Mg-protoporphyrin-IX and delta-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. delta-Aminolevulinic acid sysnthesis

A cell-free chloroplast preparation obtained from greening cucumber cotyledons catalyzes the biosynthesis of δ-amino[¹⁴C]levulinic acid from [¹⁴C]glutamate. The reaction is greatly enhanced by the inclusion of ATP, glutathione, and levulinic acid (an inhibitor of δ-aminolevulinate dehydratase). The presence of NAD, light, and α,α′-dipyridyl stimulated the reaction slightly. Oxygen was not required for the reaction. Synthesis was linear for the first 15 min of the reaction at 6.6 pmol · min^?1 · (mg of protein)^?1 and then slowed to a rate of 0.75 pmol · min^?1 (mg of protein)^?1, which was sustained for at least 1.5 h. The optimum temperature for this conversion is 37 °C. Although the activity was drastically diminshed by lysing, intactness was not an absolute requirement. Possible mechanisms of reaction and regulation are discussed.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells

The membrane-bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Millipore filters with pores 0.22 μm in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5′-guanylyl-imidodiphosphate at a concentration of 1 · 10^?6 M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5′-guanylyl-imidodiphosphate.     

The influence of light on copper‐limited growth of an oceanic diatom,Thalassiosira oceanica (Coscinodiscophyceae)

Thalassiosira oceanica (CCMP 1005) was grown over a range of copper concentrations at saturating and subsaturating irradiance to test the hypothesis that Cu and light were interacting essential resources. Growth was a hyperbolic function of irradiance in Cu‐replete medium (263 fmol Cu′ · L^?1) with maximum rates achieved at 200 μmol photons · m^?2 · s^?1. Lowering the Cu concentration at this irradiance to 30.8 fmol Cu′ · L^?1 decreased cellular Cu quota by 7‐fold and reduced growth rate by 50%. Copper‐deficient cells had significantly slower (P < 0.0001) rates of maximum, relative photosynthetic electron transport (rETR_max) than Cu‐sufficient cells, consistent with the role of Cu in photosynthesis in this diatom. In low‐Cu medium (30.8 fmol Cu′ · L^?1), growth rate was best described as a positive, linear function of irradiance and reached the maximum value measured in Cu‐replete cells when irradiance increased to 400 μmol photons · m^?2 · s^?1. Thus, at high light, low‐Cu concentration was no longer limiting to growth: Cu concentration and light interacted strongly to affect growth rate of T. oceanica (P < 0.0001). Relative ETR_max and Cu quota of cells grown at low Cu also increased at 400 μmol photons · m^?2 · s^?1 to levels measured in Cu‐replete cells. Steady‐state uptake rates of Cu‐deficient and sufficient cells were light‐dependent, suggesting that faster growth of T. oceanica under high light and low Cu was a result of light‐stimulated Cu uptake.     

CRITICAL IRRADIANCE LEVELS AND THE INTERACTIVE EFFECTS OF QUANTUM IRRADIANCE AND DOSE ON GAMETOGENESIS IN THE GIANT KELP,MACROCYSTIS PYRIFERA1

Gametophytes of Macrocystis pyrifera (L.) C. Ag. were cultured under a series of quantum irradiances in three photoperiod regimes. The quantum irradiances in each photoperiod were adjusted to provide equal daily irradiation dosages between photoperiods which allowed a critical examination of the interactions between quantum irradiance and quantum dose in determining gametophyte fertility. The lowest quantum irradiance which stimulated gametogenesis in more than 50% of the female gametophytes was 5 μE·m^?2·s^?1. The saturating irradiance was ca. 10 μE·m^?2·s^?1 at photoperiods of 12 h or greater. In terms of daily quantum dose, the lowest dose at which greater than 50% gametogenesis occurred was 0.2 E·m^?2·d^?1. However, this critical quantum dose was higher (0.4 E·m^?2·d^?1) when instantaneous irradiances were less than 5 μE·m^?2·s^?1. The saturation quantum dose was also affected by the rate at which the quantum dose was received and varied from 0.4 to 0.8 E·m^?2·d^?1. Gametophytes in all three photoperiods reached 100% fertility at quantum irradiances above 5 μE·m^?2·s^?1. Photoperiod effects were small and could be accounted for by quantum dosage effects.     

Raman spectroscopic elucidation of DNA backbone conformations for poly(dG-dT).poly(dA-dC) and poly(dA-dT).poly(dA-dT) in CsF solution

Raman spectra have been recorded for poly(dG-dT) · poly(dA-dC) and poly(dA-dT) · poly(dA-dT) in low salt and at high concentrations of CsF. Poly(dG-dT) · poly(dA-dC) shows no change in the 682-cm^?1 guanine mode, demonstrating the absence of the Z-structure at high salt. The 790-cm^?1 phosphodiester symmetric stretch, however, shifts up 5 cm^?1 in 4.3M CsF, suggesting a slight conformational change, associated with ion binding or hydration changes. Poly(dA-dT) · poly(dA-dT) shows an additional broad band at 816 cm^?1, attributed to the phosphodiester modes associated with the C3′-endo deoxyribose units in the alternating B-structure. In this case, both the 841- and the 816-cm^?1 asymmetric phosphodiester stretches, associated with the C2′- and C3′-endo units, shift down on addition of CsF in a sequential manner. Correlation of this sequence with that previously observed for the two ³¹P-nmr resonances, establishes that the phosphodiester stretching frequencies depend on the conformation of the 5′-sugar, and not on the 3′-sugar.     

Raman spectroscopic study on low-frequency collective modes in self-associates of guanosine monophosphates

Low-frequency Raman spectra of the self-associates of guanosine monophosphates (GMPs) Na₂ · 5′GMP, K₂ · 5′GMP, Na₂ · 3′GMP, and K₂ · 3′GMP, and polyribonucleic acid K · poly(rG), were obtained. In acidic gels and dried fibers, GMP molecules are known to form helical stacks of hydrogen-bonded tetramers. Some low-frequency collective modes specific to the helically stacked structures were observed. We examined the dependence of these modes on counterions and water content. The lowest frequency mode at ca. 20 cm^?1 is sensitive to the water content of the sample and is clearly visible in solid-state samples, so it works as a marker band of the environmental condition of the helices. The intensity and the peak frequency of this mode in solid-state samples depend on the helical structure and counterions. The broad peaks in the vicinity of 100 cm^?1 are influenced by cations and are independent of water content.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

A procedure is described which unequivocally demonstrates the presence of adenosine 3′:5′-cyclic monophosphate in Phaseolus vulgaris. Its concentration was determined spectrophotometrically at 2·6–9·2 nmol g^?1 of tissue (dry wt) for 6-day-old seedlings and about one-tenth of this in 13-day-old plants.     

Stimulation of mammalian adenylate cyclase activity with guanosine 5′-tetraphosphate and other guanine nucleotides

Guanosine 5′-tetraphosphate (GTP₄) stimulated mammalian adenylate cyclase activity at concentrations down to 1 μM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP₄ stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3′, 5′-monophosphate and 5′-guanylylimidodiphosphate (GMP · PNP) also stimulated mammalian adenylate cyclase activity. GMP · PNP irreversibly activated, whereas GTP₄ and GTP reversibly activated adenylate cyclase. Adenosine 5′-tetraphosphate (ATP₄) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP₄. The formation of cyclic AMP by GTP₄-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H⁺), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP₄ was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The β-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP₄, thus, suggesting that GTP₄ and β-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP₄ or GMP. PNP, thus indicating that GTP₄ resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to β-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2′-monophosphate, guanosine 3′-monophosphate or guanosine 2′,5′-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.     

Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

The periodic change in adenosine 3′,5′-monophosphate concentration in sea-urchin eggs

The level of adenosine 3′,5′-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0·10^?7 M and 1.5·10^?6 M, respectively. The activity of adenylate cyclase in a 105 000 × g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl₂ increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5·10^?6 M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Far-infrared absorption of nucleotides and poly(I)·poly(C) RNA

We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ～25 to ～500 cm^?1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm^?1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm^?1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm^?1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.