Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.Abbreviations GOGAT glutamate synthase - MS Murashige-Skoog (1962) medium - PMSF phenylmethylsulfonyl fluoride     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Temporal Profiles of Proteins Responsive to Transient Ischemia

The responses of long and short half-lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four-vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25-50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half-lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2-3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6-15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.     

Effect of Acute Hypoxia on Ascorbate Content of Plasma, Cerebral Cortex, and Adrenal Gland

Levels of ascorbic acid (AA) in the plasma, brain, and adrenal gland of rats were determined after 15 min of hypoxia (PaO2 less than 25 mm Hg) and following asphyxia. In rabbits, AA plasma levels were followed up to 75 min of reoxygenation following 15 min of hypoxia of the same severity. A significant increase (approximately 70%) in AA levels was found in plasma of rats and rabbits after hypoxia and asphyxia. This increase was found to be transient, with a return to normal levels within 1 h after resumption of normal oxygenation. Pretreatment with dexamethasone reduced the increase in AA level in both rabbits and rats. Adrenalectomy in rats, performed 24 h before the experiment, abolished the response to hypoxia. Ascorbate levels in the cerebral cortex, hypothalamus, and adrenal gland of awake rats subjected to hypoxia or asphyxia were found to be the same as in normoxic rats. Our results suggest that the observed changes in plasma AA levels are probably mediated through adrenocorticotropic hormone and that the adrenal gland is the major source of ascorbate efflux into the circulation during oxygen deprivation.     

Difference in the Pattern of Soluble Proteins from Rat Brain Regions

The electrophoretic pattern of soluble proteins from seven rat brain regions (amygdala, cerebellum, corpus striatum, cortex, hypothalamus, medulla, and midbrain) was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Although the number of protein bands (36) was identical in all brain regions studied, there were differences in their relative densities, the greatest variation occurring in the low-molecular-weight region of the electrophoretogram. The bulk of the soluble proteins had molecular weights between 23,000 and 90,000 daltons. The medulla and amygdala showed the greatest range of protein band concentration. A large number of protein bands in the midbrain and corpus striatum showed a greater concentration of protein compared to the same bands in the other regions. A protein band that migrated with the same characteristic as albumin was found. It was consistently high in all regions, the midbrains showing a 1.5-fold greater concentration compared to other regions. Linear regression analysis of wet weight of regional brain tissue against protein concentration yielded a regression coefficient (r2) of 0.77. Midbrain and corpus striatum showed a relatively higher protein concentration: weight ratio than other regions.     

Involvement of an "Antizyme'' in the Inactivation of Ornithine Decarboxylase

DL-Allylglycine causes a marked increase in mouse brain ornithine decarboxylase (ODC) activity. The amount of immunoreactive enzyme protein increases concomitantly with the activity, but the enzyme protein decreases more slowly than that of the activity. The amount of immunoreactive ODC in brain is many hundred times that of the catalytically active enzyme. The fact that mouse brain cytosol contains high amounts of dissociable antizyme (an inactivating protein) indicates the existence of an inactive, immunoreactive ODC-antizyme pool. The total antizyme content does not change markedly, but instead there are significant changes in different antizyme pools. Putrescine concentrations start to increase 8 h after treatment with allylglycine and concomitantly with this increase, antizyme is released to inhibit enzyme activity. These results indicate the involvement of antizyme in the inactivation process of ODC.     

Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity

We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6-hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d-amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.     

Quinolinic Acid Phosphoribosyltransferase in Rat Brain

Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 +/- 0.30 microM for quinolinic acid and Km = 65.13 +/- 13.74 microM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 +/- 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 +/- 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a greater than 20-fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippo-campus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve-ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at -80 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.