not mimic the distribution of any individual receptor or channel. Brain regions with high synaptic density displayed the greatest degree of specific binding, consistent with transmission being an important functional target of volatile anesthetics. These results suggest a remarkably widespread individual target, or more likely, similar binding sites in multiple targets, and are consistent with the notion that anesthesia is the result of action at multiple sites.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Biochemical and immunocytochemical characterization of GRIP, a putative AMPA receptor anchoring protein, in rat brain

The mechanisms by which glutamate receptors are concentrated in brain excitatory synapses are believed to involve interactions between receptor subunits and postsynaptic anchoring or scaffolding proteins. Recently GRIP, a protein containing seven PDZ domains, was identified as an AMPA receptor binding protein and implicated in the synaptic targeting of AMPA receptors. Here we show that GRIP mRNA is also expressed in some tissues outside of the brain, including testis and kidney. Specific antibodies were raised to study GRIP protein. On Western blots, GRIP protein appears as a heterogeneous band (approximately 130 kilodaltons) which is expressed in widespread brain regions and throughout postnatal development. Biochemical studies reveal that GRIP is largely membrane associated and enriched in the postsynaptic density (PSD), though not as highly concentrated in the PSD as is PSD-95. By immunohistochemistry, GRIP is distributed in a somatodendritic pattern in neurons of adult rat brain, with especially prominent expression in a subset of interneurons.     

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.     

Purification and characterization of a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells

Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.     

Repetitive transcranial magnetic stimulation activates specific regions in rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique to induce electric currents in the brain. Although rTMS is being evaluated as a possible alternative to electroconvulsive therapy for the treatment of refractory depression, little is known about the pattern of activation induced in the brain by rTMS. We have compared immediate early gene expression in rat brain after rTMS and electroconvulsive stimulation, a well-established animal model for electroconvulsive therapy. Our result shows that rTMS applied in conditions effective in animal models of depression induces different patterns of immediate-early gene expression than does electroconvulsive stimulation. In particular, rTMS evokes strong neural responses in the paraventricular nucleus of the thalamus (PVT) and in other regions involved in the regulation of circadian rhythms. The response in PVT is independent of the orientation of the stimulation probe relative to the head. Part of this response is likely because of direct activation, as repetitive magnetic stimulation also activates PVT neurons in brain slices.     

The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays

Western blot analysis was carried out using crude antigens of Echinococcus multilocularis and 33 serum samples from the People's Republic of China to differentiate patients with alveolar hydatid disease (AHD) with active lesions from those with inactive lesions, those with other parasitoses including cystic hydatid disease (CHD), and healthy persons. This analysis was based on the detection of antibody responses against two polypeptides, Em18 and Em16, which appeared to be species specific and good indices for the differentiation of AHD with active lesions from AHD with inactive lesions. Twenty-one of the 33 samples showed antibody responses against Em18 and/or Em16. Fifteen were antibody-positive for both Em18 and Em16, five were antibody-positive for Em18 only, and one was antibody-positive for Em16 only. Of the subjects from which the 33 samples were obtained, 12 patients had AHD with active lesions, two had AHD, but their lesions were resolved by surgical resections, 14 had CHD with active lesions, and five were apparently healthy people. Western blot analysis confirmed all 14 cases of AHD, three of the 14 cases of CHD, and showed that one of the five apparently healthy individuals had a positive serologic response for AHD. This apparently healthy person with a positive serologic response for AHD by Western blot analysis was found to be positive for the disease on re-examination. Thus, Western blot analysis based on the antibody responses against Em18 and Em16, especially against Em18, was extremely reliable for the detection of AHD with active lesions, although it failed to differentiate two of the 14 patients with CHD.(ABSTRACT TRUNCATED AT 250 WORDS)     

A specific binding protein for cardiac glycosides exists in bovine serum

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.     

A convenient method for affinity purification of maltose binding protein fusions

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; pI, 9.47) was homologous to the C1 and C2 components of prostatein ('estramustine-binding protein'), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; pI, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid-regulated and steroid-binding molecules.     

Partial purification and substrate specificity of flavin-containing monooxygenase from rat brain microsomes

Flavin-containing monooxygenase (FMO) was partially purified from rat brain microsomes through two successive chromatographies on columns of DEAE Sepharose and 2',5'-ADP Sepharose. The specific activity, benzydamine N-oxidation of partially purified brain FMO, was 122-fold higher than that of microsomes. A single band of 60 kDa was recognized by Western blotting analysis with anti-rat liver FMO. The Km value of brain FMO for thiourea was 4-fold lower, but that for cysteamine was 10-fold higher than that of liver FMO. The enzymatic activity for n-octylamine was detected in neither brain nor liver FMO. Kinetic analysis for neurotoxins also revealed that Km values of brain FMO for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and N-methyl TIQ (NMTIQ) were lower than those of liver FMO. These results indicate that rat brain FMO catalyzes several substrates of liver FMO involving neurotoxins, but it seems likely that the kinetic properties of brain FMO are somewhat different from those of liver FMO.