Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.     

Effect of acylphosphates on Ca2+ uptake by sarcoplasmic reticulum vesicles

The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca²⁺ and Mg²⁺ dependent: the lowering of the Ca⁺ concentration in the medium from 10^?4 to 10^?5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg²⁺, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg²⁺ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg²⁺ dependence; furthermore, in the absence of free Ca²⁺ and in the presence of 5 mm Mg²⁺, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiatio from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22°–24°C when the injury was located 800 μm from the soma. Elevating the temperature to 32°C or moving the lesion site to 400 or 1500 μm from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 × 10^?5 M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10^?5 M) did. The calcium channel blocker lanthanum (10^?6–10^?4 M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10^?3–10^?5 M), and the calcium ionophore A23187 (10^?5 M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH₄Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10^?5 M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10^?5 M) had no effect. Inhibition of protein synthesis with anisomycin (2 × 10^?4 to 2 × 10^?6 M) did not significantly suppress sprouting 24 h after axotomy. Both d and l isomers of glutamate (300 μM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.     

Calcium accumulation by isolated sarcoplasmic reticulum of skeletal muscle during development in tissue culture

The calcium uptake and ATPase activities of isolated sarcoplasmic reticulum were studied during the first six days of chick skeletal muscle maturation in tissue culture. Statistically significant increases in these activities were observed between the second and the sixth day of maturation. Increases in oxalate-dependent calcium uptake were demonstrated at concentrations of 2.5 × 10^?5 M calcium and 10^?4 M calcium. Calcium-binding determinations conducted in the absence of oxalate displayed changes manifested by an increase at day 5 followed by a significant decrease at day 6. Increases in total ATPase activity during maturation paralleled the sequential increases in calcium uptake. Calcium-stimulated ATPase activity, however, did not change significantly during periods of marked increase in calcium uptake, suggesting that these activities are dissociated during development of the sarcoplasmic reticulum. These data demonstrate that calcium uptake and total ATPase activity increase during muscle maturation in tissue culture and that these activities are present prior to spontaneous contractions.     

SHORT‐TERM EFFECT OF TEMPERATURE ON THE PHOTOKINETICS OF MICROALGAE FROM THE SURFACE LAYERS OF ANTARCTIC PACK ICE1

Microalgae growing within brine channels (85 psu salinity) of the surface ice layers of Antarctic pack ice showed considerable photosynthetic tolerance to the extreme environmental condition. Brine microalgae exposed to temperatures above ?5°C and at irradiances up to 350 μmol photons·m^?2·s^?1 showed no photosynthetic damage or limitations. Photosynthesis was limited (but not photoinhibited) when brine microalgae were exposed to ?10°C, provided the irradiance remained under 50 μmol photons·m^?2·s^?1. The highest level of photosynthetic activity (maximum relative electron transport rate [rETR_max]) in brine microalgae growing within the surface layer of sea ice was at approximately 18 μmol electrons·m^?2·s^?1, which occurred at ?1.8°C. Effective quantum yield of PSII and rETR_max of the halotolerant brine microalgae exhibited a temperature‐dependent pattern, where both parameters were higher at ?1.8°C and lower at ?10°C. Relative ETR_max at temperatures above ?5°C were stable across a wide range of irradiance.     

Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

Ultramicroassay for plasma renin concentration in the rat using the antibody-trapping technique

Using the antibody-trapping technique, picogram quantities of angiotensin-I generated during 24 hr of incubation at 37°C were stable and fully protected against peptidases. The method employs purification of angiotensin-I antisera on DEAE-cellulose and purification of renin substrate by affinity chromatography using specific antirenin antibodies in order to remove endogenous renin. The assay was performed in a single tube without a transfer step in a total volume of 30 μl at pH 6,5 with incubation for 24 hr at 37°C. With a normal rat plasma renin concentration of 5 × 10^?4 GU ml^?1, the detection limit was 10 nl or a total of 5 × 10^?9 GU. In the range 20–125 nl, precision was ±10%.     

Characterization and comparisons of five N2-fixing Azolla-Anabaena associations,I. Optimization of growth conditions for biomass increase and N content in a controlled environment*

Abstract Biomass increase, C and N content, C₂H₂ reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A. filiculoides Lam., A. mexicana Presl., and A. pinnata R. Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study. Under a 16 h, 26°C day at a light intensity of 200 μmol m^?2 s^?1 and an 8 h, 19° C dark period, there was no significant difference in the growth rates of the individual species on the five nutrient solutions employed. Growth was comparable from pH 5 to pH 8, but decreased at pH 9. Using the same photoperiod and light intensity but constant growth temperatures of 15–40°C, at 5°C intervals, the individual species exhibited maximum growth, nitro-genase (N²ase) activity and N content at either 25° or 30°C. There was no difference in the temperature optima at pH 6 and pH 8. The tolerance of the individual species to elevated temperature was indicated to be A. mexicana> A. pinnata> A. caroliniana> A.filiculoides. At the optimum temperature, growth rates increased with increasing photoperiod at both pH 6 and pH 8 but N₂ase activity was usually highest at a 16 h light period. At photon flux densities of 100, 200, 400 and 600 μmol m^?2 s^?1, during a 16 h light period and optimum growth temperature of the individual species, N₂ase activity was saturated at less than 200 μmol m^?2 s^?1 and growth at 400 μmol m^?2 s^?1.No interacting effects of light and pH were noted for any species, nor were light intensities up to 1700 μmol m^?2 s^?1 detrimental to the growth rate or N content of any species in a 5 week glasshouse study with a natural 14.5 h light period and a constant temperature of 27.5°C. Using the optimum growth temperature, a 16 h light period, and a photon flux density of at least 400 μmol m^?2 s^?1, the Azolla species all doubled their biomass in 2 days or less and contained 5–6% N on a dry weight basis.     

EFFECT OF TEMPERATURE,LIGHT AND pH ON GROWTH,PHOTOSYNTHESIS AND RESPIRATION OF THE DINOFLAGELLATE PERIDINIUM CINCTUM FA. WESTII IN LABORATORY CULTURES1

Optimum light, temperature, and pH conditions for growth, photosynthetic, and respiratory activities of Peridinium cinctum fa. westii (Lemm.) Lef were investigated by using axenic clones in batch cultures. The results are discussed and compared with data from Lake Kinneret (Israel) where it produces heavy blooms in spring. Highest biomass development and growth rates occurred at ca. 23° C and ≥50 μE· m^?2·s¹ of fluorescent light with energy peaks at 440–575 and 665 nm. Photosynthetic oxygen release was more efficient in filtered light of blue (BG 12) and red (RG 2) than in green (VG 9) qualities. Photosynthetic oxygen production occurred at temperatures ranging from 5° to 32° C in white fluorescent light from 10 to 105 μE·m^?2·s^?1 with a gross maximum value of 1500 × 10^?12 g·cell^?1·h^?1 at the highest irradiance. The average respiration amounted to ca. 12% of the gross production and reached a maximum value of ca. 270·10^?12 g·cell^?1·h^?1 at 31° C. A comparison of photosynthetic and respiratory Q₁₀-values showed that in the upper temperature range the increase in gross production was only a third of the corresponding increase in respiration, although the gross production was at maximum. Short intermittent periods of dark (>7 min) before high light exposures from a halogen lamp greatly increased oxygen production. Depending on the physiological status of the alga, light saturation values were reached at 500–1000 μE·m^?2·s^?1 of halogen light with compensation points at 20–40 μE·m^?2·s^?1 and I_k-values at 100–200 μE·m^?2·s^?1. The corresponding values in fluorescent light in which it was cultured and adapted, were 25 to 75% lower indicating the ability of the alga to efficiently utilize varying light conditions, if the adaptation time is sufficient. Carbon fixation was most efficient at ca. pH 7, but the growth rates and biomass development were highest at pH 8.3.     

Displacement and Return Movement of Chloroplasts in the Marine Dinophyte Pyrocystis noctiluca. Experiments with Optical Tweezers

Infrared laser traps (optical tweezers) were used to study laser-induced organelle movements in the marine alga Pyrocystis noctiluca (Dinophyta). These cells are highly suitable for optical micromanipulation due to their large size and extensive vacuole. Experiments were done with plastids held by optical tweezers and moved from the nuclear area into the vacuole. The subsequent retraction movement was analysed for speed. The displaced organelles remained connected to their original position by a thin cytoplasmic strand, often less than 1 μm in diameter. When the organelles were released they rapidly returned at an initial rate of 81.7 ± 7.8 μm . s^?1 (overall displacement 50 μm, measured distance 20 μm, 25 °C ± 1 °C, number of cells 22), slowing down with progressive retraction of the connecting strand. The return movement was reduced to 4.2 ± 0.2 μ .s^?1 (n = 10) when the organelles were displaced and held for 1 min. Displacement to a longer distance increased the rate of return movement. A change from a high to a low environmental temperature significantly reduced movement from 94.5 ± 9.0 . s^?1 (30 °C ± 1 °C, n = 22) to 34.5 ± 2.7 μm .s^?1 (5°C ± 1 °C, n = 22). Nocodazole and N-ethylmaleimide (NEM), inhibitors of microtubules and acto-myosin, respectively, did not affect the retraction of the connecting strand, but at high concentrations of NEM it became increasingly difficult to move organelles away from the nuclear area. We suggest that the return movement of organelles within laser-induced artificial strands mainly depends on the viscoelastic properties of the tonoplast. The quantification of these properties by optical tweezers allows determination of reactions of plant cells to temperature changes.     

The Effect of Quinidine on Calcium Accumulation by Isolated Sarcoplasmic Reticulum of Skeletal and Cardiac Muscle

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg⁺⁺, oxalate, and ⁴⁵Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10^-4 M quinidine to 90% with 10^-3 M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10^-6 M quinidine to 100% with 10^-3 M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca⁺⁺-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.     

The effect of temperature on photosynthetic and respiratory electron transport system activity in the shallow and deep-living phytoplankton of a subalpine lake

SUMMARY. 1. The influence of temperature on in vivo photosynthetic and in vitro respiratory electron transport system (ETS) activity was determined over the season for the 3 m (warm-water) and a 20m (cold-water) phytoplankton communities in Castle Lake. The optimum temperature of photosynthesis at 3 m (X?=20.8°C) was significantly higher than the average optimum at 20 m (X?=14.8°C). 2. Seasonally, the photosynthetic temperature optimum increased when the blue-green alga Chroococcus limneticus Lemm. was present. The temperature characteristics of this organism were maintained even after it had settled into the cold water of the hypolimnion. 3. Temperature optima were not significantly different in experiments conducted under limiting or saturating photosynthetic photon flux densities (PPFD). 4. Short-term (1 h) preincubations with dissolved inorganic nitrogen (DIN) (?80 μg NH₄NO₃-N l^?1) had little effect on the temperature characteristics of photosynthesis while the longer (>24 h) incubations provided by a whole-lake epilimnetic DIN addition (?75 μg NH₄NO₃- N l^?1) significantly lowered the photosynthetic temperature optimum to 12.5°C. Once this epilimnetic DIN was depleted the optimum roseto25°C, a value higher than that present before the enrichment, which coincided with the growth of C limneticus. 5. Respiratory ETS activity usually began to inactivate between 19 and 20°C. However, when C. limneticus was abundant the inactivation temperature was often greater ihan 25°C. 6. The average energy of activation (E) and Q₁₀ value for the 3 m community (15.9 kcal mol^?1 and 2.6 respectively) were significantly higher than those at 20 m (14.2 kcal mol^?1 and 2.4 respectively). Seasonally, the highest E and Q₁₀ values of ETS activity occurred during the late-summer bloom of C. limneticus. 7. These results demonstrate that the epilimnetic and hypolimnetic phytoplankton communities in Castle Lake are physiologically distinct with regards to their temperature characteristics.     

EFFECTS OF LIGHT AND TEMPERATURE ON NET PHOTOSYNTHESIS AND DARK RESPIRATION OF GAMETOPHYTES AND EMBRYONIC SPOROPHYTES OF MACROCYSTIS PYRIFERA1

The rates of net photosynthesis as a function of irradiance and temperature were determined for gametophytes and embryonic sporophytes of the kelp, Macrocystis pyrifera (L.) C. Ag. Gametophytes exhibited higher net photosynthetic rates based on oxygen and pH measurements than their derived embryonic sporophytes, but reached light saturation at comparable irradiance levels. The net photosynthesis of gametophytes reached a maximum of 66.4 mg O₂ g dry wt^?1 h^?1 (86.5 mg CO₂ g dry wt^?1 h^?1), a value approximately seven times the rate reported previously for the adult sporophyte blades. Gametophytes were light saturated at 70 μE m^?2 s^?1 and exhibited a significant decline in photosynthetic performance at irradiances 140 μE m^?1 s^?1. Embryonic sporophytes revealed a maximum photosynthetic capacity of 20.6 mg O₂ g dry wt^?1 h^?1 (25.3 mg CO₂ g dry wt^?1 h^?1), a rate about twice that reported for adult sporophyte blades. Embryonic sporophytes also became light saturated at 70 μE m^?2 s^?1, but unlike their parental gametophytes, failed to exhibit lesser photosynthetic rates at the highest irradiance levels studied; light compensation occurred at 2.8 μE m^?2 s^?1. Light-saturated net photosynthetic rates of gametophytes and embryonic sporophytes varied significantly with temperature. Gametophytes exhibited maximal photosynthesis at 15° to 20° C, whereas embryonic sporophytes maintained comparable rates between 10° and 20° C. Both gametophytes and embryonic sporophytes declined in photosynthetic capacity at 30° C. Dark respiration of gametophytes was uniform from 10° to 25° C, but increased six-fold at 30° C; the rates for embryonic sporophytes were comparable over the entire range of temperatures examined. The broader light and temperature tolerances of the embryonic sporophytes suggest that this stage in the life history of M. pyrifera is well suited for the subtidal benthic environment and for the conditions in the upper levels of the water column.     

Citrate transport in liposomes reconstituted with Triton extracts from mitochondria

The exchange between external [¹⁴C] citrate and internal citrate, malate or phosphoenopyruvate can be reconstituted with a Triton extract of submitochondrial particles from rat liver. The reconstituted activity is dependent on the phospholipid composition of the liposomes and is influenced by the simultaneously incorporated Triton. The kinetic properties, the substrate and tissue specificity, and the inhibitor sensitivity of citrate transport in liposomes are similar to those described for the tricarboxylate transport in mitochondria. The maximal rate of citrate exchange in the reconstituted system (13.5 μmol × min^?1 × g^?1 at 25°C and pH 7.5) accounts for 12% of the original mitochondrial activity.     

Photosynthesis of Marine Macroalgae from Antarctica: Light and Temperature Requirements

The photosynthetic performance of macroalgae isolated in Antarctica was studied in the laboratory. Species investigated were the brown algae Himantothallus grandifolius, Desmarestia anceps, Ascoseira mirabilis, the red algae Palmaria decipiens, Iridaea cordata, Gigartina skottsbergii, and the green algae Enteromorpha bulbosa, Acrosiphonia arcta, Ulothrix subflaccida and U. implexa. Unialgal cultures of the brown and red algae were maintained at 0°C, the green algae were cultivated at 10°C. I_K values were between 18 and 53 μmol m^?2 s^?1 characteristic or low light adapted algae. Only the two Ulothrix species showed higher I_K values between 70 and 74 μmol m^?2 s^?1. Photosynthesis compensated dark respiration at very low photon fluence rates between 1.6 and 10.6 μmol m^?2 s^?1. Values of α were high: between 0.4 and 1.1 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the brown and red algae and between 2.1 and 4.9 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the green algal species. At 0°C P_max values of the brown and red algae ranged from 6.8 to 19.1 μmol O₂ g^?1 FW h^?1 and were similarly high or higher than those of comparable Arctic-cold temperate species. Optimum temperatures for photosynthesis were 5 to 10°C in A. mirabilis, 10°C in H. grandifolius, 15°C in G. skottsbergii and 20°C or higher in D. anceps and I. cordata. P: R ratios strongly decreased in most brown and red algae with increasing temperatures due to different Q₁₀ values for photosynthesis (1.4 to 2.5) and dark respiration (2.5 to 4.1). These features indicate considerable physiological adaptation to the prevailing low light conditions and temperatures of Antarctic waters. In this respect the lower depth distribution limits and the northern distribution boundaries of these species partly depend on the physiological properties described here.     

Characterization of the Galactose-Specific Binding Activity of a Purified Soluble Entamoeba histolytica Adherence Lectin

ABSTRACT. We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffnity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4°C by radioimmunoassay; the dissociation coefficient (Kd was 2.39 × 10^?8 M^?1 with 5.97 × 10⁴ lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 × 10⁵/cell) rather than a significantly reduced dissociation constant (4.93 × 10^?8 M^?1). At 4°C, there was no measurable Gal-specific binding of the adherence protein to the Lec and IdlD.Lecl CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 μg/ml) to CHO cells was equivalent at 4°C and 37°C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 μg/ml). Gal-specific binding of the lectin at 4°C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells. In summary, these findings indicate that the purified E. histolytica adherence lectin demonstrates saturable Gal-specific binding to 1–6 branched-N-linked and not O-linked galactose terminal cell surface carbohydrates; however, apparently only a small percentage of purified amebic lectin molecules actually possess galactose binding activity.