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This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40 % of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7 %, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for ’brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core α1-6-linked and outer-arm α1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewisx and NeuAcα2-3 substituted Galβ1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal β1-3-linked galactose residues was observed. The presence of β1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAcα2-3Galβ1-3(NeuAcα2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one β1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues.... [ABSTRACT FROM AUTHOR]

Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However. in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosomal membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X-100, and was partially purified by gel filtration on a Sephacryl S-200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrata specificity towards proline-containing neuropeptides. The partially purified enzyme was inhibited by certain thiol-protease inhibitors and was also found to be sensitive to the metal chelator 1,l0-phenanthroline. [ABSTRACT FROM AUTHOR]

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ilc-GIy-Leu-MetNH2 (eledoisin). Using (Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase. [ABSTRACT FROM AUTHOR]

A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNasas Y, A and R. which produce acid-soluble products when incubated at near-neutral pH in the absence of metal ions. RNases Y and A yield products with the monoesterified phosphate at the 3′ position, through 2′,3′-(cyclic)phosphate intermediates. Oligonncleotides terminating with a 2′,3′-(cyclic)phosphate are the end-products of the action of RNase R, Double-stranded substrates are highly resistant to the action of all enzymes. On the basis of limited hydrolysis of end-labelled 5S RNA, the three enzymes differ in their preference for the susceptible phosphodiester bond. Thus, RNase Y hydrolyses preferentially the YpN bond, RNase A the ApN bond and RNase R the RpU bond where R is guanosine in most cases. The advantages and disadvantages of using homopolyribonucleotides and dephosphorylated dinucleotides and trinucleotides in determining various aspects of the specificity of RNases are discussed. [ABSTRACT FROM AUTHOR]

A method is described for the isolation of CDP-diglyceride from bovine brain. Yields of the product ranged from 9.2–15.5 μmol per kilogram of tissue, which corresponds to about 1% of the level of phosphatidic acid. Mild alkaline hydrolysis of the product gave three water-soluble phosphate esters which had the same electrophoretic mobilities as CMP, CDP-glycerol and glycerol 3-phosphate, The liponuclentide was quantitatively hydrolysed by CDP-diglyceride hydrolase from Escherichia coli to phosphatidic acid and CMP. No dCMP was recovered in enzymatic or alkaline hydrolysates and it is concluded there can be little or no dCDP-diglyceride in bovine brain. Brain CDP-diglyceride was similar to phosphatidylinositol in that in both lipids stearate was the major saturated fatty acid and arachidonate the most abundant unsaturated fatty acid. This differed significantly from the fatty acid patterns of other metabolically related phospholipids. phosphatidic acid and cardiolipin. Brain CDP-diglyceride was hydrolysed with phospholipase C from Clostridium welchii with the liberation of the diglyceride moiety in high yield. Treatment of the diglyceride with pancreatic lipase showed CDP-diglyceride with the asymmetric distribution of fatty acids characteristic of most mammalian phospholipids, saturated fatty acids being found mostly at position 1 and polyunsaturated fatty acids at position 2. The derived diglyceride acetates were separated into different molecular species by argentation thin-layer chromatography. These analyses showed that l-stearoyl, 2-arachidonoyl was the major species of brain CDP-diglyceride. [ABSTRACT FROM AUTHOR]

Mortality in the host armyworm larvae Pseudaletia separata parasitized by the parasitic wasp Cotesia kariyai was dramatically increased when they were simultaneously infected by the entomopathogen Serratia marcescens . Previous studies have shown that this strong insecticidal effect is due to a metalloprotease-like insecticide (MPLI) released from S. marcescens enterobacter. This study was conducted to elucidate the exact cause of the mortality resulting from MPLI. Injection of MPLI caused a sharp increase in hemolymph dopamine concentration followed by elevated levels of brain dopamine in armyworm larvae. [3 H]Dopamine injected into the hemocoel, was incorporated into the brains of MPLI-injected larvae to a level eight times greater than in BSA-injected control larvae. Transmission electron microscopy showed an obvious decrease in thickness and density of the brain sheath in insects injected with MPLI. This was probably due to the MPLI-induced elevation of hemocyte metalloprotease activities. Further, electron microscopic and TUNEL staining analyses showed a significant increase in apoptotic cells in the brain 12 h after the injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase inhibitor) before MPLI completely prevented the increase in hemolymph dopamine in test larvae and their following death. From these observations, we conclude that MPLI-injected larvae may have suffered mortal damage through increased apoptosis of brain cells caused by an influx of dopamine from the hemolymph. [ABSTRACT FROM AUTHOR]

The brain fatty acid-binding protein (B-FABP) is involved in brain development and adult neurogenesis. We have determined the sequence of the gene encoding the B-FABP in zebrafish. The zebrafish B-FABP gene spans 2370 bp and contains four exons interrupted by three introns. The coding sequence of zebrafish B-FABP gene is identical to its cDNA sequence and the coding capacity of each exon is the same as that for the human and mouse B-FABP genes. A 1249 bp sequence 5′ upstream of exon 1 of the zebrafish B-FABP gene was cloned and sequenced. Several brain development/growth-associated transcription factor binding elements, including POU-domain binding elements and the proposed lipogenic-associated transcription factor NF-Y elements, were found within the 5′ region of the B-FABP gene. RT-PCR analysis using mRNA extracted from different tissues of adult zebrafish demonstrated that the zebrafish B-FABP mRNA was predominant in brain with lower levels in liver, testis and intestine, but not in ovary, skin, heart, kidney and muscle. Quantitative RT-PCR revealed a similar tissue-specific distribution for zebrafish B-FABP mRNA except that very low levels of B-FABP mRNA, normalized to β-actin mRNA, were detected in the heart and muscle RNA, but not in liver RNA. Zebrafish B-FABP mRNA was detected by RT-PCR in embryos beyond 12 h postfertilization, suggesting a correlation of zebrafish B-FABP mRNA expression with early brain development. Radiation hybrid mapping assigned the zebrafish B-FABP gene to linkage group 17. Conserved syntenies of the zebrafish B-FABP gene and the human and mouse orthologous B-FABP genes were observed by comparative genomic analysis. [ABSTRACT FROM AUTHOR]

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are ‘hybrid-type’ which comprise combinations of α-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593–604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1. [ABSTRACT FROM AUTHOR]

Examines the functional significance of calcium-activated potassium channel variants expressed in rat brain tissues. Use of library screening for the isolation of rslo cDNA; Expression of single rslo channels in Xenopus oocytes; Highlights of molecular cloning and expression pattern of rslo in rat brain.

Examines the cloning and expression of succinic semialdehyde reductase from human brain. Implication of enzyme in the detoxification of xenobiotic carbonyl compounds; Functions of succinic semialdehyde reductase; Occurrence of mental retardation to patients with hydroxybutyric aciduria.

Examines the generation of a novel form of human neuropsin, a brain-related serine protease. Expression of neuropsin in human adult brain; Role of neuropsin in the development of the nervous system; Similarity between the amino acid sequences of the two types of human neuropsin.

We previously reported that the histone-H1 kinase activity bound to p13suc1 increased dramatically during development of the rat brain. In the present work, an in situ kinase assay in an SDS/polyacrylamide gel that contained substrate proteins was employed to characterize the enzyme. Two major proteins of 45 kDa and 100 kDa were found to have p13suc1-bound histone-H1 kinase activity. The former (p45) exhibited strong activity towards histone H1 and had weak autophosphorylation activity, whereas the latter (p100) acted on myelin basic protein or histone H1, and underwent autophosphorylation. p45 was further purified from the nuclear-enriched fraction of rat brain to near homogeneity through sequential column chromatographies. The purified enzyme retained its ability to bind specifically to p13suc1, which suggests that this binding does not require a cofactor. The immunochemical and enzymatic properties of p45 revealed that it differs from Cdk that are known to bind to p13suc1 with high affinity. However, in vitro p45 acted on the peptide motif that is conserved among substrates for cyclin-dependent kinases (Cdk) and mitogen-activated protein kinases, which implies that this protein might belong to the large family of proline-directed kinases. The evidence obtained in this study suggest that p45 is a nuclear p13suc1-bound kinase that has unique functions in the mature brain. [ABSTRACT FROM AUTHOR]

Two soluble forms of novel glutamate dehydrogenase isoperoteins, designated GDH I and GDH II, have been purified from bovine brain. GDH I and GDH II were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50mM for GDH I and GDH II, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57 500 Da. Differences between the biochemical properties of GDH I and GDH II, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability,were extensively examined in both reductive amination of α-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate GDH I and GDH II, isolated from bovine brain, are novel and distinct polypeptides. [ABSTRACT FROM AUTHOR]

Reports on the primary structure of the neuronal clathrin-associated protein auxilin and its expression in bacteria found in cow brain. Support of assembly in interaction with clathrin; Combination of open reading frames to predict molecular mass for auxilin; Similarity of the region of positions in the actin-binding protein tensin.

A ll5-bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/N′, and B. The 5′ A/A′ box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Spl in fibroblasts (box A′) and a different complex in brain (box A), Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A′ by a Spl consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3′, box B is composed of a 5" direct repeat and a 3′ GC box consisting of overlapping Spl and Krox20/Krox24 binding sites, Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C-rich subregion. [ABSTRACT FROM AUTHOR]

Reports that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with streptolysin O. Impairment of the ability of GTP[S] to stimulate PLD if the cells are previously permeabilized such that the majority of the cytosol has leaked out; Ability of rat brain and HL60 cytosols to restore GTP[S]-stimulated PLD activity in a reconstitution assay.

Describes three monoclonal antibodies, which were raised against amphophilic detergent-soluble acetylcholinesterase from human brain caudate nucleus. Monoclonal antibodies against brain acetylcholinesterases which recognize the subunits bearing the hydrophobic anchor; Selection and subcloning of the monoclonal antibodies, specific for brain DS-AChE.

A rat brain cytosolic 3,5,3′-triiodo-u-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-SepharOse-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3′-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 μM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2–4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin. [ABSTRACT FROM AUTHOR]

The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific antiserum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB- COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed. [ABSTRACT FROM AUTHOR]

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A -- Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56 and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to α1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product. [ABSTRACT FROM AUTHOR]

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Bergiund et al. (1987) J. Neurochein. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosyiphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins. [ABSTRACT FROM AUTHOR]

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, o-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2+-regulated enzyme. [ABSTRACT FROM AUTHOR]

Based on the liberation of proline from ProLeuGlyNH2 (MIF-1, melanostatin) manganese-activated prolyl aminopeptidase activities were purified from rat brain and kidney cytosolic fractions. They were distinguished from other di- and tripeptidases and an arylamidase liberating N-terminal proline. Purified prolyl aminopeptidase from both sources had identical molecular properties (native Mr 300000, subunit Mr 54000) and very similar catalytic properties. The action of the purified enzymes was not restricted to proline. Other, in particular lipophilic, amino acids were cleaved from di-, tri- and oligopeptides with even higher velocities. Peptides with N-terminal penultimate proline residues were not degraded. From a comparison of molecular data, action on peptides, influence of pH values, inhibitors and activators, it is concluded that the activity is identical with leucyl aminopeptidase (EC 3.4.11.1) and that a separate prolyl aminopeptidase (EC 3.4.11.5) does not exist in rats. [ABSTRACT FROM AUTHOR]

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0. and at 5°, 15°, and 25°C. The two sites in a molecule were equivalent on Mg2+-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25°C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2+-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236–2240]. Although no major conformational changes were noted between Ca2+- and Mg2+-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2+-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2+-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2+-Ca2+ exchange. [ABSTRACT FROM AUTHOR]

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258–268]. The paramagnetic effect of enzyme-bound Mn(II) on the spinlattice relaxation rates (T1-1) of ligand nuclei (¹H and 3P) in E · Mn(II) · Glc and E · Mn(II) · Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-Halpha;, C1-Hβ and C2-Hβ of glucose in the E · Mn(II) · Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5–30°C at 500 MHz. The ligand nuclear relaxation rates in E · Mn(II) · Glc are field-dependent and the Arrhenius plot yields an activation energy (ΔE) of 16.7– 20.9 kJ/mol. Similar measurements have also been carried out on C1-Hα, C1-Hβ and C6-31P at 270 MHz (¹H) and 202.5 MHz (31p) for the E · Mn(II) · Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5–30°C, which yielded ΔE = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (τc), determined from the field-dependent measurements of proton relaxation rates in the E · Mn(II) · Glc complex, is 0.22–1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are &assymp; 1.1 nm and &assymp; 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492–15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P · Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of &assymp; 0.5–0.6 nm from the cation. [ABSTRACT FROM AUTHOR]

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Acα2→3Galβ1→3GalNAc-R:α2→6 sialyltransferase previously described [Baubichon-Cortay, H., SerresGuillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Galβ1→3GalNAc-protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:β1→3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in α2→3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Acα2→3Galβ1→3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Galβ1→3GalNAc-R:α2→3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Acα2→3Galβ1→3GalNAc-R:α2→6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the α2→3 sialyltransferase regulates the α2→6 sialyltransferase activity since it synthesizes the α2→6 sialyltransferase substrate. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence (186 amino acid residues) of a basic cytosolic protein from bovine brain has been determined. It was previously described as a phosphatidylethanotamine binding protein. Computer analyses have been used to calculate its hydropathy profile and to predict its secondary structure. Comparison with other proteins did not detect any significant sequence similarity, except for a short region which presents 53% sequence homology with bovine phosphatidylcholine transfer protein. [ABSTRACT FROM AUTHOR]

Neuronal nuclei have a low H1 content. A stoichiometry of 0.47 molecule/nucleosome, on average, is calculated for rat brain cortical neurons by comparing its HI content with that of liver nuclei. The H1 fraction of rat cerebral cortex neurons has been resolved into five subtypes, Hla-e, that have the same mobility as the unphosphorylated H1 forms of other rat tissues. The subtypes Hla-d decay exponentially during postnatal development and are substituted to different extents by Hle. The higher replacement rate is shown by Hla with an apparent half-lifetime of about 5 days. The corresponding values for Hlb, Hic and Hld are 11, 21 and 15 days, Several conclusions can be drawn from the observation of postnatal changes in Hi subtype proportions. (a) The low H 1 content of neuronal nuclei does not imply the presence of notable peculiarities in subtype composition or in subtype substitution pattern. (b) There is turnover of H1 in differentiating neurons once cell proliferation and DNA replication have ceased. (c) The relative rates of synthesis and/or degradation of the subtypes differ in germinal cells and in neurons. (d) Comparison with previous results on H1° accumulation also shows that in cortical neurons the regulation of the subtypes H1a-e differs from that of H1°. [ABSTRACT FROM AUTHOR]

This work describes a method for the purification of the elongation factors (EF) from calf brain. The elongation factor responsible for the binding of aminoacyl-tRNA to the ribosome is found in this organ as a light form (EF-α) and as a component of heavy, polydispersed aggregates (EF-1H). EF-β, the factor enhancing the EF-lα GDP/GTP exchange, is part of EF-1H and of smaller aggregates. The fraction of EF-1α and EF-1β not associated with EF-1H, and EF-2 have been purified to homogeneity after several chromatographic steps. EF-1H consists of many proteins; among them, EF-1α, EF-1β and an EF-1γ-like protein represent three of the major components. This conclusively shows that EF-1H from calf brain is not a polydispersed aggregate of only EF-1α. EF-1β has also been purified to homogeneity from EF-1H. The property of EF-1β to aggregate with other proteins suggests that this factor plays an important role in the organization of EF-1H. The relative molecular mass of the purified factors have been determined as: EF-1α, 50000; EF-1β, 30000; the EF-1γ-like component, 49000; EF-2, 85000. Some cross-reactivity with the antibodies against the prokaryotic counterparts has been shown for EF-1α, EF-1β and EF-2 by functional and immuno-precipitation methods, suggesting the existence of structural homologies. [ABSTRACT FROM AUTHOR]

The properties of EF-1α from calf brain have been investigated and compared with those of EF-Tu. EF-1α binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K′d = 2 − 4 μM). EF-1β strongly enhances the dissociation rate of the EF-1α · GDP complex and to a lesser extent of the EF-1α · GTP complex. Aminoacyl-tRNA (aa-tRNA) stabilizes EF-1α · GTP much less efficiently than the EF-Tu · GTP complex. Unlike EF-Tu, EF-1α sustains the binding of aa-tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF-1α · GTP and EF-1α · GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF-1α-bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimicks that of EF-1β, the antibiotic is incapable of increasing the EF-1α · GDP/GTP exchange rate when aa-tRNA and ribosomes are present. Therefore, unlike EF-1β, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP-like conformation of EF-1α · GDP, as in the case of EF-Tu · GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system. [ABSTRACT FROM AUTHOR]

Studies the efflux and exchange of β-alanine in synaptic membrane vesicles from rat brain. Mechanism of β-alanine translocation; Comparison of the ion dependence of net efflux to that of exchange; Indication that the release of β-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions.

Reports on a study in which calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly. Indication that phenoxybenzamine-alkylated calmodulin and calmodulin-melittin complex inhibit the endogenous calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum; Observation that the alkylated calmodulin is actually an activator with lower affinity than the unmodified calmodulin.

Binding of the peptide neurotrausmitter substance P to brain tubulin in vitro inhibits self-assembly of the protein into microtubules and disrupts preassembled inicrotubules. This cooperative inhibition of the maximum extent of self-assembly by substance P is explicable in terms of preferential binding to the protomer state as compared to the polymer state of tubulin. The inhibition is relieved by the microtubule-associated protein MAP2, which evidently acts in a mixed competitive-noncompetitive fashion. Substance P interacts directly with the isolated C-terminal 4-kDa peptide fragment of tubulin, which appears to contain the specific binding area for MAP2, but is without effect on the self-assembly of the larger (48-kDa) part of the tubulin molecule called S-tubulin. The results are consistent with the C-terminal fragment having a binding site for the cationic substance P as well as for MAP2. However, factors other than electrostatic interaction must be operative, since the suifoxide of substance P, a derivative with oxidized methionine but similar electrostatic characteristics, is inactive in inhibiting the extent of microtubule assembly. [ABSTRACT FROM AUTHOR]

Thyroid spectrin has been obtained pure from pig thyroid glands. This protein, composed of two non-identical polypeptide chains of 240 kDa and 235 kDa, appears to possess the same structural and immunological properties as well as the same calmodulin and actin-binding properties as brain spectrin. Through cross-linking of actin filaments it is a potent gelation factor for F-actin solutions. It represents one of the major protein of the cystoskeleton underlying the thyroid plasma membrane together with myosin, α-actinin and actin. [ABSTRACT FROM AUTHOR]

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B, and 2C. The antibody was used to establish the following points. 1. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. 2. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complex with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. 3. The catalytic subunits of protein phosphatases-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. 4. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. 5. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. 6. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylate, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis. [ABSTRACT FROM AUTHOR]

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5′-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform. [ABSTRACT FROM AUTHOR]

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 × 107 mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted withe the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The balding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar. [ABSTRACT FROM AUTHOR]

An exodeoxynuclease with single-stranded DNA specificity has been isolated from rat brain and purified to electrophoretic homogeneity. Approximately 1100-fold purification with a yield of 16% has been achieved by chromatography on DNA-agarose, hydroxyapatite and Sephadex G-200. The enzyme has a pH optimum at 8.4, requires Mg2+ or Mn2+ as a cofactor and has a molecular weight of about 60000. It hydrolyzes homologous, heterologous, synthetic and depurinated substrates at the same rate liberating nucleoside 5′-monophosphates but does not attack ultraviolet-irradiated polydeoxyribonucleotides. This DNase is localized predominantly in neuronal cell nuclei and appears to be lacking in rat liver tissue. [ABSTRACT FROM AUTHOR]

The MRC OX 2 monoclonal antibody recognises antigens present on rat thymocytes, brain, follicular dendritic cells in lymphoid organs, vascular endothelium. some smooth muscle and B-lymphocytes. The OX 2 antigens recognised by this antibody were purified from brain and thymus by solubilisation with sodium deoxycholate affinity chromatography with MRC OX 2 antibody and gel filtration. The purified brain and thymocyte OX 2 antigens were glycoproteins with apparent AA 41 000 and 47000 respectively as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Rabbit antisera raised against the purified antigens were analysed by radioimmunoassay and immunoperoxidase-staining of tissue sections. The brain and thymocyte OX 2 antigens were antigenically very similar to those on the other tissues. This indicates that the unusual pattern of distribution was not the result of fortuitous cross-reaction of the MRC OX 2 antibody, as the rabbit sera would be expected to recognise more determinants on the antigen than that recognised by the monoclonal antibody. The amino acid compositions of brain and thymus OX 2 antigens were very similar but with no distinguishing features. Carbohydrate compositions showed that the OX 2 antigens were highly glycosylated. with brain OX 2 antigen containing 24% and thymocyte OX 2 antigen 33% by weight of carbohydrate. Both OX 2 antigens contained carbohydrate residues typical of structures N-linked to asparagine but lacked galactosamine, indicating the absence of O-linked structures. Thymocyte OX 2 contained higher levels of galactose and sialic acid but less fucose than brain OX 2. Similar differences had been observed for brain and thymocyte Thy-1 antigens and were also observed in pooled glycoproteins purified by lentil lectin affinity chromatography from these tissues, reflecting overall differences in the patterns of glycosylation in the two tissues. The OX 2 antigens showed many similarities to Thy-1 antigens in their odd patterns of distribution, characteristic migration on polyacrylamide gels in sodium dodecyl sulphate, and carbohydrate compositions. It is possible that OX 2 antigens, like Thy-1 antigens, have homologies with immunoglobulin domains. A possible role for OX 2 antigens in cell interactions necessary for tissue organisation is discussed. [ABSTRACT FROM AUTHOR]

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GDta by the membrane-bound enzyme has been studied under various conditions. The reaction rate decreased with increasing ionic strength in the incubation mixture, and was progressively enhanced by increasing concentrations of the primary alcohols n-pentanol to n-octanol. This stimulation correlates quantitatively with an increase in membrane ‘fluidity’ caused by these alcohols as measured by fluorescence depolarization employing l,6-diphenyl-l,3,5-hexatriene as probe. The dependence of the reaction rate on the amount of enzyme in the incubation mixture was linear only with water-soluble substrates but not with the lipophilic ganglioside substrate. Evidence is presented that lipophilic substrate and enzyme interact mainly within the plane of the membrane presumably by lateral diffusion. Taking this into consideration Michaelis-Menten theory was modified accordingly. As predicted, apparent Km values increased linearly with the amount of membrane-bound enzyme added and decreased with the concentration of n-hexanol in the incubation mixture. In the presence of varying n-hexanol concentrations the apparent Km-value decreased with increasing membrane ‘fluidity’, as measured by fluorescence depolarization of l,6-diphenyl-l,3.5-hexatriene. On the other hand, as expected, V values were not affected by membrane 'fluidity" and increased linearly with the amount of membrane protein. [ABSTRACT FROM AUTHOR]

Poly(A)-containing mRNA was isolated from the electric lobe, cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell-free protein-synthesizing system from rabbit reticulocytes: no brain-specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purely neuronal mRNA is a preferred template in the protein-synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two-dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor nerve terminals. The majority of the synaptosomal proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identifies were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity. A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non-cholinergic, revealed, as expected, some common translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of proteins specific for neurones of different transmitter types. [ABSTRACT FROM AUTHOR]

1. A rat brain supernatant and microsomal fraction contained a phosholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase. A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range ( 7 - 8.5) as well as at pH 5 - 5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH. [ABSTRACT FROM AUTHOR]

Initial rate studies have been used to investigate the kinetic mechanism followed by the purified high-Km (AR1) form of rat brain aldehyde reductase at pH 7.0. The effects of varying the aldehyde and NADPH concentrations, together with the inhibition given by the products of the reaction, are consistent with the reduction of succinic semialdehyde and p-nitrobenzaldehyde following an ordered reaction mechanism involving the formation of an intermediate ternary complex and in which NADPH is the first substrate to bind to the enzyme. Both these aldehyde substrates inhibit the enzyme at higher concentrations. This inhibition, which is uncompetitive with respect to NADPH, suggests that many previous studies on the specificity of this enzyme, that have been based on the activity determined at a single arbitrary concentration of each substrate, may have given erroneous results. [ABSTRACT FROM AUTHOR]

Reports on kinetic studies with rat-brain succinic-semialdehyde dehydrogenase. Enzyme purification; Exhibition of a relatively low Km value for succinic semialdehyde; Inhibition by p-hydroxybenzaldehyde; Metabolism of aldehydes.

Examines the effect of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain. Protection against denaturation by isocitrate; Inhibition by iodoacetate; Reduction of the affinity of the enzyme; Enhancement of protection by adenosine diphosphate.

Reports on kinetic comparisons between soluble and membrane-bound glutaminase preparations from pig brain. Dependence of activities on pH and in their inhibition by borate and some glutamine analogs; Responses to phosphate and their inhibition by glutamate; Sigmoid dependence of initial velocity on glutamine concentration; Inhibition by ammonium ions; Catalysis of the hydrolysis of L-glutamine by the enzyme glutaminase to form L-glutamate plus ammonium ions.

Characterizes a membrane-bound enzyme which degrades substance P from human brain. Enzyme activity; Possible role of the enzyme in the physiological inactivation of the peptide by neural tissues; Enzyme purification; Enzyme inhibition by metal chelating agents; Thermolability; Molecular weight.

Levels of liver alcohol dehydrogenase, liver mitochondrial aldehyde dehydrogenase and brain (Na + K)-stimulated ATPase were determined in control rats and in rats fed an ethanol liquid diet for 7 weeks, The alcohol dehydrogenase level did not change with the diet; however, the levels of mitochondrial aldehyde dehydrogenase and of the (Na + K)-ATPase were increased 2.5-fold and 2-fold respectively. Also, the levels of these enzymes from brain and liver fractions of rats given a 20% ethanol solution as the sole drinking fluid were determined at intervals from 4 to 18 weeks. A progressive increase was found in the level of aldehyde dehydrogenase and of ATPase. To ascertain whether synthesis or degradation of these enzymes were responsible for the marked increase in activity the turnover of several liver fractions and of the indicated enzymes were measured by the dual-labelled isotope technique. Long-term ethanol administration did not affect the rate of turnover of the liver cell fractions except for the mitochondrial fraction. This fraction showed a marked decrease in the rate of degradation. The degradation constant of the liver alcohol dehydrogenase did not change; however, the rate of degradation of liver mitochondrial aldehyde dehydrogenase (low-Km) and of brain (Na + K)-ATPase was decreased. [ABSTRACT FROM AUTHOR]

DNA polymerase γ and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase γ and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems: essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements for Mn2- (0.1 mM), Mg2+ (5 mM) and pH (8.0): template preferences, poly(A) - (dT)20-25 > activated DNA > poly(dA)-(dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA : molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pl 5,4). We therefore conclude that brain nuclear DNA polymerase γ and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical. [ABSTRACT FROM AUTHOR]

An isolation procedure for rat brain aldolase C has been developed which also permits the isolation of aldolase C from experimental hepatomas. Certain enzymatic properties (specific activity and Michaelis constant towards the two specific substrates: fructose 1,6-bisphosphate and fructose 1-phosphate) and physico-chemical properties (molecular weight, N-terminal amino-acid) of the two enzymes have been studied and compared. Moreover, an amino-acid analysis has been carried out for rat brain aldolase C. Within experimental errors, the two enzymes appear to be identical. [ABSTRACT FROM AUTHOR]