PHOTOCHEMICAL ACTIVITIES OF SONICATED LAMELLAE OF SPINACH CHLOROPLASTS WITH SPECIAL REFERENCE TO THE ACTION OF CRS (CYTOCHROME C REDUCING SUBSTANCE)

The photooxidation of reduced DPIP with NADP and O₂ as finalelectron acceptors was studied with sonicated lamellae of spinachchloroplasts, with special reference to the possible role ofCRS, a newly discovered factor of photochemical system I. Acorrelation between inactivation of NADP-photoreduction anddissolution of CRS was observed on treatment of the lamellaewith various organic solvents. The treatment also suppressedthe O₂-linked photooxidation of reduced DPIP, although the suppressionwas not so marked as in the former reaction. The suppressedphotooxidation of reduced DPIP (with O₂ and NADP as electronacceptors) was partially restored on addition of dyes of negativeredox potential, such as methyl viologen. The experimental resultsindicate that a factor participates in the reactions of photochemicalsystem I, probably functioning as an electron carrier couplingthe photooxidation of P700 with the reduction of ferredoxinor O₂. The finding that the above mentioned activity of thefactor is destroyed by treatments which are effective in solubilizingCRS from the chloroplasts suggests the identity of the factorwith CRS. ¹ Present address: Nomura Research Institute of Technology andEconomics, Kamakura, Kanagawa.     

Photoreduction of ferredoxin-NADP in the presence and absence of ferredoxin-reducing substance (FRS)

Preparations of ferredoxin-reducing substance (FRS) were obtained from spinach chloroplasts within the elution volume range and with the spectral characteristics described by Yocum and San Pietro (8). However, no support was found for the view that FRS is the primary electron acceptor of Photosystem I. The FRS-depleted chloroplast fragments retained their Photosystem I activity, which was not enhanced by the addition of FRS. No evidence was found for a prior photoreduction of FRS by chloroplasts followed by a dark reduction of ferredoxin and NADP by reduced FRS. The FRS-depleted chloroplast fragments were found to retain and to photoreduce bound ferredoxin upon illumination by Photosystem I light at 25°K. These results suggest that the role of a primary electron acceptor of Photosystem I ascribed to FRS may belong to bound ferredoxin.     

Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.     

Response of nitrogenase to altered carbon supply in a Frankia-Alnus incana symbiosis

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro ( Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that, in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.     

Dark starvation and plant metabolism

Summary The fixation pattern of radioactive labelled photosynthetic intermediates was followed under steady state conditions during prolonged dark starvation of spinach plants (Spinacia oleracea L.). It is suggested that the considerable increase of radioactive dihydroxyacetonephosphate is correlated with a specific leakage of the outer chloroplast envelope induced by dark starvation. The primary fixation product, phosphoglyceric acid, followed the same decreasing tendency as observed for the net CO₂ fixation. In contrast, the relative label in other intermediates is the same as in the controls. When after several days of dark starvation the plants were again transferred into light, a regeneration of the CO₂ fixation accompanied by the appearance of a normal fixation pattern was observed. Since the regeneration was prevented by the addition of lincomycin, the net increase is considered to be due to a new protein synthesis rather than a reactivation.Abbreviations GAPDH glyceraldehyde phosphate dehydrogenase (E.C. 1.2.1.13) - DHAP dihydroxyacetonphosphate - FDP fructose 1,6-diphosphate - glol glycolic acid - PGA 3-phosphoglyceric acid - S-D-P sugar diphosphates - S-M-P sugar monophosphates Part I: Postius and Jacobi (1971).     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Enrichment of a 50-kilodalton polypeptide in a photosystem II-phycobilisome particle from Porphyridium cruentum

A 50-kDa polypeptide was obtained from photosynthetically active phycobilisome-photosystem II preparations from the red alga Porphyridium cruentum after removal of phycobiliproteins. Removal of phycobiliproteins caused destabilization of the structure of the phycobilisome-photosystem II preparations and was accompanied by a decline in photosystem II activity (oxygen-evolution and dichlorophenol-indophenol (DPIP) reduction). The treatments in increasing relative effectiveness were: addition of EDTA (10 mM), lowering the pH (6.8----4.4), and lowering the ionic strength (to ca. 1 mM phosphate). The lowering of the ionic strength by dialysis resulted in a preparation highly enriched in a 50-kDa polypeptide (apparent molecular mass on SDS-PAGE). This preparation retained photosystem II activity as evidenced by the photoreduction of DPIP in the presence of diphenylcarbazide (222 mumol DPIP/mg chlorophyll/h). Also it had a 698-nm (77K) fluorescence emission maximum, as compared to a 668-nm emission in the unfractionated preparation, which indicates enrichment of the photosystem II reaction center. Comparing our results with those obtained from green plants and a cyanobacterium leads us to suggest that the reaction center II polypeptides are highly similar in all chlorophyll alpha-containing plants.     

Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules

Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv `Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark-stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence.     

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.     

Characterization of bovine liver cytosolic 3 alpha-hydroxysteroid dehydrogenase and its aldo-keto reductase activity.

1. 3 alpha-Hydroxysteroid dehydrogenase was purified to homogeneity from bovine cytosolic fraction, which was monomeric and its molecular weight was estimated to be about 35 kDa. 2. The enzyme had ability to catalyze NADP(H)-dependent oxidoreduction of position 3 alpha-hydroxy and keto group of steroids and also could catalyze the reduction of some ketones and quinones. 3. In addition, benzenedihydrodiol was one of the substrates of dehydrogenase activity with NADP+. 4. Indomethacin, synthetic steroids and SH-reagents were potent inhibitors for this enzyme. 5. Inactivation of the enzyme by GSSG-treatment was restored to its original activity by the addition of DTT. 6. The presence of coenzyme, 0.33 mM NADP+, completely protected from the DTNB-inactivation. 7. Bovine liver cytosolic enzyme immunologically crossreacted with rat liver 3 alpha-hydroxysteroid dehydrogenase.     

Participation of the iron-containing pterine-protein complex in NADP reduction and electron transport]

A factor of protein nature, containing pteridines and iron ions was isolated from pea leaves. The compound was shown capable of activating NADP reduction during chloroplasts illumination in the absence of ferredoxin. The compound was termed "NADP-reducing factor" (NRP). Freshly isolated NRF in combination with the protein possessing the NADP-reductase activity, reduces NADP in the dark. The factor accepts the electron from the reaction site of the first photosystem and activates hydrogen liberation in the systems, containing hydrogenase. A possibility of an existence of an additional site of NADP reduction in chloroplasts is discussed.     

Photoreduction of NADP(+) in photosystem II of higher plants: requirement for manganese

The effects of reversible managanese extraction on NADP(+) photoreduction were studied with higher plant subchloroplast preparations of photosystem II (PS II). Under anaerobic conditions, when the reaction centers (RCs) of PS II are "closed" (i.e. in the state [P680 Pheo] Qā), and in the presence of ferredoxin-ferredoxin-NADP(+) reductase, NADP(+) reduction is observed at a rate of 0.8-1.1 μmol/mg x chlorophyII x h. After complete removal of manganese from PS II, the rate of NADP(+) reduction is reduced 40-5- fold. Upon the addition of Mn at a concentration of approx. 4 Mn atoms per reaction center, the NADP reduction is restored up to 85-90% of the initial value, When half of this amount of Mn is combined with about 40 times of the equivalent concentration of other divalent ions (Ca2?, Sr2?, Mg2? etc) the reaction is also reactivated. Dinoseb (10??M) an inhibitor of electron transfer in PS II prevents NADP(+) photoreduction. It is concluded that under conditions when the first quinone acceptor, Q(A), is in its reduced state (Qā), electrons are transferred from reduced pheophytin (Pheo(-)) to NADP(+), indicating that PS II can reduce NADP(+) without the participation of PS I. On the basis of these and literature data, and alternate pathway for electron phototransfer in PS II reaction centers of higher plants is suggested. Some problems concerning the Z-scheme are discussed.     

Reduction of 2,6-dichlorophenol-indophenol (Hill Reaction) by Chloroplasts with Different Chlorophyll a/b Ratios Under monochromatic Light

Chloroplasts with different chlorophyll a/b ratios were isolated from 7 to 8 days old wheat seedlings and the activities of reduction of 2,6-dichlorophenol-indophenol (DPIP) by these chloroplasts as function of the chlorophyll a/b ratios were studied under mono-chromatic light (650 m μ, 670 mμ, 680 mμ, 707 mμ). It was found that the DPIP reducing activities by these chloroplasts varied with their chlorophyll a/b ratios, and these variations are affected by the wavelengths of the illuminating light. Under 650 mμ, at the a/b ratios of 2.2 to 2.82, the activities of DPIP reduction in- creased with the a/b ratios, but decreased when the a/b ratios were higher than 2.82. Under 670 mμ, the DPIP reducing activities also varied with the a/b ratios of isolated chloroplasts. However, the variation was more gradual and steadier. Under 680 mμ, the DPIP reducing activities increased with the a/b ratios over 3.0, but decreased rather suddenly at a/b ratio of 3.30. Essentially the same relation held for 707 mμ, but the Hill reaction activities ceased to decline farther when a/b ratio rose to 3.40 at 707 mμ. When the results were analyzed in terms of the “relative activities” of the chloroplasts of the above mentioned wave lengths, it was found that the values of the "relative activity" (a/a+b) declined steadily from 1.47–1.29 at an a/b ratio range of 2.05–3.40, while the values of the "relative activity" (b/a+b)increased steadily from 3.10--4.40 at the same range of a/b ratios. But it is to be noted that, under 650 mμ, the (a/a+b) was 1.36 at the a/b ratio range of 2.63–2.82. Interesting enough, the DPIP reducing activities were the highest of all with these a/b ratios. Activities of DPIP reduction by isolated chloroplasts kept at 0 ℃, 20 ℃, 30 ℃, and 45 ℃ diminished with time when illuminated at all wavelengths. However, for those kept at 45 ℃ their activities were lost after 20 minutes, except those illuminated with the wave length of 680 mμ which still maintained 30 % of the initial activity. When kept at the above mentioned temperatures, the chlorophyll a/b ratios of all batches of chloroplasts declined steadily with time. The above results are interpreted as being indicative of the possibility that the pigment systems for the partial reaction (Hill reaction) of the over-all photosynthesis process consisted mainly of Chlb650 and Chla670. And the correlation of temperature and abolition of activity reduction of DPIP of chloroplasts varied under monochromatic light of different wave lengths.     

Mechanism of photoinactivation in photosynthetic systems II. The occurrence and properties of two different types of photoinactivation

Photoinactivation of the activity of NADP photoreduction withreduced DPIP or with reduced TMPD as the electron donor wasinhibited by the absence of oxygen in the atmosphere or by thepresence of photosynthetic inhibitors (CMU, DCMU, o-phenanthroline)in the preillumination mixture. Photoinactivation of the photoreductionof NADP or DPIP with water as the electron donor was not affected,or even accelerated, by these conditions of preillumination.The concentrations of inhibitors required for maximum inhibitionin the former case corresponded to those required for inhibitionof photosynthetic electron transport. The results indicatedthe occurrence of 2 different types of photoinactivation, eachspecifically affecting photosystems I and II, and differingin behaviours; including their requirement for oxygen in theatmosphere and their responses toward the presence of photosyntheticinhibitors during the preillumination period. (Received July 30, 1969; )     

Reversible dark-induced senescence of soybean root nodules

Nodule senescence was induced in intact soybean [Glycine max. (L.) Merr., cv Woodworth] plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod.     

NADP regulates the light activation of NADP-dependent malate dehydrogenase

The chloroplastic NADP-dependent malate-dehydrogenase (EC 1.1.1.82) activity is modulated by light and dark. The enzyme is activated upon illumination of intact or broken chloroplasts or by incubation with dithiothreitol, whereas dark has the opposite effect. The present communication shows an additional regulation of the light modulation: in isolated intact pea chloroplasts, light activation was inhibited in the presence of electron acceptors such as sodium bicarbonate, 3-phosphoglycerate or oxaloacetate, which consume NADPH₂ and produce NADP. With broken chloroplasts, addition of NADP resulted in a pronounced lag phase of NADP-dependent malate dehydrogenase light activation, while NADPH₂ was without any effect. The extent of the lag phase was correlated to the amount of NADP added. When light was replaced by dithiotreitol, the inhibition effect was even more pronounced. It was assumed that NADP inhibits the modulation reaction directly: reduced thioredoxin, a potent mediator of activation by light, or dithiotreitol appear to counteract NADP in a competitive manner. The results indicate a physiological role of NADP in the regulation of chloroplastic NADP-dependent malate dehydrogenase which is capable of removing electrons from the chloroplast, via oxaloacetate reduction and malate export. Thus an NADP concentration sufficient for continuous photosynthetic electron flow may be achieved.     

Light Dependent Increase of Triosephosphate Dehydrogenase in Pea Leaves

Data from 3 lines of investigation were presented indicating that chlorophyll is not necessary for the increase in the triphosphopyridine nucleotide-requiring triosephosphate dehydrogenase accompanying the illumination of etiolated pea plants. These include A) the kinetics of the development of chlorophyll and enzyme activity, B) the presence of enzyme activity in leaves grown in the dark on normal plants and C) the high specific enzyme activity in leaves of a chlorophyll-less mutant.It was also shown that the light-initiated increase of enzyme activity continues for several days after removal from the light and that illumination with far-red light before the dark period inhibited, but did not abolish, this increase. The ability of green plants to continue to produce the enzyme in the dark was eventually lost with time, for after 7 days in the dark a stimulation in leaf protein formation was not accompanied by an increase in enzyme activity.     

Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the k_max/K_s ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the k_max/K_s value. {k_max is the apparent catalytic rate constant of peptidyl transferase, and K_s is the dissociation constant of the encounter complex between acetyl (Ac)[³H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.     

The effects of starvation and refeeding on egg laying and the synthetic activity of the albumen gland in Bulinus truncatus, a snail vector of urinary schistosomiasis

1. The effects of starvation (for 1, 2, 3, 6, 9 and 12 days, respectively) and refeeding (12 days starvation and 1, 2 and 3 days refeeding, respectively) on egg laying and albumen gland activity in the freshwater snail, Bulinus truncatus were studied. 2. The egg laying of starving snails rapidly decreased and ceased by day 6 of starvation. Egg laying was restored 24 hr after refeeding. The recorded decrease in albumen gland wet weight was proportional to the starvation periods. The DNA contents of the glands of the different experimental groups was not statistically different from the controls. 3. Albumen gland synthetic activity expressed as 14C-glucose incorporation into galactogen/microgram DNA and 3H-amino acids into total protein was determined. The glands showed an abrupt decrease in synthetic activity after 1 day of starvation and gradually decreased further until days 9-12. The decrease in activity of the glands was more rapid than that of egg laying. Upon refeeding, the activity of the glands recovered rapidly, simultaneous with the increase in wet weight and egg laying. 4. In conclusion, there is a correlation between egg laying and the in vitro activity of albumen glands. The results show a short-term effect of starvation on the fecundity of the snails. Such studies could be useful in field studies as well as snail control by applying molluscicides under optimal conditions.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.