Differences in the Structure of A and B Forms of Human Monoamine Oxidase

Abstract: [³H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [³H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [³H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS-PAGE of [³H]pargyline-bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS-solubilized, [³H]pargyline-labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [³H]pargyline-labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

The Synthesis and Release of [3H-Tyrosine1]Methionine5-Enkephalin from Guinea Pig Brain Slices

Abstract: Stores of methionine-enkephalin were labelled on the N -terminal by incubation of whole brain slices with [³H]tyrosine (10 °Ci/ml). The ³H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD₂ and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [³H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N -terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [¹⁴C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N -terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 m M ) elicited a Ca²⁺-dependent release of the synthesised [³H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [³H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.     

Immobilization Antigens from Tetrahymena thermophila Are Glycosyl-Phosphatidylinositol-Linked Proteins

We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [³H]ethanolamine or [³H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [³H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [³H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors.     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .     

THE SPECIFICITY AND SULFHYDRYL SENSITIVITY OF THE INOSITOL TRANSPORT SYSTEM OF THE CENTRAL NERVOUS SYSTEM

Abstract— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro , the uptake of [³H]scyllitol or [³H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [³H]scyllitol or [³H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [³H]scyllitol accumulation and both isomers had a comparable affinity (K_t= 0.1 m m ) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo , in unanesthetized rabbits. the transport of unmetabolized [³H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d -chiroinositol. Similarly, the transport of unmetabolized [³H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.     

FAST AXOPLASMIC TRANSPORT OF A CALCIUM-BINDING PROTEIN IN MAMMALIAN NERVE

Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of ⁴⁵Ca²⁺ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported ⁴⁵Ca²⁺ found to be bound to a protein of 15,000 dalton. Using [³H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound ⁴⁵Ca^{2 +}. The level of [³H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [³H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca²⁺ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed.     

PROTEIN SYNTHESIS IN THE BRAIN OF OCTOPUS VULGARIS, LAM

Abstract— The process of protein synthesis in the brain of Octopus vulgaris Lam has been examined after systemic administration of [³H]leucine and upon incubation of the tissue in sea water containing the radioactive precursor. After injection of [³H]leucine in the branchial heart, the radioactivity of the TCA-soluble fractions of the three main brain divisions reached a maximum in about 30 min and decreased thereafter, while incorporation into the protein fractions was complete in approx. 2 h. Per unit wet weight the radioactivity of brain proteins was higher than that of most other organs. In vitro the rate of incorporation of [³H]leucine in the protein fraction of the optic lobe remained low for more than 1 h, but increased several fold thereafter. Preincubation of the tissue in sea water abolished the lag period. Similar effects were observed in the vertical lobe as well as in the optic lobe of young and adult octopuses but not in the white body, a non-nervous organ. The process of protein synthesis in the optic lobe is markedly inhibited by puromycin, cycloheximide and chloramphenicol. Electrophoretic analysis on polyacrylamide gels indicated that the soluble proteins labelled in vitro and in vivo are similar.     

VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [³H]acetate as a precursor of ACh. Little vesicular [³H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [³H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [³H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.     

Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two-dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two-dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [³H]fucose as a precursor or by separating [³⁵S]methionine-labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.     

Plasmid-associated bacteriocin production in a JK-type coryneform bacterium

Abstract The outer membrane of Escherichia coli K-12 has a variety of proteolytic activities. We were able to label several outer membrane proteins with [³H]diisopropylfluorophosphate (DFP). This suggests that they are serine proteases. The number of labelled proteins detected varied with the E. coli K-12 strain used. Strains bearing a tolC mutation, in addition, gave better labelling and/or had more labelled proteins. A previously described [³H]DFP-labelled outer membrane protein was shown not to be the TolC protein since it has a slightly lower M _r, it is not labelled more intensely in a TolC-overproducing strain, and it is still labelled in tolC mutant strains.     

ANALYSIS OF PROTEINS UNDERGOING AXONAL TRANSPORT IN NIGRO-STRIATAL NEURONS IN THE RAT

Abstract— An analysis of proteins undergoing axonal transport in nigro-striatal neurons, after the stereotaxic injection of [³H]leucine into the substantia nigra of rat brain was performed. As early as 6 h after the injection [³H]proteins appeared in the caudate-putamen. The maximum accumulation was at 5 days and there was still residual protein radioactivity present at 30 days. About 70 per cent of the total radioactive protein in the caudate-putamen was solubilized by homogenization in 0–5%, (v/v) Triton X-100 and remained in the supernatant on centrifuging for 1 h at 100,000 g. The supernatant fraction, when chroma-tographed on a DEAE-cellulose column, was resolved into four protein peaks (A, B. C and D) which were found to be labelled differently as a function of time after the injection of [³H]leucine. Peak A was substantially labelled in a first phase (6–24 h) and reached its maximum in a second phase (5 days). The proteins comprising this peak appeared to undergo both fast and slow axonal transport. Although some labelling in peak B was evident at 6 h, maximal activity did not occur until 5 days. No radioactivity could be detected in peaks C and D at 6 h. Maximal labelling of these two peaks also occurred at 5 days. These data suggest that the proteins of peaks B, C and D were transported primarily by slow axoplasmic flow. The radioactive protein peaks A and B from the second phase of the transport were excluded from a Sephadex G-200 column, pointing to their high molecular weights (13,000–200,000). Peak B. which had the highest specific radioactivity (c.p.m./mg protein) at 5 days, contained a significant level of tyrosine hydroxylase, an important component of dopaminergic neurons.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.     

MELATONIN: DEACETYLATION TO 5-METHOXYTRYPTAMINE BY LIVER BUT NOT BRAIN ARYL ACYLAMIDASE

Abstract— Rat liver and brain slices were incubated in vitro with [³H]melatonin. Liver slices synthesized small amounts of [³H]5-methoxyindoleacetic acid ([³H]5-MIAA) along with other melatonin metabolites including 6-hydroxymelatonin. Pretreatment of animals prior to killing with the irreversible monoamine oxidase inhibitor pargyline allowed [³H]5-methoxytryptamine ([³H]5-MT) to be recovered from the incubation. No [³H]5-MIAA or [³H]5-MT could be detected in incubations with hypothalamic slices or following intraventrieular injection of [³H]melatonin. The possibility that the deacetylase aryl acylamidase was in part responsible for the deacetylation occurring in liver slices was examined. Liver aryl acylamidase was able to utilize [³H]melatonin as substrate to produce [³H]5-MT. Furthermore, the liver enzyme was inhibited by melatonin ( K_i. 1 m m ) when tested with the alternate substrate o -nitroacetanalide. Brain aryl acylamidase did not generate any detectable [³H]5-MT nor was it inhibited by melatonin. These results suggest that 5-MT is not formed in brain from melatonin although trace amounts of 5-MT in the periphery could be derived from this precursor.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Potassium-Stimulated Release of Radiolabelled Taurine and Glycine from the Isolated Rat Retina

Abstract: The release of preloaded [³H]glycine and [³H]taurine in response to a depolarising stimulus (12.5-50 m M KCl) has been studied in the superfused rat retina. High external potassium concentration immediately increased the spontaneous efflux of [3H]glycine, the effect of 50 m M K⁺ apparently being abolished by omitting calcium from the superfusing medium. In contrast, although high potassium concentrations increased the spontaneous emux of [³H]taurine from the superfused rat retina, this release was not evident until the depolarising stimulus was removed from the superfusing medium. The magnitude of this "late" release of [³H]taurine was dependent on external K⁺ concentrations, and appeared immediately after cessation of the stimulus irrespective of whether it was applied for 4, 8, or 12 min. Potassium (50 m M )-induced release of taurine appeared partially calcium-dependent, being significantly reduced (p < 0.01) but not abolished by replacing calcium with 1 mM EDTA in the superfusate. High-affinity uptake systems for both [³H]glycine and [³H]taurine were demonstrated in the rat retina in vitro ( K _m values, 1.67 μ M and 2.97 μ M ; V_max values, 19.3 and 23.1 nmol/g wet weight tissue/h, respectively). The results are discussed with respect to the possible neuro-transmitter roles of both amino acids in the rat retina.