Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

The chemotactic activity of various sirenins and analogues and the uptake of sirenin by the sperm of allomyces

Optimal response of the sperm of Allomyces from the highly male strain M16 to the chemotactic agent, sirenin, was shown to occur when the sperm suspension contained 2 mm piparazine-N′, N-bis[2-ethane sulfonic acid] buffer, 3 mm CaCl₂, and chelated trace elements. For the male strain M3, the CaCl₂ needed was 3.5 mm with the other two components the same as for M16. The inclusion in the sperm suspension of MgCl₂, KH₂PO₄, or NH₄Cl was without effect, except that under certain conditions phosphate was detrimental. The variability of 10 replicate assays was substantially reduced by using sperm in the bioassay at a concentration of 500,000 per ml rather than the former concentration of 100,000 per ml with a concomitant reduction in the concentration of sirenin above the membrane to which the sperm were attracted.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Isolation of active mitochondria from tomato fruit

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl₂, 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 × g for 15 minutes. The supernatant is then centrifuged at 11,000 × g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl₂, 10 mm tris-HCl, 10 mm KH₂PO₄ and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and `Honey Dew' melon.     

Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores'' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores'' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca²⁺ divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP⁺ spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 μM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores'' inner membrane.     

Light and calcium interactions in chlorella inhibited by sodium chloride

Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na⁺ and Cl⁻, more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O₂ evolution, and decreased rates of CO₂ fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca²⁺ extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm² increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm² added Ca²⁺ reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca²⁺. Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO₄ and maintenance of the light intensity at 9.4 mw/cm². The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm² for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm² and 8.2 doublings/day at 9.4 mw/cm² with 10 mm CaSO₄ added.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Nucleoside Diphosphate-sugar 4-Epimerases I. Uridine Diphosphate Glucose 4-Epimerase of Wheat Germ

Uridine diphosphate (UDP)-glucose 4-epimerase (EC 5.1.3.2) has been purified over 1000-fold from extracts of wheat germ by MnCl₂ treatment, (NH₄)₂SO₄ fractionation, Sephadex column chromatography, and adsorption onto and elution from calcium phosphate gel. The enzyme has a pH optimum of 9.0. Km values are 0.1 mm for UDP-d-galactose and 0.2 mm for UDP-d-glucose. NAD is required for activity; K_a = 0.04 mm. NADH is an inhibitor strictly competitive with NAD; K_i = 2 μm. Wheat germ also contains UDP-l-arabinose 4-epimerase (EC 5.1.3.5) and thymidine diphosphate (TDP)-glucose 4-epimerase which are distinct from UDP-glucose 4-epimerase.     

An Anomaly in Potassium Accumulation by Barley Roots: II. Effect of Calcium Concentration and Rubidium-86 Labeling

Excised barley roots accumulated 40 to 50% more K⁺ from 0.04 mm than from 0.06 mm KCl when incubated for 24 hours in KCl solutions containing 0.2 mm CaSO₄. This phenomenon was not markedly influenced by the rate of absorption of the counteranion. The presence of Na⁺ in the treatment solutions decreased total K accumulation but did not alter the K⁺ concentration at which the accumulation peak occurred. Short interval studies indicated that this phenomenon is easily observable after 4 hours and begins to become apparent within 2 hours. In comparison with barley, accumulation of K⁺ by excised wheat roots decreased as KCl concentration was increased from 0.02 to 0.06 mm; but K⁺ accumulation curve for corn roots showed no peaks or depressions in the concentration range of 0.01 to 0.1 mm. A normal hyperbolic curve was noted for the accumulation of Na⁺ from 0.01 to 1 mm NaCl by barley roots.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar

Several weakly transported sugars were tested for transport by the Na⁺-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na⁺ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K_i for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na⁺-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na⁺-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydrolyase (deaminating) in a species of Corynebacterium

1. Three bacterial isolates capable of growth on l-threonine medium only when supplemented with branched-chain amino acids, and possessing high l-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that l-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of l-threonine 3-dehydrogenase or l-threonine aldolase in threonine catabolism. 3. l-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [S_0.5], was 40mm and the Hill coefficient (h) was 2.0. l-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60μm, but did not affect the Hill constant. At the fixed l-threonine concentration of 10mm, the effect of l-valine was biphasic, progressive activation occurring at concentrations up to 2mm-l-valine, but was abolished by higher concentrations. Substrate-saturation plots for the l-valine-activated enzyme exhibited normal Michaelis–Menten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the `biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of l-threonine dehydratase was constitutive and was not subject to multivalent repression by l-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of l-threonine, apparently initiated by a `biosynthetic' l-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Endogenous metabolism of fungus spores: stimulation by physical and chemical means

Endogenous respiration of spores of the fungus Myrothecium verrucaria can be stimulated up to over-10 fold by diverse chemicals or by physical treatments. Greatest effects were caused by azide (12-fold at 250 μm) and by 2,4-dinitrophenol (7-fold at 300 μm). Marked stimulation was also caused by 10 μm silver (5-fold), 30 μm pentachlorophenol (6-fold), 10 μm carbonyl cyanide m-chlorophenyl hydrazone (4.5-fold) and 10 μm merthiolate (4-fold). Physical treatments such as heat (50 C), freezing, and sonication at sublethal levels were also stimulatory. Stimulation by azide or dinitrophenol was much greater in young than in old spores, whereas response to other chemicals and to freezing was relatively unaffected by spore age. In older spores the effect of azide was no greater than some other inhibitors. During incubation with azide, the endogenous trehalose reserves decreased and changes in free amino acids occurred, both increases and decreases. Thus anabolic as well as catabolic changes occur as evidenced also by the germination of a few (up to 5%) spores. The mechanisms of stimulation must be varied and complex. Permeability changes in the membrane confining endogenous reserves are proposed as a common initial cause. Additional changes in characteristics of membranes of other subcellular particles, as well as enzymic phenomena such as uncoupling of oxidative phosphorylation, are presumably involved in instances where greater stimulation occurs. The data are consistent with the hypothesis that dormancy in these spores results from separation of substrates from metabolic enzymes and more specifically that metabolites are sequestered rather than enzymes.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

Electrically induced protoplast fusion using pulse electric fields for dielectrophoresis

The formation of protoplast chains in suspensions of isolated pea (Pisum sativum cv Ran 1) mesophyll protoplasts induced by electric fields was studied. The chain formation induced by a sine-wave field (2 V, peak to peak; 500-0.1 kHz) was compared to that induced by an alternating pulse field (1 V, amplitude; 0.1-0.4 kHz). An increased number of dielectrophoretically paired protoplasts, formation of protoplast chains in the presence of CaCl₂ up to 5 mm, and protoplast fusion in the presence of 3 mm CaCl₂ were found when the pulse field was applied. The present results suggest the possibility of electrically induced protoplast fusion at cation concentrations that prevent fusion when sine-wave fields are applied.     

Photoinhibition in Vitis californica: The Role of Temperature during High-Light Treatment

Leaves of Vitis californica Benth. (California wild grape) exposed to a photon flux density (PFD) equivalent to full sun exhibited temperature-dependent reductions in the rates or efficiencies of component photosynthetic processes. During high-PFD exposure, net CO₂ uptake, photon yield of oxygen evolution, and photosystem II chlorophyll fluorescence at 77 Kelvin (F_m, F_v, and F_v/F_m) were more severely inhibited at high and low temperatures than at intermediate temperatures. Sun leaves tolerated high PFD more than growth chamber-grown leaves but exhibited qualitatively similar temperature-dependent responses to high-PFD exposures. Photosystem II fluorescence and net CO₂ uptake exhibited different sensitivities to PFD and temperature. Fluorescence and gas exchange kinetics during exposure to high PFD suggested an interaction of multiple, temperature-dependent processes, involving both regulation of energy distribution and damage to photosynthetic components. Comparison of F_v/F_m to photon yield of oxygen evolution yielded a single, curvilinear relationship, regardless of growth condition or treatment temperature, whereas the relationship between F_m (or F_v) and photon yield varied with growth conditions. This indicated that F_v/F_m was the most reliable fluorescence indicator of PSII photochemical efficiency for leaves of different growth conditions and treatments.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.