Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Lysine biosynthesis and the evolution-of pennate marine diatoms

The classification of lysine biosynthetic pathways in various organisms have been used to investigate their descent in evolution. We have attempted these determinations in the diatoms Amphora coffeaeformis var:perpusilla (Grunow Cleve.) and Phaeodactylum tricornutum (Bohlin). Additionally, we have verified earlier results of Vogel in a green alga, Chlorella pyrenoidosa strain Tx 71105 (Texas Culture Collection). Our research indicates that the diaminopimelic acid route is involved in all three organisms. While these studies do not exclude the possible co-existence of the α-aminoadipic acid route, the results imply a closer evolutionary relationship of pennate diatoms to bacteria and “classical” photosynthetic plants rather than to heterotrophic or mixotrophic fungi and atypical algal strains such as the Euglenophyta.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Enhancement of Hypophysectomy-Induced Dopamine Receptor Hypersensitivity in Male Rats by Chronic Haloperidol Administration

It has been reported that hypophysectomy (HYPOX) would antagonize the development of a neuroleptic-induced dopamine receptor hypersensitivity, and suggested that the neuroleptic-induced dopamine receptor hypersensitivity may be mediated by the neuroleptic-induced hyperprolactinemia. Conversely, we and others have reported on the ability of HYPOX animals to develop a neuroleptic-induced dopamine receptor hypersensitivity. The present study was undertaken to define the possible role(s) of prolactin in the modulation of striatal dopamine receptor sensitivity. The data from these studies indicate: that HYPOX alone will result in the development of a striatal dopamine receptor hypersensitivity; that the HYPOX-induced dopamine receptor hypersensitivity could be increased by the chronic administration and withdrawal of haloperidol; that administration of prolactin to HYPOX rats would partially antagonize the development of the neuroleptic-induced dopamine receptor hypersensitivity; and that the administration of prolactin alone had minimal effects on the apomorphine-induced behavior or neurochemistry of the HYPOX animals. These results suggest that the neuroleptics do not require the presence of a pituitary secretion (specifically, prolactin) to induce a striatal dopamine receptor hypersensitivity; however, they do indicate that a pituitary secretion, perhaps prolactin, may have the ability to modulate striatal dopamine sensitivity.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

Evidence for Feedback Regulation of Glutamate Decarboxylase by γ-Aminobutyric Acid

Abstract: The concentration of γ-aminobutyric acid (GABA) in the human ovary and the capacity of a membrane preparation from the same organ to bind [³H]GABA specifically were examined. The GABA concentration in the ovary was found to be 214 ± 66 nmol/g frozen tissue (mean ± SEM of six independent determinations). Moreover, a single population of high-affinity GABA binding sites has been identified in the ovarian membranes. The apparent dissociation constant ( K _d) and maximum binding capacity ( B _max) were 38.3 n M and 676 fmol/mg protein, respectively. The specific binding of [³H]GABA was displaced by muscimol, unlabelled GABA, or (+)bicuculline, but was unaffected by (±)baclofen and picrotoxin. The present results show that GABA and an extremely high density of GABA_A receptor binding sites are present in the human ovary, indicating a physiological significance of this amino acid in the female reproductive system.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

Factors influencing algal species assemblages on reef and cobble substrata off Ghana

Subtidal algal assemblages were studied on two substrata, rocky reefs and calcareous cobbles. Although the two occur together at comparable depths, their vegetation differs in species composition and richness, and in patterns of plant size, life form, and longevity. The reef bears a species-rich, patchy cover of small filamentous and crustose forms, with occasional clumps of more robust species. The cobbles support a sparse cover of large leafy and dendritic species in addition to many of the smaller species found on the reef. The floristic separation arises from differential establishment and survival of species under conditions of (1) grazing by fish and urchins (on the reef only), and (2) seasonal physical disturbance during storms leading to the removal of most algae (on the cobbles only). Both substrata show a seasonal floristic cycle, but the trend is more pronounced on cobbles. Species do not depart from randomness in their patterns of co-occurrence on individual cobbles or reef fragments. Interspecific competition appears comparatively unimportant in determining species composition on either substratum.     

Peptidoglycans as promoters of slow-wave sleep. II. Somnogenic and pyrogenic activities of some naturally occurring muramyl peptides; correlations with mass spectrometric structure determination

The structures of components of the sleep-promoting material purified from human urine were established by fast atom bombardment-mass spectrometry, as reported in the accompanying paper (Martin, S. A., Karnovsky, M. L., Krueger, J. M., Pappenheimer, J. R., and Biemann, K. (1984) J. Biol. Chem. 259, 12652-12658). We report here that two substances isolated from that preparation, viz. N-acetylglucosaminyl-1,6 -anhydro-N-acetylmuramyl-Ala-gamma-Glu-diaminopimelyl-Ala) and that compound lacking the terminal alanine, are active as somnogens. Cerebro-intraventricular administration of 1 pmol of the glycotetrapeptide was sufficient to induce prolonged excess sleep in rabbits. A similar substance obtained from Brevibacterium divaricatum in which the free carboxyls of the glutamic and diaminopimelic moieties, indicated above, were amidated (N-acetylglucosaminyl-1,6-anhydro-N-anhydromura-myl-Ala-iso- Gln- epsilon-amido-diaminopimelyl -Ala-Ala) was not active as a promoter of slow-wave sleep. Deamidation of this peptide to a mixture of the free dicarboxylic forms produced a somnogenic substance. Our findings show that in addition to the muramyl form of peptidoglycan monomers, the anhydro muramyl form, with no reducing end, is compatible with somnogenic activity. Furthermore, the data obtained with a natural product amplify our earlier observations with smaller synthetic molecules of the importance of amidation/deamidation in the structure-activity relationships of muramyl peptides.